Department of Cell & Developmental Biology

Eighteenth Annual Retreat October 22, 2021

JOE C. DAVIS YMCA OUTDOOR CENTER YMCA CAMP WIDJIWAGAN

YMCA OF MIDDLE TENNESSEE 3088 Smith Springs Road, Nashville, TN 37013 P (615) 360-2267 https://www.campwidji.org/retreats-and-events/facilities/nelson-andrews-leadership-lodge-great-room

Street entrance to Camp Widjiwagan

Aerial view of Camp Widjiwagan and the Nelson Andrews Leadership Center

Front Entrance to the Nelson Andrews Leadership Center

Session I.A

The InsP3R coordinates mitochondrial behaviors to promote longevity Feng, GF, Burkewitz, KB

ABSTRACT Mitochondrial function plays a central role in aging and a variety of age-onset diseases. While interventions that mildly inhibit mitochondrial function can extend lifespan from yeast to mammals, too severe a disruption in mitochondrial function is toxic or lethal. Therefore, in order to therapeutically leverage the beneficial effects of mitochondrial reprogramming in contexts of age or mitochondrial disease, we must understand how cells control this delicate balance between mitochondrial protection and dysfunction. Previously, we discovered that inter-organelle calcium signaling from the endoplasmic reticulum (ER) to mitochondria regulates lifespan in C. elegans. Whether thesecalcium signaling pathways play important roles in mitochondrial longevity paradigms, however, is little explored. We now reveal that lifespan extension via mild inhibition of electron transport chain (ETC) requires the inositol triphosphate receptor (InsP3R), a conserved calcium release channel on the ER surface. The InsP3R mediates both direct flux of calcium into the mitochondrial matrix as well as into the cytosol, and we find that InsP3R function regulates diverse mitochondrial processes, including bioenergetics, and fission/fusion dynamics. Furthermore, we have identified that the InsP3R plays novel roles in regulating retrograde transcriptional responses from the mitochondrial network. We are currently identifying which of these InsP3R-dependent processes are critical in longevity, and mitochondrial dynamics appear to be particularly important. Collectively, these results suggest that the ER InsP3R plays a central role in reprogramming mitochondrial functions and promoting longevity when ETC function is impaired.

Session I.B

Nanoscale organization and molecular assemblies of transcellular nanocolumns in chemical synapses

Sun, R; Wilson, L; Haider, M; Wang, XY; Alten, B; Zhou, QJ

ABSTRACT As key functional units in neural circuits, neuronal synapses play important roles in brain information processing, learning and memory. Synaptic abnormalities are believed to underlie various neurological and psychiatric disorders. Resolving the structure of synapses will lead to better understanding of synaptic functions in the brain. Here, by using the cutting-edge cryo-electron tomography technique in synapses of cultured neurons and isolated synaptosomes, we are able to visualize in situ three-dimensional organization of synaptic organelles and macromolecules in their native states. Quantitative analyses of the synapses reveal the transcellular nanocolumns in the synapses of cultured neurons. Furthermore, high-resolution tomograms obtained from the synaptosome sample allows us to detect the similar transcellular nanocolumns, as well as glutamate receptor-like particles and putative adhesion molecules. These results clarify unresolved issues regarding the ultrastructural features of synapses and support the idea that trans-synaptic alignment exists to carry out synaptic functions.

Session I.C

Synergistic action of WDR5 and HDM2 inhibitors in SMARCB1-deficient cancer cells

Florian, AC; Woodley, CM; Wang, J; Guerrazzi, K; Chih-Yuan Hsu, C; Matlock, BK; Flaherty, DK; Lorey, SL; Liu, Q; Fesik, SW; Howard, GC; Weissmiller, AM; Tansey, WP

ABSTRACT Rhabdoid tumors (RT) are rare and deadly pediatric cancers driven by loss of SMARCB1, which encodes the SNF5 component of the SWI/SNF chromatin remodeling complex. Loss of SMARCB1 is associated with a complex set of molecular and phenotypic changes, including vulnerability to inhibitors of protein synthesis and of the p53 ubiquitin-ligase HDM2. Recently, we discovered small molecule inhibitors of the "WIN" site of WDR5, which in MLL-rearranged leukemia cells decrease the expression of a set of genes linked to protein synthesis, inducing a translational choke and causing p53-dependent inhibition of proliferation. Here, we characterize how WIN site inhibitors act in RT cells. As in leukemia cells, WIN site inhibition in RT cells causes the rapid and comprehensive displacement of WDR5 from chromatin, resulting in a decrease in protein synthesis gene expression. Unlike leukemia cells, however, the response of RT cells to WIN site blockade occurs independent of p53. Exploiting this observation, we demonstrate that WIN site inhibitor synergizes with an HDM2 antagonist to block RT cell proliferation in vitro. These data reveal a p53-independent action of WIN site inhibitors, and forecast that future strategies to treat RT could be based on dual WDR5/HDM2 inhibition.

Session II.A

Apoptotic Find-me Signals are an Essential Driver of Stem Cell Conversion to The Cardiac Lineage

Fort, L, Gama V., Macara I.G

ABSTRACT Pluripotent and embryonic stem cells (iPSCs, ESCs) can be driven by manipulation of Wnt signaling through a series of states similar to those that occur during early embryonic development, transitioning from an epithelial phenotype into the cardiogenic mesoderm lineage and ultimately into functional cardiomyocytes. Strikingly, we observed that iPSCs and ESCs undergo widespread apoptosis upon Wnt activation, followed by a synchronous epithelial-mesenchymal transition (EMT). The EMT requires induction of transcription factors SNAI1/SNAI2 downstream of MESP1 expression, and double knock-out of SNAI1/2, or loss of MESP1 in iPSCs blocks EMT and prevents cardiac differentiation. Remarkably, blockade of early apoptosis chemically or by ablation of pro-apoptotic genes also completely prevents the EMT, suppressing even the earliest events in mesoderm conversion, including EOMES, TBX6, and MESP1 induction. Conditioned medium from WNT-activated WT iPSCs overcomes the block to EMT by cells incapable of apoptosis (Apop-), suggesting the involvement of soluble factors from apoptotic cells in mesoderm conversion. Treatment with a purinergic P2 receptor inhibitor or addition of apyrase demonstrated a requirement for nucleotide triphosphate signaling. ATP was sufficient to induce a partial EMT in Apop- cells treated with WNT activator. We conclude that nucleotides, in addition to acting as chemo-attractants for clearance of apoptotic cells can, unexpectedly, function as essential paracrine signals in mesoderm specification.

Session II.B

Characterization of the cell of origin and implication of the immune microenvironment in the progression of colorectal cancer

Rolong, A., Chen, B., Scurrah, C.R., McKinley, E.T., Simmons, A.J., Ramirez-Solano, M.A., Markham, N.O., Vega, P.N., Heiser, C.N., Coffey, R.J., Shrubsole, M.J., Lau, K.S.

ABSTRACT Colorectal cancer (CRC) is the 3rd most deadly and the 4th most commonly diagnosed type of cancer worldwide. The disease can arise from precursor polyps that differ in their cellular origins, molecular heterogeneity, and immunogenic potential. Our previous work on a single-cell transcriptomic and imaging atlas of the two most common human colorectal polyps, conventional adenomas and serrated polyps, revealed adenomas arise from WNT-driven expansion of stem cells, while serrated polyps may come from differentiated cells through gastric metaplasia. Metaplasia-associated damage is coupled to a cytotoxic immune microenvironment preceding hypermutation, which is driven partly by antigen presentation differences associated with tumor cell differentiation states. We validated these results using mouse models where a single mutation (BrafV600E), representative of the serrated type, revealed that the cytotoxic infiltration only occurs in the differentiated cell compartment ("villus regions") even though both stem and differentiated cells should have the mutation. This led us to hypothesize that the differentiation status of neoplastic cells may play a role in modulating the immune microenvironment. To investigate the effects of the differentiation state of tumor cells, we used two models of stem and non-stem cell driven tumors where the same tumorigenic process (APC mutation) was used in Lrig1- and Mist1-driven tumors, respectively. We found non-stem-driven tumors present an increased cytotoxic environment and cells derived from these tumors have higher antigen presentation capabilities and decreased stem cell potential. Collectively, our findings provide a paradigm shift into the malignant progression of CRC and the role of the accompanying tumor microenvironment which could lead to new avenues for tailored treatment based on particular tumor type.

Session II.C

Direct visualization of microvilli biogenesis Gaeta I, Meenderink L, Postema M, Cencer C, Tyska M

ABSTRACT Microvilli are actin-bundle-supported surface protrusions that play essential roles in diverse epithelial functions. To develop our understanding of microvilli biogenesis, we used live imaging to directly visualize protrusion growth at early stages of epithelial differentiation. Time-lapse data revealed that specific factors, including epidermal growth factor pathway substrate 8 (EPS8) and insulin-receptor tyrosine kinase substrate (IRTKS) (also known as BAIAP2L1), appear in diffraction-limited puncta at the cell surface and mark future sites of microvillus growth. New core actin bundles elongate from these puncta in parallel with the arrival of ezrin and subsequent plasma membrane encapsulation. In addition to de novo growth, we also observed that new microvilli emerge from pre-existing protrusions. Moreover, we found that nascent microvilli can also collapse, characterized first by loss of membrane wrapping and ezrin enrichment, followed by a sharp decrease in distal tip EPS8 and IRTKS levels, and ultimately disassembly of the core actin bundle itself. These studies are the first to offer a temporally resolved microvillus growth mechanism and highlight factors that participate in this process; they also provide important insights on the growth of apical specializations that will likely apply to diverse epithelial contexts.

Session III.A

cAMP controls a trafficking mechanism that maintains the neuron specificity and subcellular placement of electrical synapses during development

Palumbos S, Skelton R, McWhirter R, Mitchell A, Swann I, Heifner S, Von Stetina S, Miller DM

ABSTRACT Electrical synapses are established between specific neurons and within distinct subcellular compartments, but the mechanisms that direct gap junction assembly in the nervous system are largely unknown. Here we show that a developmental program tunes cAMP signaling to direct the neuron-specific assembly and placement of electrical synapses in the C. elegans motor circuit. We use live cell imaging to visualize electrical synapses in vivo and a novel optogenetic assay to confirm that they are functional. In VA motor neurons, the UNC-4 transcription factor blocks expression of cAMP antagonists that promote gap junction miswiring. In unc-4 mutants, VA electrical synapses are established with an alternative synaptic partner and are repositioned from the VA axon to soma. We show that cAMP counters these effects by driving gap junction trafficking into the VA axon for electrical synapse assembly. Thus, our experiments establish that cAMP regulates gap junction trafficking for the biogenesis of electrical synapses.

Session III.B

Spatially targeted proteomics and multimodal imaging distinguish molecular signatures of amyloid plaque-containing islets from type 2 diabetic donors Kruse, A.R.S., McMillen, J., Walker, J.T., Judd, A.M., Patterson, N.H., Gutierrez, D.B., Norris, J.L.,

Spraggins, J.M., Powers, A.C., Caprioli, R.M.

ABSTRACT The pancreas is a complex organ with a crucial role in digestion and blood glucose regulation. Approximately 2% of the pancreas are islets, which are groups of cells responsible for producing insulin and glucagon. The islets of individuals with type 2 diabetes (T2D) are dysfunctional and may undergo cell death. Amyloid plaques are found within some T2D islets. The molecular environment that supports amyloid plaque formation, islet cell death, and the relationship between these processes remains poorly understood. Since amyloid plaques vary in morphology and location, comparing molecular signatures of islets with and without plaques is essential. Assessing individual islets in situ by mass spectrometry proteomics may help unravel the molecular underpinnings of plaque formation in T2D. We combined microscopy, microLESA (micro-Liquid Extraction Surface Analysis), laser capture microdissection (LCM), and imaging mass spectrometry (IMS) to study normal and T2D pancreas tissue. Autofluorescence and polarized light microscopy were used to localize islets and amyloid plaques while eliminating washing steps. Microscopy approaches were integrated with two spatial proteomics technologies: (1) microLESA, in which trypsin droplets were targeted to individual islets for on-tissue digestion, and (2) LCM, which employed lasers to excise islets for in-solution trypsin digestion. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was performed on digested islet proteins from microLESA and LCM, generating data from non-diabetic and T2D islets. Additionally, Matrix-Assisted Laser Desorption Ionization-Imaging Mass Spectrometry (MALDI IMS) was performed to spatially analyze peptides in healthy and T2D pancreas tissue. Preliminary analyses comparing amyloid-containing versus non amyloid-containing islets in T2D pancreas suggest that the proteins prefoldin and nucleophosmin, molecular chaperones associated with protein folding, and glycine amidinotransferase, an enzyme involved in creatine biosynthesis, are spatially associated with intra-islet amyloid. We then compared islets from healthy and T2D donors and identified several key proteins including peroxiredoxin that are differentially expressed in T2D pancreata. Gene ontology and networking analyses identified proteins involved in cellular stress response and protein aggregation that were upregulated in T2D pancreata. Taken together, these findings suggest that specific enzymes and chaperones are associated with amyloid plaques, either as a stress response or as an aberrant signal involved in plaque formation. To further leverage these data, we are using machine learning to identify proteins most predictive of a plaque-containing islet environment based on spatially targeted LC-MS/MS and IMS data. This approach allows us to compare specific islets within intact tissue, with the goal of understanding molecular changes in islets and the specific role of amyloid plaques in T2D.

Session III.C

XMAP215 promotes microtubule catastrophe by disrupting the growing microtubule end

Farmer V, Arpag G, Hall S, and Zanic M.

ABSTRACT The GTP-tubulin cap is widely accepted to protect microtubules against catastrophe. The GTP-cap size is thought to increase with the microtubule growth rate, presumably endowing fast-growing microtubules with enhanced stability. It is unknown what GTP-cap properties permit frequent microtubule catastrophe despite fast growth. Here, we investigate microtubules growing in the presence and absence of the polymerase XMAP215. Using EB1 as a GTP-cap marker, we find that GTP-cap size increases regardless of whether growth acceleration is achieved by increasing tubulin concentration or by XMAP215. Despite increased mean GTP-cap size, microtubules grown with XMAP215 display increased catastrophe frequency, in contrast to microtubules grown with more tubulin, for which catastrophe is abolished. However, microtubules polymerized with XMAP215 have large fluctuations in growth rate; display tapered and curled ends; and undergo catastrophe at faster growth rates and with higher EB1 end-localization. Our results suggest that structural perturbations induced by XMAP215 override the protective effects of the GTP-cap, ultimately driving microtubule catastrophe.

Session IV.A

A tRNA processing enzyme is central to germline-to-soma regulated mitochondrial stress response

Held, J., Saunders, B., Feng, G., Pereira, C., Burkewitz, K., Patel, M

ABSTRACT According to the disposable soma theory of aging, a trade-off exists between reproduction and the preservation of the body due to the limited availability of resources. Consistent with the predictions of this theory, compelling evidence from many species indicates that limiting reproduction extends lifespan. Nevertheless, how the reproductive status is communicated with the rest of the soma remains a fundamental mystery. Excitingly, using Caenorhabditis elegans as a model system, we have discovered that the loss of reproductive capacity triggers, in somatic cells, a conserved stress pathway called the mitochondrial unfolded protein response (UPRmt). The UPRmt is predominantly activated in the intestine and entails expression of hundreds of genes aimed at restoring mitochondrial function, thereby preserving somatic integrity when challenged with mitochondrial stress. We found through a targeted screening approach that the tRNA processing enzyme HOE-1 (homolog of ELAC2) is required for UPRmt induction in the intestine. Hyper-activation of HOE-1, via both gain-of-function mutations and overexpression, is sufficient to robustly induce intestinal UPRmt, even in the absence of mitochondrial stress. Mechanistically, HOE-1 functions to generate tRNA species required to trigger UPRmt. Blocking tRNA biogenesis and tRNA export from the nucleus are both sufficient to suppress HOE-1 dependent UPRmt induction. Importantly, HOE-1 induces UPRmt only in sterile animals. Thus, the ability of HOE-1 to preserve somatic integrity is contingent on limiting reproduction. These data also place HOE-1 at the heart of germline-to-soma communication. Consistent with this hypothesis, HOE-1 induces UPRmt in the intestine only after animals reach reproductive maturity. Furthermore, HOE-1 does not induce UPRmt in the soma of males. These data are consistent with the prediction that the reproduction/preservation-of-the-soma trade-off is more pronounced in females as they carry the greater burden of reproduction. Taken together, we have identified a critical molecular player involved in preserving somatic integrity in response to compromised reproductive status. At the CDB retreat I plan to present this work, which has been favorably reviewed at eLife.

Session IV.B

The role of epithelial cell integration in mammary gland development Pfannenstein A, Macara IG

ABSTRACT The mammary gland is a branched network of ducts and alveoli that function to produce milk for mammalian offspring. While the luminal epithelium is organized into a cellular monolayer, it originates from multilayered structures present during development called terminal end buds (TEB). The TEB leaves behind a duct of monolayered epithelial cells as it borrows through the fat pad during development. It is currently thought that cavitation of the ductal lumen through apoptosis is the primary means by which the lumen of the mammary duct is formed behind the multilayered TEB. While this hypothesis supports a plausible mechanism for cavitation, it does not accurately account for the elongation of the duct behind the TEB. Spatial calculations suggests that cells in the TEB integrate towards the outer luminal layer and that this migration of cells is the primary mechanism for cavitation of the duct rather than apoptosis. This integration of cells into a monolayer also promotes the elongation of the duct behind the TEB as it grows into the surrounding stroma. There is little known about how cells in the TEB rearrange to form the mature mammary gland. To study the progression of multilayered to monolayered epithelium as seen in the mammary gland, we developed a quantitative cell culture assay that determines the efficiency of cellular integration into a monolayer. Using this methodology, we have verified that loss of adherens junctions prevents stable integration of cells into monolayers, which is consistent with previous data in cultured cells and in primary tissue. Interestingly, we found that loss of tight junction proteins reduces integration. This may be due to the inability of integrating cells to adhere with intact monolayer tight junctions, a likely first step in integration. It remains to be seen if tight junctions promote integration in the developing mammary gland, but this assay provides a tool for investigating mechanisms of mammary epithelial organization. Overall, we show support for an altered model of mammary gland development where cellular integration promotes ductal elongation and organization of the epithelium into a monolayer.

Session IV.C

ER membrane contact sites regulates biogenesis of RNA-containing EVs Barman, B, Sung, BH; Ping, J; Krystofiak, E; Millis, B; Ramirez, M; Allen, R; Prasad, N; Vickers, K;

Patton, J; Liu, Q; Weaver, AM.

ABSTRACT Extracellular RNAs have emerged as a novel mechanism for cell-to-cell communication and drive many physiological and pathological processes including cancer. The major vehicle for transmission of these extracellular RNAs is extracellular vesicles (EVs). However, the underlying mechanisms by which these RNA-containing EVs are generated are largely unknown. Here, we show that membrane contact sites (MCS) between the endoplasmic reticulum (ER) and organelles that form EVs (e.g., the plasma membrane and endosomes) are critical for this process. Indeed, KD of a key MCS linker molecule VAP-A affects biogenesis of a distinct subset of EVs that are enriched with RNAs and RNA-binding proteins. We also find that this subset of EVs is critical for transmission of miR-100 between cells and for growth of tumors in mice. Since VAP-A directly binds lipid transporters on organelles contacted by the ER, we carried out a lipidomics analysis of control and VAP-A-KD EVs. Indeed, VAP-A-KD EVs had reduced concentrations of ceramides, which are known to impact EV biogenesis. Likewise, we find that KD of the ceramide transporter and VAP-A-binding protein CERT reduces the number of RNA-containing EVs. Altogether, we discover that VAP-A-CERT linkages between the ER and endosomes are key subcellular sites where EV biogenesis takes place.

Posters (Odd) Poster number 1 Ahmed, SM 3 Anthony, CC 5 Arceneaux, D 7 Arpag, G 9 Rodgers, NC* 11 Baum, T* 13 Bhattacharjee, R 15 Blackburn, J 17 Bryant, J 19 Caplan, LR 21 Cencer, CS 23 Chalkley, MBL* 25 Chatterjee, S 27 Chen, L 29 Colley, M 31 Contreras-Panta, E 33 Cullati, S 35 Dbouk, N 37 de Caestecker, C* 39 Djambazova, K 41 Donahue, E 43 Fitz, GN 45 Fye, M 47 Gailey, CD 49 Geben, LC 51 Goswami, S 53 Grub, L 55 Hayes, J 57 Ho, K-H 59 Islam, M 61 Jarvis, B 63 Jimenez L 65 Jung, Y-J 67 Jyotsana, N 69 Kassel, S 71 Narasimhan, S* *Breakout session speakers

Posters (Even) Poster number 2 Lee, G 4 McAtee, C Meegan, J. (see P36) 6 Min, J 8 Molina, P 10 Morales, EA* 12 Mulligan, AG* Narasimhan, S* (see p71) 16 Neumann, E K 18 O’Connor, JT 20 (Murobushi) Ozawa, PM 22 Peebles, K 24 Pereira, CV 26 Ramirez, S* 28 Blackburn, JB 30 Riedmann, K 32 Rivera, E 34 Robertson, G Rodgers, NC* (see P9) 36 Meegan, J. 38 Saleh, N 40 Schaaf, KR 42 Siladi, AJ 44 Sohn, Y 46 Stanchfield, M 48 Stricker, A 50 Trinh, LT 52 Vega, P* 54 Waghmare, I 56 White, J 58 Zhao, G 60 Wilson, L 62 Won, Y 64 Yang, M 66 Sanchez, Z 68 Ziehm, E 70 Yarboro, MT *Breakout session speakers

P1

Mutations in the exocyst component EXOC2 cause severe defects in human brain development

Syed Mukhtar Ahmed, Nicole J. Van Bergen, Christian De Caestecker, Ian Macara and John Christodoulou

ABSTRACT The exocyst, an octameric protein complex, is an essential component of the membrane transport machinery required for tethering and fusion of vesicles at the plasma membrane. We report pathogenic variants in an exocyst subunit, EXOC2 (Sec5). Affected individuals have severe developmental delay, dysmorphism, and brain abnormalities; variability associated with epilepsy; and poor motor skills. Family 1 had two offspring with a homozygous truncating variant in EXOC2 that leads to nonsense-mediated decay of EXOC2 transcript, a severe reduction in exocytosis and vesicle fusion, and undetectable levels of EXOC2 protein. The patient from Family 2 had a milder clinical phenotype and reduced exocytosis. Cells from both patients showed defective Arl13b localization to the primary cilium. The discovery of mutations that partially disable exocyst function provides valuable insight into this essential protein complex in neural development. Since EXOC2 and other exocyst complex subunits are critical to neuronal function, our findings suggest that EXOC2 variants are the cause of the patients’ neurological disorders.

P3

Characterization of STK38, a Novel Nuclear Regulator of Wnt signaling Christin C Anthony, Leif R Neitzel and Ethan Lee

ABSTRACT The canonical Wnt/β-catenin signaling pathway induces cellular responses such as proliferation and cell fate specification, and improper Wnt- pathway activation leads to diseases such as cancer. While much is known about the regulation of canonical Wnt/β-catenin signaling at the level of the receptor and β-catenin degradation, regulation downstream of the destruction complex is poorly understood. Additionally, Wnt signaling results in a wide variety of cellular responses, and how cells determine when to express specific genes also remains poorly understood. Our lab has identified a serine/threonine kinase, STK38, as a novel regulator of Wnt signaling. Work with our collaborators in animal models suggests STK38 is involved in heart development, and the Wnt proteins Pygopus2 and BCL9 are requisite components of Wnt-mediated heart development. STK38 co-immunoprecipitates with Pygo2, suggesting a role for STK38 in Wnt-mediated heart development. Future studies will determine if STK38 functions as a kinase in the Wnt pathway or as a scaffolding protein in the nucleus to regulate context-dependent Wnt ,target gene transcription.

P5

Optimizations for High Quality scRNA-seq data Arceneaux, D. Simmons, J. Heiser, C. Chen, B. Southard-Smith, A. Lau, K.

ABSTRACT Single-cell RNA sequencing (scRNA-seq) is a transcriptomic approach that is useful for studying cellular responses, revealing rare cell populations, and tracking the trajectories of developing cell lineages. However, an ongoing challenge in single-cell analyses is distinguishing true biological discoveries as opposed to artifacts from methodology. Previous work focuses on technical challenges in library preparation and on computational analysis pipelines to better address quality control, nevertheless, these tools all address post capsulation drawbacks, and thus don't combat artifacts introduced during single-cell encapsulation. In our work, we investigate optimizing single-cell dissociation and inDrop specific encapsulation, which has decreased the plethora of apoptotic cells and droplets containing doublets, which has led to a reduction in the abundance of artifacts previously associated with scRNA-seq datasets. We describe a series of experiments in which we optimize not only cell dissociation methods but also methods of encapsulation and show that the alteration of dissociation and microfluidics in inDrop encapsulation decreases the amount of ambient RNA present in our datasets. We expect that these optimizations that take place during experimental setup will reduce the number of artifacts present, and hence lead to sounder biological interpretation.

P7

Determination of the tubulin GTP-hydrolysis rate through combination of image analysis and computational modeling

Arpag, G; Farmer, V; Wang, S; Zanic, M

ABSTRACT Microtubules are dynamic polymers essential for a variety of cellular processes. Microtubules polymerize by the addition of GTP-bound tubulin heterodimers to the ends of the microtubule polymer. Incorporation of GTP-tubulin is followed by GTP hydrolysis, forming a polymer lattice composed of GDP-tubulin, with only a 'cap' of GTP-tubulin maintained at the growing polymer end. The GTP-cap is a stabilizing structure ensuring persistent microtubule growth; when GTP-cap is lost, microtubules switch to depolymerization. The size of the GTP-tubulin cap is a result of a balance between the polymerization rate and the GTP-hydrolysis rate. However, whether GTP-hydrolysis rate is constant, or changing in a manner coupled to the microtubule polymerization rate is not understood. Simultaneous measurements of the GTP-cap size and the microtubule polymerization rate can, in principle, allow determination of the GTP-hydrolysis rate within the microtubule polymer. Standard methods to determine the GTP-cap size employ microtubule end-binding EB protein, whose comet-like localization marks the GTP-cap at the end of a growing microtubule. Here, we use a combination of EB-comet image analysis and computational simulations to improve the measurements of the GTP-cap size and directly determine the GTP-hydrolysis rate. Our findings bear direct relevance for the understanding of the molecular regulation of microtubule dynamics.

P9

CLASP2 facilitates co-organization of microtubules and actin

Rodgers, N C Lawrence, E J Zanic, M

ABSTRACT Coordination between the microtubule and actin cytoskeletons is essential in cell motility, neuronal growth cone guidance, and wound healing. In a cellular context, this coordination is controlled by crosslinking proteins and when disrupted can lead to disease. While studies in cells have identified important crosslinking proteins, the mechanisms underlying the interactions between microtubules and actin filaments remain largely unknown. Members of the CLASP (Cytoplasmic Linker-Associated Protein) family of proteins have been implicated in the cytoskeletal crosstalk. However, the specific dynamic interactions between microtubules and actin mediated by CLASPs are less understood. Here, we demonstrate the direct interaction of CLASP2 with actin using biochemical pull-downs with purified protein components. We then investigate CLASP2's colocalization with microtubules and actin using an in vitro reconstitution assay, visualized by total internal reflection fluorescence microscopy (TIRFM). Our results demonstrate that CLASP2 directly interacts with actin in vitro, preferentially colocalizes with bundled F-actin, and can increase the coalignment of microtubules along F-actin bundles. Next, we characterized the different interactions between microtubules and actin filaments when CLASP2 is localized to the ends of growing microtubules and along the microtubule lattice. When CLASP2 is at the ends of growing microtubules, the frequency of microtubule zippering events along bundled F-actin increases in the presence of CLASP2. Furthermore, we find that CLASP2 bound to the microtubule lattice facilitates accumulation of multiple actin filaments along microtubules over time and may facilitate actin polymerization along microtubules. Taken together, our results elucidate the different microtubule-actin interactions that underly microtubule-actin co-organization by CLASP2, essential in several cellular processes.

P11

Understanding Mitochondrial Fission Dynamics During Neuronal

Differentiation and Maturation Baum T, Bright A, Gama V

ABSTRACT The large GTPase Dynamin-Related Protein 1 (DRP1) executes mitochondrial fission - the process of fragmentation necessary for mitochondrial trafficking in neurons. Children harboring de novo mutations in DRP1 exhibit severe neurodevelopmental deficits including developmental delay, optic atrophy, microcephaly, and intractable seizures. There is therefore a critical need to understand DRP1 malfunction and how subsequent loss of overall mitochondrial fitness impairs neurodevelopment. Though it is established that DRP1 is necessary for typical neurodevelopment, there is little understood about DRP1 dysfunction in specific types of neurons or how known mutations disrupt protein interactions during development. This project focuses on testing the mechanism of DRP1 dysfunction both at sites of fission and the downstream effects on cell-type-specific neuronal differentiation and maturation. Fission is a critical precursor to efficient mitochondrial transport within the highly branched and elongated morphology of specific types of neurons. To investigate how DRP1 dysfunction differentially effects neuronal subtypes, patient-derived iPSCs with DRP1 mutations will be differentiated into region-specific neurons of the forebrain, midbrain, and hindbrain with functional readouts of impaired motility assessed in parallel. To examine alterations in protein-protein interactions at sites of fission in cells with DRP1 mutations, we plan to examine the assembly/disassembly capacity of F-actin and ER with mutant forms of DRP1 using super-resolution microscopies in tandem with biochemical approaches to define the nature of these contacts. Overall, we aim to elucidate the protein interactions and cell-type-specific differences that make fission disruption highly pathogenic during neural development.

P13

The Cdc15 F-BAR domain bridges paxillin-like protein 1 between the cytokinetic ring and plasma membrane to promote accurate completion of

cytokinesis Bhattacharjee R, Snider CE, Igarashi MG, Gould KL

ABSTRACT Cytokinesis is the final step in cell division, in which two daughter cells physically separate. Most eukaryotes, including Schizosaccharomyces pombe, complete cytokinesis by constricting an actin-and myosin-based cytokinetic ring (CR) that is built on the plasma membrane. The F-BAR protein Cdc15 mediates attachment of the CR to the plasma membrane and is essential for cytokinesis; its N-terminal F-BAR domain is responsible for oligomerization and membrane binding, while its C-terminal SH3 domain binds other partners at a distance from the membrane. We previously demonstrated that the essential cytokinetic formin Cdc12, through a short N-terminal motif, directly binds a negatively charged region on the cytosolic face of the F-BAR domain. Here, we show that paxillin-like protein Pxl1, which is important for CR stability, contains a motif highly related to that in formin Cdc12 and it also binds the Cdc15 F-BAR domain directly. Interestingly, Pxl1 also binds the Cdc15 SH3 domain. In an effort to understand how and why Pxl1 interacts with two distinct regions of Cdc15, we mapped the interaction between paxillin and the Cdc15 C-terminus and found a single PxxP motif that binds the Cdc15 SH3. However, whereas disruption of Pxl1-Cdc15 F-BAR domain resulted in reduced Pxl1 levels in the CR, a mutation in the single PxxP motif of Pxl1 which is responsible for the protein's interaction with Cdc15-SH3, did not. Furthermore, there was no additive effect when Pxl1 mutations disrupting Cdc15 F-BAR and C-terminus SH3 binding were combined. Our data indicate that Pxl1-Cdc15 F-BAR interaction is crucial for Pxl1's localization to the CR. Interestingly, we found that disrupting Cdc15 interaction altogether, although it greatly reduced CR Pxl1, did not significantly affect cytokinesis. These data not only point to another mechanism of Pxl1 CR recruitment but indicate that very little CR Pxl1 is sufficient for its cytokinetic function. Given that the LIM domains of paxillin proteins interact with F-actin, we speculate that actin and F-BAR Cdc15 cooperate to bring sufficient Pxl1 to the CR to support cytokinesis.

P15

The Role of TAZ in airway epithelial transition states Blackburn, J; Schaff, J; Du, R-U; Gutierrez, A; Haberman, A; Calvi, C; Xin, M; Wickersham, N; Ware,

L; Banovich, N; Kropski, J; Blackwell, T; Richmond, B

ABSTRACT The airway epithelium is composed of a variety of different cells types that vary in their ability for self-renewal and trans-differentiation. Yet, the mechanisms that facilitate these transitions are not well understood. TAZ (gene name WWTR1) and its paralog YAP (gene name YAP1) are transcriptional coactivators that intersect a variety of pathways, sensing the environment outside of and within the cell to control proliferation and differentiation. While thought to operate mostly redundantly there are select differences between the two, and prior work has shown that their individual deletion from the airway epithelium in mice yields divergent results, with YAP affecting the more proximal airways and TAZ having a greater affect on the more distal regions. We hypothesized that TAZ facilitates cell type transition states, particularly in the distal lung, by temporarily "reprograming" airway cells away from mature cell gene expression when trans-differentiation is needed i.e. to recover from injury. We first performed single cell RNA sequencing (scRNA-seq) of COPD explanted lungs and deceased donor lungs that were rejected for transplantation to assess WWTR1 (TAZ) expression in the adult lung epithelium and found that WWTR1 (but not YAP1) is particularly elevated in transitional cell states (basal, terminal airway secretory cells, AT2/AT1 transitional cells) with its highest expression in non-mucin secreting secretory cells. Downstream genes of YAP/TAZ signaling were also increased in these cells demonstrating active YAP/TAZ signaling. We next used a pharmacologic activator of YAP/TAZ in a human airway secretory cell line (HBEC3-KT) and discovered that YAP/TAZ signaling suppresses secretory maturation supporting the role of this pathway in secretory "deprograming". We then assessed TAZ localization in this same cell line without manipulation and found that TAZ was abundantly localized in the nucleus at low confluency when cells are actively proliferating but decreased in both abundance and nuclear localization at confluency. Conversely, mature secretory markers were only abundantly expressed at confluency following a decrease in TAZ signaling. We then assessed TAZ localization following injury via cigarette smoke and found that TAZ shifted to a more nuclear localization. Future directions will explore TAZ's impact specifically on secretory cells via lentiviral overexpression or knockdown, with or without YAP overexpression or knockdown to isolate the individual functions of both genes utilizing both HBEC3-KT cells and primary airway epithelial cells.

P17

DYRK2 as a novel positive regulator of Wnt/Beta-catenin signaling Bryant, J, Tian, A, Robbins, D, Ahmed, Y, and Lee, E

ABSTRACT Protein kinases regulate multiple steps of Wnt signal transduction. In an expression cloning screen for proteins that perturb development in Xenopus embryos, we identified a kinase, Dual Specificity Tyrosine Phosphorylation Regulated Kinase 2 (DYRK2), as a Wnt pathway activator. DYRK2 overexpression in Xenopus embryos promotes axis duplication, whereas DYRK2 morpholino-mediated knockdown results in ventralized Xenopus embryos. In human cells, DYRK2 overexpression enhances Wnt reporter activity, increases intracellular b-catenin levels, and enhances LRP6 phosphorylation. In contrast, knockdown of DYRK2 by RNAi in human cells inhibits Wnt pathway activation and prevents LRP6 phosphorylation. In Drosophila DYRk2 is required to promote Wnt pathway activation, thereby demonstrating its conservation of function in the Wnt pathway. Preliminary studies, using a purified, recombinant reconstitution system, suggests that DYRK2 cooperates with GSK3 to phosphorylate LRP6. We propose that DYRK2 is part of a kinase complex involved in regulating Wnt receptor activation. Future experiments will be performed to confirm the role of DYRK2 in regulating LRP6 activity at the plasma membrane in cells, to determine whether DYRK2 directly promotes GSK3 activity towards LRP6.

P19

Enteroendocrine cell formation is an early event in pancreatic tumorigenesis Leah R. Caplan, Vera Vavinskaya, David G. Gelikman, Nidhi Jyotsana, Vincent Q. Trinh, Marcus C.B.

Tan, and Kathleen E. DelGiorno

ABSTRACT Pancreatic acinar-to-ductal metaplasia (ADM) is an early response to tissue injury that recapitulates many processes identified in pancreas development. Expression of oncogenic KrasG12D induces ADM, which can progress to pancreatic intraepithelial neoplasia (PanIN) and, eventually, pancreatic ductal adenocarcinoma (PDAC). Acinar cells undergoing ADM give rise to heterogenous cell populations, including enteroendocrine cells (EEC) recently described by our laboratory. The term ‘enteroendocrine cell’, however represents a lineage populated by multiple subtypes characterized by the expression, or co-expression, of various hormones including serotonin (enterochromaffin cells), somatostatin (delta cells), ghrelin (epsilon cells), and pancreatic polypeptide (gamma cells). Using IHC to detect general-EEC and hormone-specific markers, EEC subtype presence and proportion were analyzed in pancreatic lesions spanning ADM to PDAC in pancreata from 6- and 12-month old KrasG12D;Ptf1aCre/+ (KC) and KrasG12D;Pou2f3fl/fl;Ptf1aCre/+ (KPouC, tuft cell knockout) mice, and KrasG12D;Trp53R172H;Pdx1-Cre (KPC) mice with or without PDAC. Co-immunofluorescence (co-IF) was performed to examine hormone co-expression. ADM and PanIN 1a lesions contained the highest percentage of EECs, and the frequency of all EEC subtypes decreases with increasing lesion grade in all genetic mouse models analyzed. KPouC lesions contained more EECs than equivalent lesions from age-matched KC mice. Co-IF for EEC subtype hormones demonstrates promiscuity in enterochromaffin cells. These results are consistent with EEC formation as an early event in disease progression. EEC subtypes differ in abundance with disease progression, suggesting differing function(s) throughout tumorigenesis.

P21

An adhesion-based mechanism for stabilizing microvilli on the surface of epithelial cells

Cencer, CS; Silverman, JB; Tyska, MJ

ABSTRACT The evolution of actin-supported cell surface protrusions, known as microvilli, has allowed transporting epithelia to modify their apical cell surface structure to optimize their function. Such organization is a central theme to how tissues such as the kidney and small intestine expand surface area available for solute transport. Microvilli found on the surface of these epithelia exhibit a well-organized 'brush border' made up of thousands of protrusions connected to their neighbors via a tip-localized adhesion complex composed of cadherins CDHR2 and CDHR5. While it is known that the intermicrovillar adhesion complex (IMAC) plays a role in the final organization and maintenance of the brush border, how nascent microvilli accumulate on the apical surface during differentiation and the role of the IMAC in this process remain unclear. Here we show that, at time points early in differentiation, epithelial cells present two general populations of microvilli: (1) a marginal population at the edges of cells, characterized by high protrusion density, and (2) a medial population characterized by much lower protrusion density. Strikingly, marginal microvilli extend across cell-cell junctions and make direct physical contact with protrusions on neighboring cells. Based on these static observations, we set out to test the idea that the edges of cells provide a point of capture and stabilization for nascent microvilli. Two CL4 cell populations, expressing eGFP-CDHR2 and mCherry-CDHR5, were mixed and used in fluorescence recovery after photobleaching (FRAP) and microvilli tracking experiments to examine how IMAC's influence microvilli stability. Indeed, FRAP analysis reveals that transcellular adhesion complexes, formed between marginal microvilli of neighboring cells, are more stable than those bridging medial clusters of microvilli. Furthermore, tracking analysis on live CL4 cells shows that medial microvilli are more motile than marginal microvilli, suggesting that transcellular adhesion serves as an anchoring point for motile microvilli. These findings suggest a new, adhesion-based mechanism for the stabilization of microvilli on the surface of transporting epithelial cells. This process has broad implications in understanding how microvilli and other cell surface features, such as stereocilia in inner-ear hair bundles, become organized for optimal cell function.

P23

Non-Canonical Functions of TSC2 Protein in Mitotic Division Chalkley, M.B.L., Mersfelder, R.B., Sahin, M., Ihrie, R.A. & Ess, K.C.

ABSTRACT Tuberous Sclerosis Complex (TSC) is a debilitating developmental disorder characterized by a variety of clinical manifestations. While benign tumors in the heart, eyes, lungs, kidney, skin, and brain are a hallmark of the disease, often the most severe symptoms of TSC are neurological, including seizures, autism, psychiatric disorders, and intellectual disabilities. TSC is caused by a loss of function mutation in the TSC1 or TSC2 genes, which encode the hamartin/tuberin proteins respectively. These proteins function as a heterodimer that negatively regulates mechanistic Target of Rapamycin Complex 1 (mTORC1). The majority of work on TSC has been focused on the effects of mTORC1, a critical signaling hub, on regulation of diverse cell processes including metabolism, cell growth, translation, and neurogenesis. However, work focused on potential non-canonical functions of TSC2 is rare and the potential cell biological mechanisms involved are not well understood. Understanding whether mTORC1-independent functions of TSC2 contribute to patient phenotypes will be essential to tailoring treatment, as many patients are currently treated with mTOR-targeting agents. To examine neurodevelopmental phenotypes in a cell-based model of TSC, we utilized TSC patient-derived induced pluripotent stem cells (iPSCs) that harbor a disease-causing heterozygous microdeletion mutation in the TSC2 gene. Patients are heterozygous for mutations, but the most widely accepted model is that second hit mutations in TSC1/2 occur in tumorigenesis. To model this state, CRISPR was used to create a similar deletion mutation in the other TSC2 allele, producing a homozygous mutant line. TALENs was also used to correct the heterozygous mutant to wild type, creating a set of isogenic lines. We observed aberrant multipolar mitotic division, a novel phenotype, in the TSC2 mutant iPSCs. Multipolar mitotic division occurs when there are more than the typical two spindle poles during mitosis. The multipolar phenotype is not significantly affected by treatment with rapamycin, an mTORC1 inhibitor, in the TSC2 mutant iPSCs, indicating that multipolar division is an mTORC1-independent phenotype. Interestingly, the multipolar phenotype in the TSC2 mutant cells does not continue through differentiation into mixed glutamatergic and GABAergic cortical neurons. Further analyses are in progress, but our findings show a non-canonical function for TSC2 protein that can provide crucial insight into TSC2 effects on normal development, ultimately identifying additional potential avenues for therapeutic intervention in patients with TSC.

P25

Modeling microtubule interactions in vitro and in silico Chatterjee, S and Zanic, M

ABSTRACT The dynamics of the microtubule cytoskeleton play a crucial role in a myriad of cellular processes such as cell division, cell motility, and tissue morphogenesis. Individual microtubules dynamically toggle between phases of growth and shrinkage - a behavior called dynamic instability. Dynamic instability is well-studied at individual microtubule level, but how it facilitates the remodeling within a multi-microtubule network remains elusive. Previous studies, as well as preliminary analysis of our data, identify several types of inter-microtubule interactions that modulate the dynamics of individual microtubules. Microtubule-microtubule encounters can result in: (a) microtubule-microtubule crossover, (b) microtubule retraction upon encounter and (c) zippering/realignment of microtubules. The likelihoods of each of these encounter events are regulated by microtubule-associated proteins (MAPs) and molecular motors. Here, we have undertaken a combinatory approach utilizing in vitro reconstitution of microtubule dynamics with TIRF microscopy, and agent-based computational simulations to elucidate microtubule-microtubule interactions. By screening the outputs of the in silico model and comparing the predictions with experimental observations, we aim to determine how microtubule-microtubule encounters at individual-microtubule level influence the spatiotemporal remodeling of microtubule cytoskeleton as a whole, providing key insights into the mechanistic underpinnings behind a variety of cellular processes.

P27

SIN3A/HDAC complex is essential for DNA replication in medulloblastoma Lei Chen, Wen Li, and Chin Chiang

ABSTRACT Medulloblastoma (MB) is one of the most common malignant brain tumors in children. The lack of targeted, efficient, and low side-effect therapies for young patients with MB is particularly challenging. Targeting histone deacetylases (HDAC) is an appealing therapeutic approach because they do not permanently alter the genetic code but act on reversible epigenetic marks, with a lower risk of side effects. Although HDAC inhibitors have been widely used in pre-clinical studies for their anti-tumor effects in a variety of mouse models of MB, the molecular mechanism as to how HDAC inhibition represses tumor growth is not well understood. Here we show that Sin3a, a component of Hdac co-repressor complex, plays a critical role in DNA replication of MB cells. Conditional deletion of Sin3a in a mouse model of MB suppressed tumor growth and improved overall survival in a dosage dependent manner. Sin3a null MB cells displayed slower incorporation of thymidine analogs during DNA replication. Moreover, DNA combing analysis shows the velocity of DNA replication fork is significantly reduced when compared to control MB cells. Inhibition of HDAC or Sin3A using small molecule inhibitors also lead to a similar defect in DNA replication velocity in several human MB cell lines. As an epigenetic consequence of SIN3A/HDAC inhibition, a pre-deposition H4K5ac mark, which is removed during chromatin maturation, was improperly accumulated in the chromatin of MB cells. We also show that SIN3A/HDAC complex is recruited onto the DNA replisome through their interaction with PCNA. In summary, our study indicates that Sin3A/HDAC complex is essential for the progression of DNA replication in MB and that the disruption of this complex using a small molecule inhibitor of SIN3A may serve as a potential therapeutic target for MB.

P29

PASEF Imaging Mass Spectrometry: Coupling Structural Lipidomics to In Situ Tissue Mapping

Colley, M., Djambazova, K., Neumann, E., Fuettere, A., Krause, M., Caprioli, R., Spraggins, J.

ABSTRACT Lipidomics by both LC-MS and imaging mass spectrometry (IMS) has enabled a deeper understanding of biological systems. Parallel-accumulation-serial fragmentation (PASEF) applied to LC-MS has provided identification of species across four dimensions (retention time, CCS, MS1, MS2,) with high speed and confidence. However, without tailored surface sampling, LC-MS lacks spatial context which is important to structure/function relationships. IMS provides high spatial resolution maps of biomolecular species on a cellular level. Identification in IMS is traditionally made in one dimension based on MS1 mass accuracy or by fragmentation of a limited number of selected ions (<5) requiring compromised spatial resolution. PASEF elevates IMS to a four-dimensional platform that enables identification of tens of ions at each pixel while maintaining high spatial resolution. Normal human kidney portions from renal cancer nephrectomies, rat brain tissue, and rat liver homogenates were studied. A prototype MALDI timsTOF Flex mass spectrometer (Bruker Daltonics) was used to acquire the data in positive and negative ion mode with prototype timsControl software which allows for precursor ion selection and fragmentation during MALDI acquisition. Structural lipidomic images were generated from the combination of MS1, CCS, and fragment ion contribution. Mass maps of the precursor and fragment ions are spatially relevant and appear in specific cell types and functional tissue units of the human kidney and rat brain. For example, m/z 742.540 localizes to tubule structures in the cortex of the human kidney and was identified as PE(36:2) by MS1 with a 3 ppm mass error. PASEF imaging provides further structural elucidation of this species finding it to be, more specifically, PE(18:0_18:2). With this PRM-version of PASEF we are able to accumulate and perform serial fragmentation on 25 target ions per pixel at 10 µm spatial resolution. This work represents a new paradigm for how IMS experiments can be performed. PASEF imaging mass spectrometry allows for MS/MS based analysis while still maintaining the multiplexed and high spatial resolution advantages of MALDI IMS.

P31

Interleukin 13 (IL-13) effects in murine SPEM organoids Ela Contreras-Panta, Su-Hyung Lee, Eunyoung Choi, and James R. Goldenring

ABSTRACT Gastric cancer is one of the leading causes of cancer mortality worldwide mainly because the mechanisms involved in stomach carcinogenesis progression remain unclear. The onset of metaplasia phenotypes in the stomach are correlated with the development of gastric cancer. Therefore, understanding the events that lead to stomach metaplasia proliferation and progression remains a priority. Spasmolytic Polypeptide-Expressing Metaplasia or SPEM is the earliest type of metaplasia seen in the stomach. Using transgenic Mist1-Kras mice, we have established for the first-time murine SPEM organoid lines. After Immunofluorescence characterization, we have found these organoids keep their SPEM features across multiple passages in in vitro culture. Moreover, single-cell RNA-sequencing analysis revealed SPEM organoids have a distinctive transcriptional profile in comparison with mouse dysplastic organoids, established previously. These data combined suggest SPEM organoids are a novel and powerful in vitro tool. Additionally, investigations from our laboratory have demonstrated that Interleukin (IL) 13 could promote SPEM because mice lacking IL-13 did not develop metaplasia. We hypothesized that continuous exposure to IL-13 may promote SPEM proliferation and progression to more advanced lineages. To address this, we treated the mouse SPEM organoids with recombinant murine IL-13 at a concentration of 1 ng/ml for a period of 4 days. IL-13 vehicle solution (0.1% BSA) was used as a control. Phase contrast images were taken daily to study the effects on organoid growth by quantification of diameter. Additionally, samples were collected for RNA and protein isolation to analyze changes in transcription and translation between treated and control groups. Diameter measurements showed that SPEM organoids under IL-13 treatment have significantly increased diameters in comparison with control and only medium (Intesticult). Moreover, immunoblotting showed up-regulation of phosphorylation of STAT6 in organoids under IL-13 treatment; and RT-qPCR revealed significantly increased transcription of SPEM-related genes including Tff2, Muc6 and He4 (Wfdc2). These results suggest that IL-13 has a direct effect on SPEM organoid proliferation and progression. Further analyses are needed to understand the mechanism of action for promoting proliferation and progression, as well as to define the receptor to which IL-13 is binding in the cells of SPEM organoids.

P33

Kinase domain autophosphorylation rewires the activity and substrate specificity of CK1 enzymes

Cullati, S., Chaikuad, A., Chen, J., Gebel, J., Schulz, L., Zhubi, R., Navarrete-Perea, J., Guillen, R., Gygi, S., Hummer, G., Dotsch, V., Knapp, S., Gould, K.

ABSTRACT CK1s are acidophilic serine/threonine kinases with multiple critical cellular functions; their misregulation contributes to cancer, neurodegenerative diseases, and sleep phase disorders. Here, we describe an evolutionarily conserved mechanism of CK1 activity: autophosphorylation of a threonine (T220 in human CK1δ) located at the N-terminus of helix αG, proximal to the substrate binding cleft. Crystal structures and molecular dynamics simulations uncovered inherent plasticity in αG that increased upon T220 autophosphorylation. The phosphorylation-induced structural changes significantly altered the conformation of the substrate binding cleft, affecting substrate specificity. In T220 phosphorylated yeast and human CK1s, activity toward many substrates was decreased, but we also identified a high-affinity substrate that was phosphorylated more rapidly, and quantitative phosphoproteomics revealed that disrupting T220 autophosphorylation rewired CK1 signaling in Schizosaccharomyces pombe. T220 is present exclusively in the CK1 family, thus its autophosphorylation may have evolved as a unique regulatory mechanism for this important family.

P35

Identifying genes involved in germline-to-gut communication through mutagenesis screening

Dbouk, N, Held, J, and Patel, M

ABSTRACT The mitochondrial unfolded protein response (UPRmt) is essential in dealing with mitochondrial stress and preserving mitochondrial function. Work in our lab has shown that the tRNA processing enzyme, HOE-1, is necessary for the induction of UPRmt in the gut tissue of C. elegans in response to mitochondrial stress. Interestingly, hyperactivation of HOE-1 is sufficient to induce UPRmt in the gut, but only in sterile animals. These data suggest the existence of a germline-to-gut communication pathway that activates UPRmt in the gut when germline function is compromised. However, the molecular components of this inter-tissue signaling pathway are unknown. We are using a genetic screen as a broad base approach to elucidate potential candidate genes involved in this signaling. To find genes responsible for connecting UPRmt in sterile worms, we crossed a GFP reporter strain of UPRmt, zcis13, in worms that have a temperature sensitive mutation, glp-1, that causes sterility. Using this strain, we mutagenize the worms, grow them at sterility causing temperature, and screen the mutated worms for GFP induction. After many mutagenesis cycles, this systematic screening approach will help us identify genes involved in the germline-to-gut communication.

P37

Crumbs complex assembly during trafficking de Caestecker, C and Macara, I

ABSTRACT Polarity is a universal attribute of eukaryotes, and in epithelial cells establishes spatially distinct apical and basolateral domains. A key determinant of the apical domain in epithelia is Crumbs3, which organizes the Crumbs and Par polarity complexes at the cell cortex. In contrast with other apical polarity machinery, Crumbs3 is a transmembrane protein, and therefore requires vesicle transport to reach the cell surface. However, the mechanism by which Crumbs3 containing vesicles are delivered to the presumptive apical surface, and the roles accessory polarity proteins may play in this process are poorly understood. Studying anterograde trafficking dynamics at steady state poses a challenge, as Crumbs3 and its associated complexes are actively endocytosed and recycled. To address this problem, we implemented the Retention Using Selective Hooks (RUSH) system to enable synchronized anterograde trafficking of Crumbs3 in cultured epithelial cells. In this system, Crumbs3 is retained in the endoplasmic reticulum (ER) through interaction with an ER-localized streptavidin. Administration of biotin disrupts this interaction, enabling anterograde trafficking of Crumbs3. Using this method, we found that other Crumbs complex proteins (Pals1/Patj/Lin7c) associate with Crumbs3 at the ER and Golgi apparatus during transit, while Par complex components (Par3/Par6/aPKC) associate only at the apical surface. Surprisingly, live-imaging these co-trafficking events revealed that the majority of Pals1 departs the Golgi with Crumbs3 several minutes prior to departure of uncomplexed Crumbs3. This differential timing suggests that Crumbs3-Pals1 may follow a distinct trafficking itinerary from Crumbs3 alone, and that the interaction during transit is important for delivery of Crumbs3 and its accessory proteins to the apical surface. These findings challenge a prevailing theory that polarity machineries assemble only at the apical surface.

P39

Differential distributions of Disialoganglioside Isomers -GD1a and GD1b in Murine Tissue by MALDI TIMS IMS

Djambazova, K., Dufresne, M., Migas, L.,Kruse, A., van de Plas, R., Caprioli, R., Spraggins, J.

ABSTRACT Gangliosides are classified as acidic glycosphingolipids, containing ceramide moieties and oligosaccharide chains with one or multiple sialic acid residue(s). These molecules are critical components of neuronal and glial cells and have been implicated in neurological disease such as Alzheimer's and Huntington's disease. The presence of multiple sialylation sites gives rise to many possible isomeric structures, complicating ganglioside characterization. Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) enables the untargeted spatial analysis of gangliosides, among other biomolecules, within tissue. Integrating trapped ion mobility mass spectrometry (TIMS), a gas-phase separation technology, with MALDI IMS, allows for the investigation of isomeric lipid structures in situ. Here we demonstrate the partial gas-phase separation of disialoganglioside isomers GD1a and GD1b and elucidate their unique spatial distributions within murine tissues. GD1a and GD1b differ in the position of a single sialic acid, where GD1a has a sialic acid on the internal and terminal galactose units, and GD1b has both sialic acids in a chain attached to the internal galactose. Immunohistochemical approaches can reveal the localization of GD1a and GD1b species within tissues but provides no information on the ceramide moieties. MALD IMS, on the other hand, can clearly show the localization of GD1s with different ceramide compositions, such as d36:1 and d38:1, but cannot discern between GD1a and GD1b. Our results demonstrate the partial TIMS separation of GD1a(d36:1) / GD1b(d36:1), and GD1a(d38:1) / GD1b(d38:1) in a mixture of both standards, as well as in situ. The unique spatial distributions of the four ions were visualized within the murine brain and spinal cord. Although gangliosides are well studied in the context of neurological diseases, they have also been linked to infectious diseases, including Staphylococcus aureus (S. aureus). As a case study, S. aureus infected mouse kidney revealed changes in the ganglioside composition at different time points post infection. Our results suggest that several TIMS-separated isomeric monsialogangliosides - GM1a and GM1b have different localizations within S. aureus abscesses. MALDI TIMS MS/MS studies were employed to identify these structures. Our findings demonstrate that MALDI TIMS IMS can be used to discern the localization of mono- and disialogangliosides that differ in the position of the sialic acids and their ceramide compositions.

P41

Investigating the role of ER remodeling in aging Eric KF Donahue and Kristopher Burkewitz

ABSTRACT Age-related disease and physiologic decline are driven by changes at the molecular and cellular levels. Some of these age-dependent changes are shared across great evolutionary distances, including impaired proteostasis, metabolic disarray, and a loss of mitochondrial form and function. Although the endoplasmic reticulum (ER) plays a central role in regulating each of these hallmark changes of aging, whether ER form and function are also remodeled during aging is under-studied. Using the rapid-aging, transparent nematode C. elegans as a model, we have begun investigating the age-dependent dynamics of ER form and function. We reveal that ER morphology in older animals is dramatically remodeled, marked by a change in the amount and distribution of functionally specialized ER subdomains such as sheets and tubules. We find that autophagy plays a key role in this morphological remodeling, and long-lived animals are protected against it. Furthermore, ER tubule structures appear to be enhanced in aged animals. As ER tubules license sites of mitochondrial fission, we hypothesized that enhanced ER tubulation may drive the mitochondrial fragmentation commonly observed in contexts of aging and age-related disease. By genetically manipulating ER morphology, we show that suppressing ER tubulation protects mitochondrial morphology from age-related fragmentation. Together, these studies demonstrate that the ER is remodeled with age and establish ER morphology as a novel target to prevent age-related mitochondrial decline.

P43

Protrusion growth driven by myosin-generated force Fitz, GN; Weck, M; Feehan-Nelson O; Tyska, MJ

ABSTRACT Actin-based membrane protrusions such as filopodia, stereocilia, and microvilli are defining morphological features of animal cells, and support a range of biological functions including cell motility, adhesion, mechanosensation, and nutrient absorption. A central model of protrusion growth, derived from decades of investigation, indicates that actin polymerization produces the primary force to bend the membrane outward. However, force-generating myosins are also abundant residents of all types of protrusions and can interact directly and indirectly with the membrane. Whether myosin-generated forces contribute to protrusion growth through these membrane interactions remains an unanswered question. To test this concept, we devised a drug-inducible system that gives us switchable-control over the interaction between myosin motor domains and the plasma membrane. Strikingly, we found that the application of myosin-generated forces to the membrane is sufficient to drive robust protrusion growth. Using this system, our data reveal a mechanism for protrusion growth that is independent of both the cargo-carrying function of myosin and supporting actin-nucleating machinery. Additionally, we observed that different modes of membrane interactions (i.e. integral vs. peripheral) support protrusion growth albeit with varying efficiencies. Finally, we showed that multiple classes of myosin motor domains are able to drive protrusion growth in various cell types. Here, we present a novel system that provides temporal control over protrusion growth, and in turn uncover a mechanism for protrusion growth dependent on myosin-generated forces.

P45

Role of microtubules at insulin secretion hot spots Fye, M., Gu, G., Kaverina, I.

ABSTRACT Pancreatic β cells are critical for whole-body glucose homeostasis, which they achieve by synthesizing and secreting the insulin molecules necessary for cells to take up blood glucose. This prevents high blood glucose and associated diseases such as type 2 diabetes and cardiovascular disease. A complex web of microtubules in β cells regulates insulin granule transport via kinesin motors. β cells take advantage of microtubule-mediated transport to reduce insulin granule anchoring at the membrane and prevent insulin over-secretion. Our preliminary data have detected sliding microtubules parallel to the membrane and stationary minus ends perpendicular to the membrane. These microtubule arrangements may contribute to regulation of insulin granule positioning. Despite microtubules' negative effects on insulin secretion, β cells exhibit enhanced insulin granule exocytosis at local sites termed hot spots, which are marked by higher levels of insulin secretion than other areas of the β cell. Enhanced insulin granule exocytosis at hot spots allows for rapid and directed secretion of insulin to the blood. ELKS is a marker of insulin secretion hot spots in β cells and is thought to have a scaffolding function that influences cell polarity. ELKS forms a complex with the phosphoinositide-sensing protein LL5β, to anchor microtubules to the cell cortex in fibroblasts. LL5β is necessary for formation of this complex and simultaneously binds ELKS and microtubule plus ends. Thus, LL5β and ELKS provide a potential link between microtubules and insulin secretion hot spots in β cells. I hypothesize that microtubule minus ends localize away from hot spots to prevent nondirectional secretion, while LL5β and ELKS stabilize microtubule plus ends at hot spots for rapid and directed insulin secretion. Characterizing rapid and directed insulin secretion would enhance understanding of β cell dysfunction in the progression of type 2 diabetes.

P47

Identifying Key Regulators of Presynaptic Remodeling in C. elegans D-type GABAergic Motor Neurons

Gailey CD, Cuentas-Condori A, Tipps J, Taylor SR, Rodgers E, and Miller DM

ABSTRACT Neural circuits are actively remodeled during development as synapses are dismantled and reassembled elsewhere. In C. elegans, D-type GABAergic motor neurons are useful for investigating the mechanism of synaptic remodeling. C. elegans initially develops with 6 Dorsal D (DD) neurons with ventrally located presynaptic terminals. During early larval development, the DD ventral presynaptic terminals are disassembled and reconstituted on the dorsal side. Concurrently, 13 Ventral D (VD) neurons emerge and establish ventral presynaptic terminals. The IRX-1/Iroquois (homeodomain) and UNC-55 (COUP-TF) transcription factors are major regulators of synaptic remodeling. IRX-1 promotes DD synaptic remodeling, whereas UNC-55 is expressed in VD neurons where it inhibits IRX-1 to block remodeling. The key roles of IRX-1 and UNC-55 in regulating synaptic remodeling suggest that downstream effectors may be transiently expressed in remodeling DD neurons. Here, we have used single cell RNA-Seq (scRNA-Seq) to identify potential regulators DD synaptic remodeling.We used FACS to isolate D-class GABAergic motor neurons from 3 developmental stages for scRNA-Seq: (1) Before remodeling; (2) During remodeling; (3) After remodeling. UMAP plots of these data identified distinct clusters of D-class neurons corresponding to each developmental stage and confirmed developmentally regulated expression of known drivers of synaptic remodeling (e.g., irx-1, unc-8). Differential expression analysis identified 93 transiently expressed candidate remodeling genes, some of which are already confirmed. We are now using RNAi and mutant analysis to test these candidate genes for roles in DD remodeling. Our approach exploits GFP markers to monitor the location (ventral vs dorsal) of D-class presynaptic domains in live cell imaging experiments.

P49

Mapping the Time Course of mTORC1-Driven Tumorigenesis in the Developing Brain

Geben, LC; Rushing, GV; Brockman, AA; Lanaghan, ZM; Sweet, SR; Ess, KC; Irish, JM; & Ihrie, RA

ABSTRACT Subependymal giant cell astrocytomas (SEGAs) are large brain tumors that grow in patients with Tuberous Sclerosis Complex, a neurodevelopmental disorder that causes the growth of cortical lesions, learning disabilities, and epilepsy. SEGAs preferentially present near the ventral region of the lateral ventricles - a position that makes them difficult to surgically resect and can block the flow of cerebral spinal fluid - and are thought to be derived from neural stem/progenitor cells. However, the precise time window during development when a neural stem cell is susceptible to tumorigenesis is unmapped. Neural stem cells exist in heterogeneous populations that are precisely organized in space and across time. Specifically, different regional neural stem cell populations have varying levels of activation of mTOR, the master regulator of cell size and growth, and are variably proliferative or quiescent. We previously showed that different spatial populations of postnatal neural stem cells have varying levels of mTORC1 signaling, but the time when these signaling differences emerge, and their relationship to the cell cycle, is not known. Further, studies of pre- and postnatal TSC2 ablation revealed that prenatal neural stem cells are uniquely capable of generating SEGA-like tumors in the mouse, but the exact stage of prenatal development when tumor susceptibility exists has not been explored. Using per-cell measurements of mTORC1 activity in murine stem cell cultures, we found that embryonic and postnatal neural stem cells have differing responses to the induction of quiescence, including further differences observed across diverse spatial populations. Complementary in situ analysis of intact embryonic mouse brain confirms these patterns. These tools are now being applied to patient-derived induced pluripotent stem cell and brain organoid models of TSC. Collectively, these data suggest that embryonic and postnatal neural stem cells are distinct populations, possibly with distinct mechanisms of regulating cell cycle entry and thus tumor susceptibility. Results from this project may reveal the developmental time window when a prenatal neural stem cell is susceptible to SEGA growth, thereby informing the development of temporally and spatially targeted preventative treatments.

P51

Anthropoid-specific interaction of the chromatin associated protein WDR5 with the DNA helicase HELBB

Goswami S., Guarnaccia A.D., Howard G.C., Grieb B.C., Tansey W.P.

ABSTRACT WDR5 is a highly-conserved protein that performs a variety of functions in the nucleus. Its best-known role is scaffolding MLL/SET complexes that catalyze histone methylation, but WDR5 acts outside this setting to promote ribosomal protein gene transcription, recruit MYC to chromatin and bookmark genes for reactivation after mitosis. WDR5 is also overexpressed in cancer and is an auspicious target for pharmacological inhibition in malignancy. Current WDR5 inhibitors target the WIN (WDR5-interaction) site on WDR5, a deep pocket that binds to an arginine containing WIN motif [A-R- (A/S/T)] in partner proteins. The mechanism through which WIN site inhibitors function is largely unknown. Using quantitative proteomics, we identified ~25 proteins whose interaction with WDR5 is altered by WIN site inhibition. My project centers on one of these proteins, a DNA helicase known as HELB. I have identified a WIN motif at the amino terminus of HELB that is required for its interaction with WDR5. I have shown this WIN motif is post-translationally processed to generate an unusually high affinity WIN site, likely orders of magnitude tighter than most WDR5 WIN site binders. Interestingly, this WIN motif is only present in anthropoid HELB proteins, suggesting the interaction arose late in evolution for a specific molecular function in these animals. My current work is geared to understanding the functional significance of the WDR5-HELB interaction and its contribution to WIN site inhibitor function in cancer cells.

P53

Establishing C. elegans as a model system to study mitochondrial epigenetics Grub, L., Held, J., Patel, M.

ABSTRACT The metabolic state of the mother is an important health determinant of her offspring, and even subsequent generations. Epigenetic modifications provide powerful molecular means for the transmission of conditional information from parent to progeny. As a maternally inherited genome that encodes essential components of the electron transport chain, the mitochondrial genome (mtDNA) is ideally positioned to serve as a conduit for the trans-generational transmission of metabolic information. Excitingly, new evidence shows that mtDNA contains N6-methyladenosine (6mA), a type of epigenetic modification. However, 6mA has previously been shown in mammalian cell culture and there is currently no model for studying the role of mtDNA 6mA in the trans-generational transmission of information. We, therefore, set out to establish Caenorhabditis elegans as a model to study mtDNA epigenetics. C. elegans are a simple in vivo model, with a rapid generation time, and a wide variety of transgenic tools available. Here, we provide the first evidence that mtDNA of C. elegans is methylated in vivo. We first isolated an enriched mtDNA sample with limited genomic contamination by differential centrifugation of mitochondria. Using this enriched mtDNA sample, we identified methylation in the mitochondrial genome using a dot blot assay with an anti-6mA antibody. To specifically measure mtDNA 6mA, we designed a 6mA immunoprecipitation assay using an anti-6mA antibody conjugated to magnetic beads, followed by digital droplet PCR to quantify mtDNA. Immunoprecipitation of mtDNA is significantly enriched above control using anti-6mA. Combined, these assays provide compelling evidence of 6mA in C. elegans. This discovery provides an excellent basis for future studies to investigate the role of mitochondrial epigenetics in the trans-generational transmission of metabolic information.

P55

A role for “non-muscle” alpha-actinins in cardiac myocyte sarcomerogenesis James Hayes, Jr., Dylan Ritter, Abigail Neininger, Ela Knapik, Dylan Burnette

ABSTRACT The protein alpha-actinin is a dimeric actin-crosslinking protein with multiple paralogs in vertebrates. In general, each specific alpha-actinin paralog is believed to function primarily in either a “muscle-specific” context or in a more general, “non-muscle” context. I present data showing that the traditionally deemed “non-muscle” alphaactinins, alpha-actinin-1 and alpha-actinin-4, may have unexpectedly important – and opposing – roles during the formation of the cardiac sarcomere, a muscle-specific structure that is fundamental in driving the mammalian heartbeat. In a sarcomerogenesis assay, human cardiac myocytes depleted of alpha-actinin-1 assemble fewer sarcomeres than controls, while those depleted of alpha-actinin-4 are hypertrophic and assemble more sarcomeres. Restoring the levels of alpha-actinin-1 and -4, respectively, with overexpression rescues control phenotypes. Furthermore, alpha-actinin-1-depleted myocytes have fewer and smaller adhesions, which normally anchor tension-producing sarcomere precursors to the extracellular matrix as they mature into sarcomeres. Recently, I have begun performing experiments in vivo, using zebrafish embryos as a model organism for heart development. My overall goal is to define the mechanistic underpinnings of actinin-1 and -4-dependent influence on sarcomerogenesis at the level of the individual cell and, subsequently, to establish whether the mechanistic phenomena observed in cells hold true within the context of a developing organ in vivo.

P57

The microtubule minus end-binding protein CAMSAP2 is required for insulin vesicle trafficking and secretion of pancreatic β-cells

Kung-Hsien Ho, Gu Guoqiang, and Irina Kaverina

ABSTRACT The regulation of microtubule dynamics in β-cells is coupled to glucose metabolism and is critical for glucose-stimulated insulin secretion (GSIS). CAMSAP2 is a microtubule minus-end binding protein, which stabilizes and positions microtubules in cells. In the mouse insulinoma MIN6 cells, CAMSAP2 forms small stretches at microtubule minus ends in the cytoplasm as expected. Surprisingly, primary islet β-cells do not have CAMSAP2 stretches in the cytoplasm but have CAMSAP2 highly enriched at the Golgi. This unique localization of CAMSAP2 is specific to β-cells but not in other islet endocrine cells, and it is independent of microtubule-binding. CAMSAP2 depletion by shRNA impairs GSIS but does not affect the insulin secretion when cells are depolarized by potassium chloride. Furthermore, we found that CAMSAP2 is required to maintain the total insulin level and proper Golgi-ER trafficking in islet beta cells. Biochemical analyses suggest that islet β-cells express a smaller CAMSAP2 isoform than the CAMSAP2 expressed in MIN6 cells. We proposed that this differential isoform may confer its distinct subcellular localization at the Golgi and is required to support robust insulin production in primary islet β-cells.

P59

Tob2 marks a population of quiescent intestinal stem cells Mirazul Islam, Matthew E. Bechard, Paige Vega, Vishal M. Shah, Janney Wang, Won Jae Huh, Cody N.

Heiser, Bob Chen, Robert J. Coffey, Ken S. Lau

ABSTRACT Intestinal stem cells (ISCs) reside at the base of crypts and self-renew, giving rise to differentiated epithelial cell types at both steady-state and after tissue injury. Lrig1 is one of the ISC markers with a tumor-suppressive function that marks cells at the base of crypts. Here, we reveal the heterogeneity of the Lrig1+ population and found that a subset of Lrig1+ cells (Lrig1-Mid) is quiescent in nature. We identified a marker gene called Transducer of Erbb2.2 (Tob2) that marks this population. We noticed that Tob2+ cells are relatively rare and mostly found at the crypt base in the colon and at the +4 position in the small intestine. We performed Mki67 staining and found that most of the Tob2+ cells are Mki67 negative, implying quiescent state of this population. An orthogonal approach, using homing CRISPR barcoded mouse, also confirmed slow-cycling nature of these cells. Next, we explore injury-inducible property of these cells using whole-body irradiation (10 Gy) and DSS treatment (2.5% in drinking water) in mice. We found that Tob2+ cells increase in both injury models, indicating Tob2+ cells are injury inducible. We performed colony formation assay of different Lrig1+ cells using Lrig1-Apple reporter line and found that both Lrig1-Hi and Lrig1-Mid populations have almost equal colony-forming efficiency. This data confirms that Lrig1-Mid population has stem cell properties. Additionally, we found that TOB2 overexpression by DOX induction significantly reduced the growth rate over time, indicating strong anti-proliferative role of TOB2 protein. Further characterization of Tob2+ population is ongoing. Collectively, these data suggest that Tob2+ population is quiescent intestinal stem cells.

P61

Clearing and 3D Imaging of Large Tissue Blocks: Perils, Pitfalls and Success Jarvis B, Ziehm E, Booth D, Ursu V, Ray M, Wright CVE

ABSTRACT 2D tissue section analysis is limited in some ways, for example, for following fine filamentous structures from cell body to terminus, defining all axons on a neuron, or all filopodia of an exploratory macrophage. Resolving 3D structural volume in large tissue blocks obtains overviews and subcellular details, cellularly or molecularly relevant to normal organogenesis, physiological dysfunction, aging and cancer, amongst others. Biopsy-sized samples are imaging-processed with confocal microscopy while whole tissues use light-sheet imaging. There are many 3D imaging approaches, such as CLARITY, SWITCH, and iDISCO. Notably, these protocols were originally developed for brain and not more fragile tissues or organs, such as the pancreas, where fixation and autodigestion are notorious hurdles. We carried out a comparative survey on several protocols highlighting a number of problems with several existing methods. Problem 1: SDS for delipidation causes loss of tissue structural integrity. Problem 2: SDS either does not respect the antigen or incomplete SDS washout causes problems with antigen-antibody interaction. Problem 3: Antibody penetration issues – some antibodies present species-specific inability to penetrate the tissue. Problem 4: Substantial tissue autofluorescence over the 405-488 nm range. Some hurdles seem particularly applicable to the pancreas, and other tissues and organs are much less affected. We study the pancreas to evaluate early susceptibility to T1DM, mechanisms of spreading of endocrine-cell dysfunction across the organ in T2DM, and the nascent neoplastic niche in pancreatic cancer. We believe we have developed a protocol designed for these challenges. Our 3D-imaging process is non-SDS based, heavily reliant on a novel combination of detergents, additional “blocking” reagents, antigen stabilizers and autofluorescence minimizers. We have established a 3D toolkit of commercially available antibodies that work in various tissues. We have also verified that numerous antibodies that work perfectly in regular 2D IF (10 micron) or thick sections (50 micron) fail in large tissue blocks. Our process works for both mouse and human tissue blocks. As human tissue is extremely valuable and difficult to acquire, mouse tissue is used for troubleshooting and protocol optimization. The result is a combination process for staining a whole tissue block followed by imaging in confocal microscopy or light-sheet microscopy. Acquired data is then quantitatively analyzed via surface-rendering and other methods in applications such as Arivis, Imaris, and even “Oculus”-based virtual personal internal tours of the pancreas.

P63

Evaluation of cell culture conditions on the presence of Ago2 and miRNAs in small extracellular vesicles

Jimenez L, Barman B, Saffold C, Pelletier R, and Weaver A

ABSTRACT Extracellular vesicle (EV)-carried miRNAs have been shown to influence gene expression and functional phenotypes in recipient cells. Many investigators have found Ago2 in EVs and it is postulated that Ago2 is a major transporter of miRNAs into small EVs (SEVs), such as exosomes. Others have reported extracellular Ago2 that is non-vesicular. We set out to evaluate the effect of growth factor signaling and serum contamination on the detection of Ago2 in SEVs. Wildtype KRAS colorectal cancer cells, DKs8, were conditioned with 3 different culture media (Serum-free DMEM, EV-depleted FBS in DMEM, and Opti-MEM). In all conditions, we found the highest abundance of SEVs in cushion-density gradient fractions 6 and 7, as assessed by Western blot analysis. Ago2 was detected in the same fractions as SEVs in both the Serum-free DMEM and Opti-MEM conditions. In contrast, Ago2 was present in both vesicular and non-vesicular fractions in the EV-depleted FBS in DMEM condition. No significant differences were observed in the size and number of SEVs collected in the three conditioning methods. However, Western blots of equal vesicle numbers showed that Ago2 levels were significantly reduced in SEVs obtained from Opti-MEM and EV-depleted FBS in DMEM conditions compared to the Serum-free DMEM condition. Analysis of SEVs by dot blots showed Ago2 primarily on the inside of SEVs. Select miRNAs are on the inside of DKs8 Serum-free DMEM and Opti-MEM SEVs, as they only were sensitive to the RNase A treatment in the presence of Triton X-100. In contrast, the miRNAs appear to be predominantly on the outside of SEVs in the EV depleted FBS in DMEM condition, as the RNase A treatment in the absence of Triton X-100 abolished the detection of select miRNAs. Since Opti-MEM contains growth factors, we also collected SEVs from DKs8 cells conditioned with Serum-free DMEM supplemented with EGF. We did not observe that Ago2 levels in the Serum-free DMEM with EGF condition were altered compared to the Serum-free DMEM condition, as assessed by Western blot analysis. In summary, multiple factors may affect the ability to detect vesicular Ago2, including serum and growth factors in the conditioned media that may provide sources of extravesicular Ago2 and regulate the trafficking of Ago2 into vesicles.

P65

Enhanced extracellular vesicle production via hollow fiber bioreactor Youn Jae Jung, Jefferey L. Franklin, John T. Wilson and Alissa M. Weaver

ABSTRACT Extracellular vesicles (EVs) have emerged as a promising strategy to deliver effector molecules for intercellular signaling. Current approaches for EV production typically rely on 2D cell culture due to lack of a scalable biomanufacturing platform. However, the low EV production yield from 2D cell culture remains a challenge. In this study, we used hollow fiber bioreactors, which allows cells to grow to high density under 3D-like conditions to produce EV-enriched conditioned media without serum contamination. We investigated the production yield and the characteristics of small EVs (sEVs) purified from DKO-1 cells and hTERT-MSCs via hollow fiber bioreactors. sEVs were collected by iodixanol gradient ultracentrifugation and characterized by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA) and Western blot analysis. The number of sEVs purified from bioreactors was increased approximately 20-fold compared with that purified from an equivalent volume of conditioned medium from cells cultured in 2D. DKO-1 and hTERT-MSC derived sEVs from hollow fiber bioreactor displayed the expected round shape and size (50-200 nm in diameter) along with EV marker proteins, including Flotillin-1 and CD63. In addition, we evaluated the expression levels of miRNAs in sEVs by RT-qPCR. Small ncRNAs in sEVs collected from bioreactors, including U6, miR-100, miR-125b and let-7a, were well expressed and protected from RNase. Our results demonstrate that hollow fiber bioreactors can enhance EV production from cells while also preserving the integrity.

P67

Lipid nanoparticle mediated co-targeting of genetic and metabolic dependencies in pancreatic cancer

Nidhi Jyotsana, Dmitry S Koktysh, Kenny T Ta, Kathleen E DelGiorno

ABSTRACT

Background & Aims: Nearly all cases of PDAC are caused by mutation of the KRAS gene. However, despite intense efforts, mutant-KRAS oncoprotein remains undruggable and demands novel cross-disciplinary approaches for its direct targeting. Emerging evidence suggests that KRAS-mutant cancers are particularly vulnerable to inhibition of cystine import via SLC7A11 (Solute Carrier Family 7 Member 11). Therefore, we combined the latest lipid nanoparticle (LNP) delivery systems with siRNAs targeting SLC7A11 and/or mutant-KRAS as a novel and effective therapeutic strategy against PDAC. Methods: The metabolic dependency of PDAC cell lines on SLC7A11 was determined by varying cystine concentrations in the medium. siRNAs targeting mutant-KRAS and SLC7A11 were packaged in LNPs using microfluidic mixing technology. LNP-siRNAs were characterized and tested for their uptake efficiency using microscopy and flow cytometry. LNPs were conjugated with mesothelin/MSLN antibody for enhanced uptake in PDAC cells. The functional efficacy of LNP-siRNAs on PDAC cell lines was tested via annexin V assay, cell counting, and scratch assay. Result: Our LNP-siRNA formulations are ~ 75nm in diameter, with an siRNA encapsulation efficiency of >95%. LNP-siRNAs are efficiently and stably taken up by PDAC cells. MSLN- conjugated LNPs showed enhanced uptake in PDAC cells. Various PDAC cells show differential dependency on cystine for their survival and upregulate SLC7A11 expression levels under cystine starvation. Target specificity of LNP-mutant-KRAS siRNA and LNP-SLC7A11 siRNA was confirmed in various PDAC cell lines. Co-targeting of mutant-KRAS and SLC7A11 decreased total live cell count in various PDAC cell lines and, significantly increased cell death in MIA Paca-2 cells under lower cystine concentrations in the medium. Conclusions: LNP-siRNA mediated co-targeting of mutant-KRAS and SLC7A11 synergistically affect PDAC cell growth in vitro. Based on these results, our LNP-siRNA co-targeting approach may effectively target PDAC in vivo, with lower cystine levels present in the pancreatic tumor microenvironment.

P69

The Deubiquitylase USP47 Regulates Groucho/TLE Ubiquitylation to Promote Canonical Wnt Signal Transduction

Kassel, S., Saito-Diaz, K., Hanson, A.J., Goldsmith, L., Benchabane, H., Ahmed, Y., and Lee, E.

ABSTRACT The Wnt pathway is important for embryonic development, organ development, and tissue homeostasis. In the absence of Wnt ligand, the transcriptional coactivator Beta-catenin is phosphorylated by a "destruction complex", ubiquitylated, and targeted for proteasomal degradation. Activation of the pathway results in destruction complex inhibition, and elevated levels of Beta-catenin, which is translocated to the nucleus to drive the transcription of Wnt target genes. We identified the deubiquitylase USP47 as a positive regulator of Wnt signaling in a genome-scale screen in Drosophila S2 cells using the Drosophila Gene Collection. We found that USP47 is required for Wnt signaling in Drosophila, Xenopus, and human cells, indicating an evolutionarily conserved function. Knockdown of USP47 enhances ubiquitylation of Groucho (Gro)/TLE in cultured cells but does not affect steady state levels of Gro/TLE. Here, we report a new role for the deubiquitylase USP47 in regulating a nuclear Wnt transcriptional program.

P71

Merging of ventral fibers at adhesions drives the remodeling of cellular contractile systems in fibroblasts

Narasimhan,S; Holmes,WR; Kaverina,I

ABSTRACT Ventral stress fibers (VSFs) are higher-order, contractile actin fibers instrumental in cellular processes such as force production, polarization, and migration. Studies on the VSF network has shown that they have varying sizes and orientations in the cell- from short, thin fibers to long, thick bundles that span the length of the cell and align to the main axis. Current theories on the formation of VSFs include condensation from the cortical mesh for smaller fibers and crosslinking with other stress fiber subtypes (such as dorsal stress fibers and transverse arcs) for longer fibers. Our studies have uncovered a novel phenomenon of stress fiber merging in migratory fibroblasts, whereby two ventral fibers are rearranged into a single prominent stress fiber. This can be considered a new method used by a cell for VSF formation. The mechanism of merging involves the connection of two ventral fibers by an emerging myosin II bridge at an intervening adhesion, leading to adhesion dissolution and a single VSF formation. Our data indicate that contractile cohesion through the myosin II bridge drives the VSF remodeling and adhesion dissolution as the fibers combine. We further investigate through experiments and computational models how varied conditions such as the mode of the initial fiber crosslinking and the rate of adhesion dissolution contribute to the efficiency of the merging phenotype. Merging is used for dynamic reorganization of VSFs in steady state according to cellular needs such as forming protrusions, cell turning or simply to change the distribution of VSFs in the cell.

P2

Microtubule dynamics regulates gap junction trafficking and placement in the motor circuit

Lee, G.; Palumbos, S.; Strothman, C.; Zanic, M.; Miller, D.

ABSTRACT Gap junctions, or electrical synapses, mediate fast communication between neurons, but the mechanisms that direct gap junction assembly between specific neurons or within specific subcellular compartments are poorly understood. Gap junction localization ultimately depends on kinesins, which walk along microtubule (MT) tracks to deliver gap junction components to their cellular destinations. We are investigating gap junction assembly in VA motor neurons in which the UNC-4 transcription factor regulates the neuron specificity and placement of electrical synapses. Gap junction trafficking is disrupted in unc-4 mutants (Palumbos et al, worm meeting abstract). To investigate the mechanism of this effect, we utilized the MT plus-end marker, EBP-2::GFP, to monitor MT dynamics. We determined that unc-4 mutant MTs show a faster polymerization rate but a shorter growth distance, suggesting disrupted MT dynamics in the unc-4 mutant. This alteration in MT dynamics could be due to VAB-8, an atypical kinesin that is negatively regulated by UNC-4. VAB-8 binds MTs but lacks motor activity, and its ectopic expression has been shown to disrupt gap junction trafficking. We found that the unc-4;vab-8 double mutant shows restoration of WT-like MT polymerization rate but does not restore growth distance. We are now further testing the hypothesis that ectopic expression of VAB-8 in unc-4 mutant VA neurons disrupts gap junction trafficking by altering MT dynamics.

P4

Exosomes in filopodia formation McAtee C, Hoshino D, Hong NH, Sung BH, Maldonado A, von Lersner A, Zijlstra A, and Weaver A

ABSTRACT Introduction: Exosomes are small extracellular vesicles (SEVs) that carry a variety of cargoes and have been shown to promote tumor cell motility and metastasis. Cell motility is influenced by dynamic formation and stability of filopodia: actin-rich protrusions that extend from the leading edge and perform directional sensing. Filopodia regulators such as fascin are upregulated in multiple epithelial cancers and can promote invasive phenotypes. However, how filopodia are induced and controlled by extracellular factors is poorly understood. Here, we describe a role for SEVs in regulating filopodia formation and tumor cell motility. Methods: We utilized B16F1 melanoma cells and HT1080 fibrosarcoma cells for fixed- and live-cell imaging to quantify filopodia numbers and dynamics in control and exosome-deplete conditions. iTRAQ proteomics was used to identify SEV protein cargoes that contribute to filopodia formation. In vivo experiments were performed using a chick embryo model for metastasis. Results: Inhibition of exosome secretion in cancer cell lines, via Rab27a or Hrs knockdown, led to decreased filopodia numbers. Specificity to SEVs was demonstrated by rescue experiments in which purified SEVs but not large EVs rescued the filopodia phenotypes of exosome-inhibited cells. Live imaging of Hrs-KD cells revealed that exosome secretion regulates formation and stability of filopodia. Proteomics data and molecular validation experiments identified the TGF-beta coreceptor endoglin as a key SEV cargo regulating filopodia formation, cancer cell motility, and metastasis. Summary: In this study, we identified exosomal endoglin as a regulator of filopodia formation and in vivo metastasis. These data are relevant to cancer as endoglin expression is altered in many cancers. In addition, endoglin is the disease gene for hereditary hemorrhagic telangiectasia, and may influence angiogenesis. Overall, our data implicate SEV-carried endoglin as a key cargo regulating filopodia.

P6

Trop2+CD133+CD166+ dysplastic stem cells are de novo stem cells driving dysplasia transition to gastric cancer

Min J, Zhang C, Bliton RJ, Caldwell B, Caplan L, Presentation KS, Park DJ, Kong SH, Washington MK, Kim WH, Lau KS, Magness ST, Lee HJ, Yang HK, Goldenring JR, Choi E

ABSTRACT Background: Intestinal-type gastric cancer develops within a cascade of pre-cancerous metaplasia to dysplasia and adenocarcinoma. Gastric dysplasia is especially considered a key transition state between pre-cancer and cancer and at the greatest risk of developing adenocarcinoma. Cancer stem cells (CSCs) are known as a key subset leading cancer initiation or progression. However, it is not clear whether stem cells, which are responsible for the maintenance and progression of dysplastic cells, are actually present in dysplasia. Our previous study identified two putative dysplastic stem cell (DSC) populations, CD44v6-CD133+CD166+ (DP) and CD44v6+CD133+CD166+ (TP), which may contribute to cellular heterogeneity of dysplastic cell lineages. Hypothesis: Although the dysplastic cells might be gastric cancer-initiating cells and critical targets for intervention in the early induction of cancer, different risks of the heterogeneous dysplastic cell lineages remain largely unknown. We therefore hypothesized that DSCs are responsible for maintaining dysplastic cell lineages and evolution of dysplasia to adenocarcinoma. Methods: DP- and TP-DSCs were isolated from dysplastic organoids established from active Kras-induced mouse stomachs and utilized for RNA-seq to compare transcriptome profiles and to evaluate differentiation capacity. Subcutaneous injection of DSCs in immunodeficient nude mice was conducted to examine their tumorigenic potential. Single cell RNA-seq and immunostaining were performed to define molecular and cellular heterogeneity of DSC-driven tumors, and genetic changes during the DSC evolution were examined by whole-exome seq. Human tissue microarrays were used to identify the presence of DSCs in human dysplasia and dysplastic organoids were established from human dysplastic tissues to evaluate a therapeutic approach using Pyrvinium, targeting CK1α which is a downstream intermediate of Wnt signaling pathway. Results: Transcriptome profiles of the two DSC populations displayed high similarity of molecular characteristics as CSCs, but DP-DSCs showed more dynamic differentiation capacity than TP-DSCs. Growth and survival of dysplastic organoids were controlled by inhibition of Wnt signaling pathway using Pyrvinium. DP-DSCs evolved to heterogeneous types of tumors including high-grade invasive adenocarcinoma in mice, and additional genetic mutations related to human gastric cancers were acquired during the tumor formation. We also confirmed the presence of DSCs in human dysplasia and the effect of Wnt inhibition involved in the maintenance of human dysplastic organoid survival. Conclusions: We conclude that DSCs are de novo CSCs which lead dysplasia evolution to gastric cancer and can be targeted by Wnt inhibition. This study will provide important insights to guide the future therapeutic clinical strategies by targeting DSCs as cancer-initiating cells in patients with gastric cancer.

P8

Genome-wide screens to identify genes that regulate epithelial integrity and extrusion

Molina, P , Macara, I

ABSTRACT Cell extrusion is a mechanism used by epithelial tissues to maintain homeostasis in the presence of oncogenic signals, cell death, or during overcrowding. However, the mechanisms that regulate extrusion of epithelial cells are not fully understood. Global approaches have not yet been employed to interrogate the collective behavior of cell extrusion. My poster will show the two approaches we have developed to better understand the regulation of cell extrusion, using mouse mammary epithelial cells as the model system. The first approach will use a pooled CRISPR library to conduct a genome-wide knockout screen that identifies genes required to maintain epithelial integrity. Loss of any such gene will disrupt cell-cell adhesion and promote escape of the cell harboring the gene deletion from the epithelial monolayer. These extruded cells can be captured and sequenced to identify the gRNA and its target gene. To validate the approach, I have shown that knockout of E-cadherin results in efficient extrusion of Ecad-negative cells. In the second approach, I am studying the mechanism by which epithelial cell monolayers promote the extrusion of cancer cells. I have adapted a novel method called the G-baton (GFP-based touching nexus) system to identify genes that promote the extrusion from mammary epithelia of cells that harbor a mutant H-Ras oncogene. The system transfers a fluorescent tag from a "sender" cell specifically to neighboring cells with which it is in direct contact ("receiver" cells). This powerful new method will allow us to sort selectively for wild type cells that are touching Ras-transformed cells, then conduct RNAseq analysis to identify transcriptional changes in this subpopulation of wild type cells, as compared to wild type cells that have not been in contact with the cancer cells.

P10

MISP is an actin bundler that selectively stabilizes the rootlets of epithelial microvilli

Morales EA, Arnaiz C, Krystofiak ES, Zanic M, Tyska MJ

ABSTRACT Microvilli are conserved actin-based surface protrusions that have been repurposed throughout evolution to fulfill diverse cell functions. In the case of transporting epithelia, microvilli are supported by a core of actin filaments bundled in parallel by villin, fimbrin, and espin. Remarkably, microvilli assembly persists in mice lacking all three of these factors, suggesting the existence of unknown bundlers. We identified Mitotic Spindle Positioning (MISP) as an actin binding factor that localizes specifically to the membrane-free proximal end of the core bundle, referred to as 'rootlet'. Gain- and loss-of-function studies revealed that MISP promotes rootlet elongation and protects this end from membrane wrapping. Purified MISP exhibits potent bundling activity of actin filaments in vitro with a center-to-center spacing comparable to that of bundles produced by fimbrin. As fimbrin preferentially associates to the rootlet end of the microvillus similar to MISP, we investigated the potential interplay of these factors in cells. We found that MISP-bundled filaments recruit fimbrin, which further elongates and stabilizes core bundles in a cooperative fashion. The exaggerated elongation of core bundle rootlets driven by MISP and fimbrin also allowed us to visualize the temporal details of their formation, which preceded the assembly of microvillar protrusions. Furthermore, consistent with MISP specific targeting to rootlets, we found that the membrane-cytoskeleton linker ezrin confines MISP to the membrane-free ends of core bundles, which prevents MISP decoration of the membrane-wrapped distal end. Collectively, our results indicate that MISP is a new actin bundler specific to microvillar rootlets and exhibits mutual exclusive targeting with ezrin to tune proper membrane coverage of core bundles. Thus, these discoveries offer insight on the remarkable robustness of microvilli assembly and further reveal how epithelial cells optimize apical membrane surface area.

P12

Investigating How Dietary Restriction Remodels ER Structure to Promote Longevity

Mulligan, AG., Donahue, EKF., Burkewitz, K.

ABSTRACT Age-dependent disease, including cancer, neurodegeneration, cardiovascular disease and diabetes, represents an increasingly major threat to public health. Progress in the biology of aging, however, has revealed that interventions such as dietary restriction (DR) can ameliorate the underlying causes of aging. Because DR regimens are too extreme for widespread therapeutic use, however, our work aims to identify potential drug targets among the cellular mechanisms by which DR delays aging. One key way by which cells adapt to the altered nutrient and metabolic conditions of DR is the remodeling of organelle structures. For example, it has been shown that DR enhances fusion of mitochondrial networks, and that reprogramming mitochondrial metabolism through its morphology is critical for promoting healthier aging. Mitochondrial morphology is regulated through physical interactions with tubules of the endoplasmic reticulum (ER). ER structure is also very dynamic and is defined largely by its balance of smooth tubules, which perform roles in lipid biosynthesis and inter-organelle signaling, with rough cisternal subdomains, which are hubs for protein homeostasis and secretion. While we know these structures correlate closely with the function of the cell, our understanding of ER dynamics during aging and DR remains limited. Preliminary data suggest that aged C. elegans exhibit a loss of ER sheets and an expanded tubular domain, and that DR reverses or modulates these effects on ER structure. Here I will present my hypothesis, that DR promotes longevity by reducing ER tubulation. I propose that protecting ER structures from age-dependent shifts preserves its function, including calcium homeostasis, through modulation of the ER's unfolded protein response (UPRER). I will explore these possibilities through a combination of live fluorescence imaging and 3D ultrastructural analysis of organelle structures via focused ion beam scanning electron microscopy (FIB-SEM). I will also present our plans and genetic tools in C. elegans to analyze the UPRER's effect on ER morphology as well as how morphology influences calcium homeostasis.

P16

Building a Multimodal Molecular Atlas of the Human Kidney Neumann, E. K., Patterson, N. H., Tideman, L., Migas, L., Allen, J. L., Sharman, K., Yang, H., Romer, C. E., Gutierrez, D., Brewer, M., deCaestecker, M. P., Harris, R., Fogo, A. B., Van de Plas, R., Caprioli, R.

M., Spraggins, J. M

ABSTRACT The human kidney is composed of over 26 cell types that actively coordinate to form higher order structures, such as glomeruli and tubules. While the scientific community generally understand the roles of these cell types and structures, it is not known how these cells vary molecularly and numerically throughout a single organ or between organs within the human population, particularly as a function of demographic. Understanding both the underlying similarities and differences between these demographics has major ramifications for functional efficiency, transition to disease, and disease severity. Here, we developed a workflow consisting of sample preparation, pathological assessment, co-detection by indexing multiplexed immunofluorescence, matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS), liquid chromatography, and histological staining to obtain rich molecular and cellular profiles of 17 human kidneys. In total, we sampled from both men and women between the ages of 21 to 77 years old. Through these datasets, we have parsed biological differences between the sexes without confounding factors related to age, race, BMI, or technical variation. The large sample size and depth of molecular coverage enabled creation of the most extensive atlas of the human kidney to date, including >200 identified lipids and 23 antibody labels. We then used a mixture of supervised and unsupervised approaches to create molecular and cellular profiles of glomeruli and proximal tubules. By correlating our suite of analytical approaches with MALDI IMS, we have hypothesized the physiological roles of cell type defining lipid features. For example, phosphatidylinositol lipids were globally detected with lower abundance in female glomeruli with the largest differences observed in PI(34:2), PI(36:4), and PI(32:0). Based on multimodal data, we hypothesize that these molecules are critical substrates for prostaglandin synthesis in glomerular mesangial cells. The function of most lipids remains unknown because they are difficult to assess, demonstrating the importance of this data and power of the approach for understanding and hypothesizing the role of discrete lipids. Moreover, the established methodology is demonstrated for the human kidney but can be expanded and validated for use in other organ systems, such as the heart or spleen, or animal models, such as the mouse. Because the workflow is flexible, a variety of samples can be accommodated for high content atlas creation and exploration of biological systems.

P18

A protease-initiated model of wound detection James T. O’Connor, Fabiha Bushra Akbar, Erica K. Shannon,

M. Shane Hutson, Andrea Page-McCaw

ABSTRACT Wounds trigger surrounding cells to initiate repair, but it is unclear how cells detect wounds. The first known wound response of epithelial cells is a dramatic increase in cytosolic calcium, which occurs within seconds. What is the instructive signal created after damage that is detected by surrounding cells, causing them to increase their cytosolic calcium? To identify the mechanisms driving wound-induced calcium signaling, we monitored the calcium response after wounding in vivo in genetically modified Drosophila pupae. Using the Gal4/UAS system, we wounded on the border between tissue expressing a UAS-RNAi for potential genes of interest and an internal control tissue lacking knockdown, and compared the symmetry of the calcium response between the two sides. By doing this, we identified a G-protein coupled receptor acting through the Gq-signaling pathway to release calcium from the endoplasmic reticulum in epithelial cells. We also identified the pathway-initiating ligands, which are proteolytically cleaved from their inactive/pro-peptide forms into their active forms. Both the ligand and receptor are necessary for the calcium signal to be detected many cell diameters away from the wound margin. Furthermore, we discovered this same mechanism drives calcium responses of wing imaginal discs cultured in vitro when exposed to whole animal lysate. Interestingly, multiple classes of proteases are sufficient to activate the ligand and initiate this signaling cascade. We suggest that proteases released from cell lysis following damage are an instructive signal, cleaving the latent pro-ligand into its active from that diffuses extracellularly and binds to its receptor on distal cells to inform them of the presence of a nearby wound.

P20

Standardization of methods for biomarker discovery and validation based on Extracellular vesicle proteins from Plasma

Ozawa, P.M.M.; von Lersner, A.K.; Zijlstra, A; Weaver, A.M.

ABSTRACT Extracellular vesicles (EVs) are attractive candidates for biomarker research because they carry secreted RNA, DNA, and proteins that are can be used as biomarkers for diagnostics, staging, and treatment monitoring. Our goal is to develop a liquid biopsy method for biomarker discovery and validation based on EVs proteins from plasma. To do that, we need to optimize EV isolation of plasma for proteomic analysis (discovery) and standardize the plasma analysis by microflow cytometry analysis (validation). EVs were isolated from plasma using Size Exclusion Chromatography (SEC), follow by concentration with amicon column filters. Additionally, Nanotracking analysis and microBCA, followed by Western Blotting were performed. We are standardizing a new methodology to identify EVs using the image-based flow cytometer CellStream, in collaboration with the Zijlstra, and it will be used in the future as our validation method. We use the lipid dye Di-8-ANNEPs and CD63 antibody to identify our EVs. Several conditions were tested: purified EVs, whole plasma, SEC-EVs from plasma, spike-in of purified EVs on whole plasma, and SEC-plasma. The data collected from this instrument was analyzed using dimensional reduction analysis. Both whole plasma and SEC plasma have EVs specific populations identified by microflow cytometry analysis. Additional analysis is being done to access the detection limit for plasma EVs using both conditions. After standardization is done, we will proceed to proteomic analysis of patient and control plasma and following validation in a larger cohort.

P22

The Mystery of the Peroxidasin Mutant: Why Does this Catalytically Dead Drosophila mutant survive?

Peebles, K., LaFever,K. , Bhave,G. , Page-McCaw, A.

ABSTRACT The basement membrane is a sheet-like extracellular matrix that underlies epithelia and surrounds muscles. In the gut of Drosophila, the stiff basement membrane surrounds the muscles used in peristalsis to keep them flat and smooth. Collagen IV is one of the main components of the basement membrane, where it adds structure and stiffness. The Peroxidasin (Pxn) gene encodes an enzyme that catalyzes the covalent bonds necessary for crosslinking collagen IV at the NC1 domain. This crosslinking supports basement membrane stability and contributes to its stiffness. Drosophila hypomorphic mutants with reduced Pxn-mediated crosslinking die before reaching adulthood, while mice mutants with reduced Pxn-mediated crosslinking survive. We expected that a mutation that eliminates the catalytic domain of Pxn in Drosophila would die at the end of embryogenesis, when collagen IV mutants die. Using CRISPR, we created a mutant (Pxn11) that deletes a portion of the catalytic domain, eliminating its activity. Further, this deletion also caused a frameshift mutation that inserted a stop codon soon after the deletion. Unexpectedly, some mutants homozygous for the deletion are still viable, albeit very few, but they live an apparently normal lifespan. These homozygotes exhibit muscle defects in the gut consistent with loss of stiffness in the basement membrane. A few hypotheses that could account for their viability will be discussed.

P24

Untangling the role of CLK-1 in mtDNA heteroplasmy dynamics Pereira, CV; Held, JP; Patel, MR.

ABSTRACT The mitochondrial genome is prone to mutations that can propagate and persist throughout generations, despite their deleterious consequences. Therefore, at any given moment, copies of wild-type and mutant mtDNA molecules can co-exist within the same cell or organism, which is termed heteroplasmy. Mitochondrial disease ensues when a certain threshold for the levels of mutation is surpassed, usually 60-90% mutation load. Since there are multiple copies of mtDNA per cell, the mtDNA copy number can vary in different cells and tissues, according to their energetic demands. Previous work from our lab suggested the existence of a homeostatic copy number regulation mechanism to maintain the wild-type mtDNA levels in the well characterized heteroplasmic C. elegans strain, uaDf5. In contrast, the mutant mtDNA escapes this control, leading to an increase in the total uaDf5 mtDNA levels. The mechanisms that dictate the proliferation of pathogenic mutations and the regulation of mtDNA copy number, remain to be elucidated. Previous studies showed that CLK-1, a protein that participates in the ubiquinone biosynthesis pathway can bind mtDNA in vitro. Recently, our group found that ubiquinone deficient clk-1(ok1247) mutant worms display an elevated total mtDNA copy number which is not normalized to control levels in the presence of the ubiquinone precursor, 2-4-dihydroxybenzoate (DHB), suggesting an additional role for this protein. Our study aims to first determine whether CLK-1 is capable of modulating mtDNA heteroplasmy frequency in uaDf5 heteroplasmic worms, by crossing clk-1(ok1247) mutant with uaDf5 worms and, evaluating the heteroplasmy frequency by quantitative ddPCR. These experiments were performed in the presence of 10mM DHB, to guarantee clk-1 mutants are supplemented with ubiquinone. Our results show that clk-1 (ok1247) mutant; uaDf5 worms have in average significantly lower mutant mtDNA frequency than wild-type; uaDf5 worms. Furthermore, we aimed to understand if CLK-1 role could be generalizable and analyzed the impact of CLK-1 absence in other heteroplasmic strains. Our work shows CLK-1 to be involved in mtDNA heteroplasmy dynamics in a mutation-specific manner and possibly, in the mechanism of mtDNA copy number regulation.

P26

Interrogating Hippo Signaling in RPE proliferation Ramirez S., Newsome W.B., Yu E., Fuhrmann S.

ABSTRACT Dysfunction and degeneration of retinal pigment epithelium (RPE) cells leads to detrimental vision diseases such as dry age-related macular degeneration (AMD). One possibility for treating dry AMD is the stimulation of endogenous RPE regeneration, but little is known about the mechanisms that could drive RPE regeneration in vivo. The Hippo signaling pathway regulates cell proliferation and regeneration in many tissues, including during retina regeneration. In the developing RPE, the Hippo pathway is required for proliferation and cell fate decisions. Here, we manipulate components of the Hippo pathway to investigate whether regeneration in the adult mouse RPE can be achieved after injury. We generated Nf2-conditional knockout (CKO) mice using the RPE-specific, doxycycline-inducible tet-on VMD2-Cre. We performed intraperitoneal or retro-orbital injections of sodium iodate (NaIO3) on 7- to 8-week-old mice to induce oxidative damage mainly to the RPE. Injury and potential regeneration were monitored over the course of 7-11 weeks post injury with electroretinography and optical coherence tomography. Proliferation and extent of regeneration were assessed by EdU incorporation and expression of OTX2 and RPE65 in the presumptive RPE layer 7.5 weeks post injury. Our preliminary results show that there can be improved tissue integrity and a trend for increased proliferation in adult Nf2-CKOs at 7.5 weeks post-injury. Furthermore, there are more OTX2-positive cells in the presumptive RPE, and several show colocalization with EdU, suggesting potential de-novo production of RPE cells. Consistent with the role of NF2 in the Hippo pathway, we detect nuclear localization of YAP in Nf2-CKO RPE. However, we also observed a reduction of photoreceptor cells in the undamaged Nf2 CKO retina, suggesting a cell autonomous, essential role of NF2. To induce a more robust activation of nuclear YAP, we included a constitutively activated Yap allele (Yaps5a) in our analysis. Unexpectedly, in undamaged RPE, we observed doxycycline-independent activation of the Yap5sa allele, associated with ectopic proliferation in the RPE of 3- to 4-week-old mice. Our results indicate a potential role for YAP activation in proliferation of mature RPE, which we hypothesize to be beneficial to promote regeneration after injury. We are currently investigating whether the proliferation observed in YAP5SA RPE can be detected after injury using another RPE Cre-driver that is not 'leaky' (Tyr-Cre), as it can be induced more specifically in the adult. Additionally, to investigate a potential mechanism, we are determining whether YAP and TEAD directly regulate RPE-specific gene expression to control RPE specification.

P28

Loss of secretory IgA in COPD small airways is pIgR-independent Jessica B. Blackburn, Jacob A. Schaff, Rui-Hong Du, David Nichols, Taylor Sherrill, Austin J. Gutierrez, Arun C. Haberman, Carla L. Calvi, Nancy Wickersham, Yong Zhang, Michael J. Holtzmann, Lorraine B

Ware, Jae Woo Lee, Nicholas E. Banovich, Jonathon A. Kropski, Vasiliy V. Polosukhin, Timothy S. Blackwell, and Bradley W. Richmond

ABSTRACT Background: Loss of secretory IgA in small (< 2 mm) airways is common in patients with chronic obstructive pulmonary disease (COPD) and is implicated in disease progression in animal models. Transport of secretory IgA across the airway epithelium is mediated by a chaperone protein called the polymeric immunoglobulin receptor (pIgR). We hypothesized that loss of secretory IgA in COPD results from fewer numbers of pIgR-expressing cells in COPD small airways - particularly those affected by goblet cell hyperplasia (GCH). Methods: Cell type-specific expression of pIgR was determined in human and murine small airways by RNA in-situ hybridization, immunostaining, single-cell RNA sequencing (scRNA-seq), and complementary in vitro studies in primary murine tracheal epithelial cells. The functional consequences of loss of pIgR in specific cell types was evaluated by examining lung sections from mice with ciliated or secretory cell-specific deletion of pIgR. Transport of secretory IgA by goblet cells was evaluated in vitro by treating primary human small airway epithelial cells with IL-13 to induce GCH and measuring transport of secretory IgA across the airway epithelium by ELISA. Results: Secretory cells are the dominant cell type responsible for pIgR expression in human and murine airways, although other cell types in the airway and epithelium are capable of lower-level expression. Deletion of pIgR in airway secretory cells but not ciliated cells reduced the global pool of pIgR in murine lungs. Contrary to our hypothesis, we found no evidence for reduced numbers of pIgR-expressing cells or reduced pIgR expression per cell in secretory IgA+ versus secretory IgA- small airways from COPD patients. Additionally, we found that IL-13-induced goblet cells express high levels of pIgR and are effective transporters of secretory IgA in vitro. Discussion: Loss of pIgR expressing cells or pIgR expression per cell does not fully account for reduced secretory IgA in the airways of patients with advanced COPD. Although loss of secretory IgA is closely associated with GCH, at least some goblet cells transcytose secretory IgA effectively. These data suggest that the relationship between GCH and loss of secretory IgA in COPD airways is indirect.

P30

Development of a Microfluidic Co-Culture System for Measurement of Endothelial-Epithelial Permeability Changes

Riedmann, K., Meegan, J., Ware, L., and Bastarache, J.

ABSTRACT Sepsis accounts for 20% of global annual deaths and has a high mortality due to the lack of effective treatments. Making matters worse septic patients often develop the acute respiratory distress syndrome (ARDS) caused by the breakdown of the pulmonary endothelial barrier allowing fluid to enter the alveolar space. While we understand the mechanisms of endothelial dysfunction, the proximal triggers remain largely unknown. Understanding the upstream triggers of endothelial dysfunction requires a system that allows for quantitative measure of permeability changes. Current methodologies either use a static culture system (Transwell) or only indirectly measure barrier permeability (electric cell-substrate sensing impedance sensing). We present an adaptation of the SynVivo 3D co-culture system that allows for real-time measurement of large-molecule diffusion under flow conditions. The microfluidic system uses three parallel channels formed inside a PDMS chip that are connected by 3 micron pores between the adjacent channels permitting diffusion between channels. In the co-culture system, both primary human lung microvascular endothelial cells and human small airway epithelial cells are grown under physiologic flow conditions to stimulate formation of tight opposing barriers. HLMVECs grown in the system show a stretched cobblestone morphology in the direction of the flow and have bright, localized staining of their adherens junctions, when stained against VE-cadherin in ICC. Permeability is measured by flowing fluorescently conjugated 60 kDa dextrans combined with treatments of interest in the outer endothelial channels and measuring diffusion into the center epithelial channel over time. We demonstrate here that cell-free hemoglobin (CFH) in the 2+ oxidative state does not induce barrier dysfunction while CFH 3+ does induce barrier dysfunction (6 vs 47, relative permeability). In addition, we used thrombin, a stimulus known to increase endothelial but not epithelial permeability, to test if the dual endothelial-epithelial barrier is intact. Thrombin treatment disrupts the endothelium, as visualized by phase contrast, but does not disrupt the epithelium allowing diffusion of the fluorescent dextran into the center channel. The development of this system opens new opportunities to evaluate septic stimuli for their ability to induce endothelial-epithelial barrier changes that underly the pathophysiology of ARDS.

P32

Intact protein imaging mass spectrometry on a MALDI timsTOF Pro: steps toward rapid analysis at high spatial and spectral resolution Rivera, E., Klein, D., Colley, M., Weiss, A., Skaar, E., Caprioli, R., Spraggins, J.

ABSTRACT Untargeted analysis of proteins in situ is often performed using MALDI imaging mass spectrometry (IMS) on time-of-flight (TOF) mass spectrometers owing to their theoretically unlimited m/z range, sensitivity, and high spatial resolution capabilities. However, the limited resolving power of linear TOF instruments results in convoluted spectra and poses a challenge to determining the exact mass of intact proteins. Implementation of FTMS platforms for protein imaging has enabled high mass resolution and accuracy measurements but requires long scan times. Here we demonstrate high spatial resolution IMS of intact proteins on a QTOF-ion mobility mass spectrometer that enables rapid analysis at spectral resolutions comparable to that obtained on a FTMS. Protein standards ranging in mass from 3.1kDa to 24kDa were analyzed by MALDI. All proteins were detected, several of which were observed in both single and multiply charged states. The 1+ and 2+ ions were detected for ubiquitin, thioredoxin, and b-lactoglobulin, demonstrating a detectable mass range of at least m/z ~18,000 and detection of proteins up to 24kDa in mass. Due to the constant mass resolving power across the mass range of QTOF platforms, all protein measurements were made with a resolution of ~40,000. This spectral performance was sufficient to baseline resolve most isotopic envelopes, even for ions with m/z > 10,000. A whole-body mouse pup tissue section was imaged at 50µm spatial resolution resulting in ~300 endogenous protein species being detected up to m/z ~15,000. These results demonstrate the capacity of the timsTOF Pro to detect a comparable number of protein species as has been previously reported using FT-ICR based platforms, with comparable resolving power at high m/z. Next, we sought to determine the limits of sensitivity and spatial resolution on the timsTOF platform by performing 10µm spatial resolution IMS on rat brain resulting in detection of ~75 proteins. This work demonstrates the utility of this platform for high spatial resolution, high mass resolving power MALDI IMS of intact proteins. Activation of trapped ion mobility spectrometry (TIMS) during analysis of the protein standard mix demonstrated charge state separation, suggesting that ion mobility will be beneficial during tissue analysis. We are currently applying this technology to the investigation of Staphylococcus aureus infected murine kidney tissues to reveal discrete protein localizations within staphylococcal abscesses and to understand the proteomic constituents found at the interface between host and pathogen.

P34

Mitochondrial fission is essential to maintain cristae morphology and bioenergetics

Robertson, G, Patel, M, Riffle, S, Marshall, A, Beasley, H, Garza Lopez, E, Hinton, A, and Gama, V.

ABSTRACT Mitochondria are dynamic signaling organelles that constantly undergo fission (fragmentation) and fusion (elongation) to adapt their structure to the bioenergetic demands of the cell. DRP1 (dynamin-related protein 1) is an essential GTPase that executes mitochondrial fission. Patients with de novo heterozygous missense mutations in the gene that encodes DRP1, DNM1L, present with encephalopathy due to mitochondrial and peroxisomal elongation (EMPF). EMPF is a devastating neurodevelopmental disease with no effective treatment. To interrogate the molecular mechanisms by which DRP1 mutations cause neurodevelopmental defects, we are using patient-derived fibroblasts and iPSC-derived models from patients with mutations in different domains of DRP1 who present with clinically disparate conditions. The G32A mutation lies in the GTPase domain of DRP1 and is associated with microcephaly. The R403C mutation lies in the stalk domain of DRP1 and causes progressively severe epilepsy. Thus far, I have aimed to uncover the impact of DRP1 mutations on mitochondrial structure and metabolic function using patient-derived fibroblasts. Using super resolution imaging, I have determined that patient cells display elongated mitochondrial morphology and lack of fission. Although I found that mutant DRP1 is still recruited to the mitochondria, DRP1 appears to display increased association with the adaptor protein on the mitochondrial membrane, FIS1, pointing to a potential dysregulation in the kinetics of DRP1 recruitment by this mitochondrial adaptor. Given that previous literature shows that mitochondrial elongation promotes oxidative phosphorylation, we explored the impact of these mutations on cellular energy production. Surprisingly, patient cells display a lower coupling efficiency of the electron transport chain, as a result of increased proton leak and aberrant cristae structure. Further, patient cells upregulate glycolysis and rely more heavily on glycolysis to feed into oxidative phosphorylation. Understanding the mechanism by which DRP1 mutations cause neurological pathology will give insight into the role of mitochondrial dynamics in neurodevelopment.

P36

The Cell-Free Hemoglobin-Oxidized LDL Axis Contributes to Microvascular Endothelial Barrier Dysfunction and Poor Outcomes During Sepsis-Induced

ARDS Jamie E. Meegan, Toria Tomasek, Alex Desco, Julie A. Bastarache, Lorraine B. Ware

ABSTRACT Disruption of the microvascular endothelial (MVEC) barrier is a driving pathological feature of sepsis-induced acute respiratory distress syndrome (ARDS). Circulating factors elevated during sepsis contribute to MVEC hyperpermeability, including oxidized products like ferric (Fe3+) cell-free hemoglobin (CFH) and oxidized LDL (oxLDL). Importantly, CFH can oxidize LDL, potentially creating a feed-forward mechanism that could perpetuate MVEC hyperpermeability. However, the mechanisms by which CFH and oxLDL contribute to MVEC barrier dysfunction during sepsis are incompletely understood. We hypothesized that elevated levels of CFH during sepsis are associated with higher levels of oxLDL and poor outcomes, and that CFH contributes to MVEC barrier dysfunction through oxidation of LDL and activation of its receptor, LOX-1. Circulating levels of oxLDL, CFH, and sVE-cadherin were measured in 25 sepsis patients at risk for ARDS. In sepsis patients, plasma oxLDL levels correlated with CFH and sVE-cadherin and were higher in patients who died in hospital (37.87 vs. 28.86 U/mL, p=0.046). Patients who developed ARDS also tended to have higher oxLDL levels. In human lung microvascular endothelial cells (HLMVECs), barrier dysfunction was assessed by Electric Cell-substrate Impedance Sensing (ECIS). CFH-mediated (p<0.0001) and oxLDL-mediated (p=0.0375) HLMVEC barrier dysfunction was attenuated by blocking LOX-1 receptor (p=0.0372 vs CFH and p=0.0156 vs oxLDL). In conclusion, increased plasma CFH and oxLDL are associated with poor clinical outcomes and MVEC barrier dysfunction in sepsis; one mechanism by which CFH may cause MVEC hyperpermeability during sepsis and ARDS is through oxidation of LDL which can drive signaling through the LOX-1 receptor. [Funding: NIH HL094296, HL103836, HL135849]

P38

Small intestinal Tuft Cell Specification and Behavior in Homeostasis and Irritable Bowel Disease

Saleh N, Banerjee A, Wang J, Vega P, Simmons J, Lau K.

ABSTRACT Crohn's disease is a debilitating disease; Patients suffer from abdominal pain, severe diarrhea, fatigue, weight loss and malnutrition. While the etiology of Crohn's disease is unclear, it is likely a confluence of genetic predisposition, environmental triggers, microbial imbalance, and immune dysregulation. Treatments for Crohn's disease have greatly improved over the last few decades, moving away from broad immunosuppression strategies and focus on targeted immunomodulation. Despite the expansion of therapeutic options, many patients fail treatment. Thus, a deeper understanding of the role of gastrointestinal epithelia in the inflammatory response is necessary. Using mouse models of Crohn's Disease, we have linked the critical role of DCLK1+ tuft cells in modulating inflammation. Upon therapeutic succinate supplement to induce tuft cell specification, the inflammatory phenotype is reversed in TNFdARE/+ mice, which spontaneously develop Crohn's-like ileitis due to excess TNF expression. This amelioration of inflammation is tuft cell-dependent, and unique to tuft cells that develop independently of the secretory master regulator ATOH1. Single-cell RNA-sequencing data reveals multiple tuft cell populations in the wildtype ileum similar to previous reports, possibly consisting of a mixture of ATOH1-dependent and ATOH1-independent tuft cells. However, only a single population of tuft cells was identified in the AtohKO ileum. We investigate the hypothesis that distinct tuft cell populations, each with different functions, arise from ATOH1 -dependent and -independent mechanisms.

P40

Influenza A virus causes shedding of the alveolar epithelial glycocalyx through activation of sheddases

Schaaf, K. R., Buggs, C. J., Putz, N. D., Jetter, C. S., Negretti, N. M., Sucre, J. M. S., Schmidt, E. P., Bastarache, J. A., Shaver, C. M.

ABSTRACT Background: The healthy alveolar epithelium is protected by a heparan sulfate rich, glycosaminoglycan layer called the epithelial glycocalyx. Our group found that the epithelial glycocalyx is shed in patients with acute respiratory distress syndrome (ARDS), likely through the activation of sheddases (membrane-bound enzymes that cleave extracellular potions of transmembrane proteins). ARDS is commonly caused by viral infections including influenza A (IAV). In murine models, IAV causes massive and persistent glycocalyx shedding into the airspace but the mechanisms by which are unknown. Since IAV is known to regulate some sheddases, we hypothesize that IAV causes glycocalyx shedding through induction of host sheddases. Methods: We examined the literature and curated a list of sheddases associated with IAV with potential to cleave the glycocalyx (MMP-7, -2, -9 and their inhibitors TIMP-1 and -2). C57BL/6 mice were infected intranasally with A/PR/8/34 (H1N1) at 30,000 PFU/mouse and bronchoalveolar lavage and lung tissue was collected at day 1, 3, and 7 post infection. Sheddase expression was assessed by RT-qPCR and RNAscope was used to localize lung sheddase expression in infected and uninfected lungs. MLE-12 mouse lung epithelial cells were infected with viable or heat-inactivated (56C for 30 min) A/PR/8/34 (H1N1) at a MOI of 1 and sheddase expression measured by RT-qPCR after 72h. Results: RNA scope reveals that MMP-7 is upregulated in infected vs. uninfected lungs at day 1 and 3 post infection, then returns to baseline levels by day 7. When upregulated, MMP-7 is only expressed in cells that are directly infected by IAV. Expression of the MMP-7 inhibitor TIMP-1 is similar to uninfected lungs on day 1, but increases 50-fold on day 3 (p=0.06). In contrast, MMP-2 and MMP-9, as well as their inhibitor TIMP-2 are not upregulated in the first 7 days after IAV infection. Preliminary studies in lung epithelial cells suggest that heat-inactivated IAV fails to upregulate MMP-7. Conclusions: Together, these data suggest that localized IAV infection increases MMP-7 in a murine model of IAV infection, but has no effect on several other sheddases. This suggests that MMP-7 may be critical for IAV-induced glycocalyx shedding. Future studies will explore the mechanisms of IAV induced glycocalyx shedding which could provide molecular targets for clinical intervention in IAV-ARDS pathogenesis.

P42

WIN site inhibition disrupts a subset of WDR5 function Andrew J. Siladi, Jing Wang, Andrea C. Florian, Lance R. Thomas, Joy H. Creighton, Brittany K.

Matlock, David K. Flaherty, Shelly L. Lorey, Gregory C. Howard, Stephen W. Fesik, April M. Weissmiller, Qi Liu, and William P. Tansey

ABSTRACT WDR5 is a highly-conserved nuclear protein which facilitates the assembly of histone-modifying complexes involved in a variety of chromatin-based, gene regulatory processes. WDR5 is best known for its role in scaffolding the assembly of MLL/SET histone methyltransferase complexes that catalyze histone H3 lysine 4 (H3K4) di- and tri-methylation (Me2/Me3), but WDR5 acts outside this setting to promote ribosomal protein gene transcription and recruit the oncogenic transcription factor MYC to chromatin. WDR5 is also a target for pharmacological inhibition in cancer, yet new compounds for degrading WDR5 have been described. Most drug discovery efforts aim to block the WIN site of WDR5, an arginine binding cavity that engages MLL/SET enzymes. Therapeutic application of WIN site inhibitors is complicated by the disparate functions of WDR5, but is generally guided by two assumptions-that WIN site inhibitors disable all functions of WDR5, and that changes in H3K4me drive the transcriptional response of cancer cells to WIN site blockade. My project tests these assumptions by comparing the impact of WIN site inhibition versus WDR5 degradation on H3K4me and transcriptional processes in Burkitt's lymphoma cells. We show that WDR5 regulates transcription widely, yet WIN site inhibition disables only a specific subset of WDR5 activity, and that H3K4me changes induced by WDR5 depletion do not explain accompanying transcriptional responses. These data recast WIN site inhibitors as selective loss-of-function agents, contradict H3K4me as a relevant mechanism of action for WDR5 inhibitors, and point to distinct clinical applications of WIN site inhibitors and WDR5 degraders.

P44

Foxl1+ Telocytes in the Stomach Yoojin Sohn, Blake FloresSemyonov, Christopher Wright, Klaus Kaestner, James Goldenring

ABSTRACT Telocytes are interstitial cells that are in the connective tissue of multiple organs, including gastrointestinal tract. Telocytes have distinct ultrastructural characteristics, with a small cell body and two to five long cytoplasmic processes, called telopodes. Telocytes have been described to have immunophenotypic heterogeneity depending on anatomical location, and recent studies have identified Foxl1+ subepithelial telocytes in the intestine to play a critical role in maintaining stem cell niche by producing Wnt signals. In this study, I examined Foxl1+ telocytes in the stomach and confirmed expression of telocytes markers in the stomach with immunofluorescence staining, which include PDGFRα and F3. I also studied whether telocytes are involved in metaplasia development in the stomach when there is a shift in proliferative cell zone. In the event of acid-secreting parietal cell loss in stomach, mature chief cells at the base of the gland transdifferentiate into mucous cell metaplasia, designated as spasmolytic polypeptide-expressing metaplasia (SPEM), and these cells become proliferative. Using mouse stomach tissues from various models of SPEM development, I observed the change in telocytes network in the stomach. In normal stomach, Foxl1+ telocytes are seen near the isthmal region of the gastric gland, where proliferative progenitor cells are present. However, when SPEM is induced, more telocytes were observed to the base of the gland, where proliferative SPEM cells arise. Using human stomach tissue, I observed the same finding that telocytes, which are present near the isthmal region in normal stomach, are found near the metaplasia surrounding metaplastic glands. These findings suggest that telocytes may play a role in metaplasia development and proliferation zone change in the stomach.

P46

Stimulating proliferation of the mammalian retina through p27Kip1 and Myc Signaling

Stanchfield, M; Jovanovic, J; Levine, E

ABSTRACT Regeneration in the mammalian retina is an aim in combating degenerative retinal diseases. In Zebrafish, damaged retinal tissue is replaced through de novo proliferation and neurogenesis that is activated in Müller glia (MG). Progress has been made in unlocking neurogenic potential in mouse MG. However, stimulating enough proliferation to replace lost tissue is largely overlooked. I predict that robust proliferation in mammalian MG can be stimulated through genetic manipulation. We have a model of MG proliferation through conditional inactivation of cyclin dependent kinase inhibitor Cdkn1b or p27Kip1 (p27Kip1cko) in adult mice. However, proliferation of MG after p27Kip1cko is transient with limited neurogenic potential. I am developing an ex vivo retinal culture system to identify and test factors to stimulate proliferation. p27Kip1 is part of a three protein complex that regulates Myc. Myc , a BHLH transcription factor, plays a major role in proliferation, cell growth, and metabolism. Likely, p27Kip1cko alone is insufficient to drive robust proliferation. Myc could be manipulated to increase proliferation of MG. I hypothesize that MG in p27Kip1cko ex vivo retinal explants will show transient proliferation and Myc overexpression will increase proliferation. I will determine the state of MG proliferation in p27Kip1cko ex vivo retinal explants vs. control retinas using proliferation markers and perform RNA-seq of FACS-sorted MG to compare expression in p27Kip1cko MG to analyze levels of proliferation-related and other genes. I will overexpress Myc in p27Kip1cko retinas and repeat the above experiments to determine if Myc effects proliferation. Factors identified from these experiments will also be manipulated to increase proliferation.

P48

Basement membrane repair dynamics in the Drosophila midgut Stricker, A., LaFever, K., Peebles, K., Hutson, M. S., Page-McCaw, A.

ABSTRACT Basement membranes are the oldest, most conserved forms of extracellular matrix and serve to separate tissue layers, provide mechanical support, direct signals to neighboring cells, and insulate tissues from signals. Further, basement membranes are subject to mechanical damage and require dynamic repair mechanisms. Faulty basement membrane repair mechanisms can aid in the progression of diseases such as asthma and diabetes, and diseases of the basement membrane itself, including Alport's syndrome and Goodpasture's syndrome. Therefore, understanding how basement membranes repair will be vital to treating these conditions. Our work utilizes the Drosophila midgut basement membrane to probe repair dynamics. In Drosophila, all major basement membrane components have been conserved but with less redundancy than mammals. Our lab has developed an assay to reproducibly damage the basement membrane and study the repair process. Previously we reported that many aspects of basement membrane repair are shared during homeostasis. Thus, it is unclear whether basement membrane damage is actively detected, or instead, passively repaired by homeostatic mechanisms. Our recent data suggests basement membrane damage is actively detected. Following damage, there is an increase in the number of enteroendocrine (EE) cells, the major secretory cell type in the gut. Additionally, the EE cells are a significant contributor of collagen IV needed to repair damage. Importantly, EE precursor cells express a mechanosensory stretch-activated ion channel, Piezo, raising the possibility that a change in stiffness of damaged basement membranes signals the initiation of repair. Excitingly, Piezo knock-out flies are able to maintain basement membrane homeostasis in the adult fly but cannot repair it after damage. This is our first evidence that there is a unique mechanism to detect basement membrane damage and initiate repair.

P50

Alternate promoters in Sox17 provide expressional plasticity during endodermal and vascular endothelial development

Linh T. Trinh, Anna B. Osipovich, Leesa Sampson, Jonathan Wong, Chris V.E. Wright, Mark A. Magnuson

ABSTRACT Sox17 expression in endothelial and endodermal cells is critical for early organogenesis. We show that divergent Sox17 mRNA forms result from the differential usage of two evolutionarily conserved promoter regions that are essential for the differential expression Sox17. Deletion of the upstream conserved region (CR1), which functions preferentially as a promoter in endothelial cells, causes a modest increase in lympho-vasculogenesis via reduced Notch signaling downstream of SOX17 in mice. In contrast, deletion of the downstream conserved region (CR2), which is located between exons 3 and 4, functions as a promoter in both endothelial and endodermal cells, and contains sequences necessary for intron excision, impairs both vascular and endodermal development causing death by embryonic day 12.5. Analysis of chromatin accessibility, transcription factor binding and histone modification data of Sox17 locus at CR1 and CR2 strongly supports the normally preferential cell-specific activities of the two promoters.

P52

Proinflammatory properties of Paneth cells in intestinal inflammation Vega P, Banerjee A, Ro J, Wang J, Islam M, Xu Yanwen, Simmons A, Lau K

ABSTRACT Paneth cells are a cell type in the intestinal epithelium that secrete various antimicrobials to regulate the microbiota that colonize the luminal compartment. Several risk alleles for Crohn's disease (CD), a chronic condition characterized by intestinal inflammation, are in genes that are important for Paneth cell function. In CD, Paneth cell defects and loss result in antimicrobial barrier disruption that is thought to promote inflammation. However, we recently observed that ablation of secretory cells, including Paneth cells, resolves ileal inflammation in a mouse model of CD. This unexpected finding may suggest an active, proinflammatory role for Paneth cells in the development of intestinal inflammation, in contrast to the idea that Paneth cell loss-of-function promotes inflammation. The field currently lacks a mechanistic investigation of proinflammatory, gain-of-function properties of Paneth cells in the context of ileal inflammation. I hypothesize that Paneth cells have proinflammatory properties that drive ileal inflammation in the TnfΔARE/+ mouse model of CD. This proposal is innovative because it challenges the conventional understanding that Paneth cell loss of function alters the microbiota to drive CD, and instead, I hypothesize the disease etiology is directly connected to the active, proinflammatory role of Paneth cells in chronic ileal disease. In Aim 1, I investigate how Paneth cells promote inflammation by examining proinflammatory Paneth cell death processes in TnfΔARE mice. In Aim 2, I investigate the effects of proinflammatory Paneth cells in TnfΔARE mice by identifying Paneth cell factor-induced inflammatory signaling pathways in mesenchymal cells. To address these aims and hypothesis, I use three independent, in vivo Paneth cell ablation models to test whether Paneth cells are drivers of ileal inflammation in the TnfΔARE mouse. Moreover, I have generated a novel mouse model to test whether Paneth cell-derived TNF is sufficient to induce ileal inflammation. Finally, single-cell techniques will be leveraged to identify Paneth cell-derived proinflammatory cytokines, and computational methods will be used to determine if Paneth cell-mesenchymal cell interactions promote ileal inflammation. Investigation of these independent, but related, aims will reveal whether they are interlinked processes, as necroptotic Paneth cells may release proinflammatory factors that signal to mesenchymal cells. Revealing mechanisms by which Paneth cells drive ileal inflammation is critical for understanding CD and will lead to novel therapeutic targets.

P54

Drosophila Matrix Metalloproteinase 2 cleaves and destabilizes Dally-like protein to attenuate Wg distribution

Waghmare I., Page-McCaw A.

ABSTRACT The extracellular availability of secreted ligands is critical for activating signaling in target cells during development and tissue homeostasis. Cell-surface glypicans are evolutionarily conserved factors that play an important role in extracellular distribution of many secreted ligands including Wnt proteins. Because glypicans play an important role in modulating extracellular ligand availibility and their activity, cell-surface glypican levels are likely regulated by other factors. In the Drosophila germarium, a tissue where oogenesis initiates, the glypican Dally-like protein (Dlp) promotes long-range extracellular Wg distribution from Wg-producing cap cells to Wg-responsive follicle stem cells inducing their proliferation, which is required for egg development. In genetic experiments, Matrix Metalloproteinase 2 (Mmp2) inhibits Dlp's long-range Wg distribution to restrict Wg signaling in follicle stem cells. Thus, Mmp2 acts as a molecular break on Dlp's long-range function. Here, we investigated the mechanism by which Mmp2 inhibits Dlp's long-range function. In cell culture, Mmp2 cleaves Dlp on the cell surface. Based on the known crystal structure of Dlp, cleavage of Dlp by Mmp2 likely induces a conformational change where the cleaved pieces remain held together by a di-sulfide bond, and the cleaved protein is endocytosed and degraded. Interestingly, in cell-culture experiments, cleaved Dlp sequesters more Wg than intact Dlp. Based on these and our previous observations, we propose a model wherein intact Dlp on the cell surface promotes long-range Wg distribution. In contrast, cleavage by Mmp2 destabilizes Dlp, and cleaved Dlp sequesters more ligand, removing the Dlp-Wnt complex from the cell surface, resulting in attenuation of ligand distribution and function. Overall, this study identifies the molecular basis of protease-mediated inhibition of cell-surface glypican, which modulates ligand distribution and function.

P56

The Role of Polyploidy During Drosophila Epithelial Wound Repair James White, Kimi LaFever Hodge, Ivy Han, Jamsine Su, M. Shane Hutson, Andrea Page-McCaw

ABSTRACT In the past decade, there has been increased acknowledgement of cellular polyploidy in development, homeostasis, and cancer. In addition to being a conserved developmentally programmed behavior, polyploidy is also induced in response to injury. Using Zebrafish epicardial explants, the Poss Lab determined changes in tension induce a wavefront of polyploid cells that lead regeneration. Work by Vicki Losick has shown adult Drosophila epithelia repair by becoming polyploid through endoreplication and cell-cell fusion. Blocking either endoreplication or cell-cell fusion in the adult delays wound closure, and blocking both inhibits closure. Although polyploidy is required for wound closure, we do not understand what endoreplication or cell-cell fusion individually contribute during repair. Using live microscopy, I have performed the first live in vivo characterization of cell-cell fusions events after wounding. I have determined that cell-cell fusions occur within the first 20-30 minutes after wounding, and it is borders between the first 2-3 rows of cells out from the wound which break down. Thus, syncytia form very rapidly and are positioned at the front lines of wound repair. Further, the majority of fusion events occur on borders shared by sequential rows of cells out from the wound, as opposed to neighbors in the same row. Previous studies in the lab have determine that within the same area syncytia form there are microtears in the plasma membrane. Thus, I hypothesize syncytia require microtears in the plasma membrane to form. Finally, I hypothesize syncytial cell formation allows distal cellular resources to be pooled at the leading edge of wound repair allowing for the construction of large migratory structures. Preliminary live-imaging studies of actin support this hypothesis, as actin label in fused cells aggregates at the wound margin in large filipodia like structures. These large structures likely allow for rapid migration during the early stages of wound repair.

P58

2'-fucosyllactose ameliorates chemotherapy-induced intestinal mucositis through protecting intestinal epithelial cells against apoptosis

Zhao, G; Williams, J; Washington, M. Kay; Townsend, Steven D.; Yan, Fang

ABSTRACT Background & Aims. Intestinal mucositis, a severe complication of antineoplastic therapeutics, is characterized by mucosal injury and inflammation in the small intestine. Therapies for prevention and treatment of this disease are needed. We investigated whether 2'-fucosyllactose (2'-FL), an abundant oligosaccharide in human milk, protected the intestinal integrity for ameliorating intestinal mucositis. Methods. 2'-FL was synthesized with 97% purity. A mouse small intestinal epithelial cell line, MSIE cells and human gastrointestinal tumor cell lines, AGS and HT29 cells, were co-treated with a chemotherapy agent, 5-fluorouracil (5-FU) and 2'-FL. Mice received intraperitoneal injection of 5-FU to induce intestinal mucositis. 2'-FL in drinking water was administered to mice before (pretreatment) or at the same time (concurrent treatment) of 5-FU injection. Bodyweight and pathological changes were analyzed. Results. 2'-FL alleviated 5-FU-inhibited cell growth in MSIE cells, but not in AGS and HT29 cells. 5-FU-induced apoptosis in MSIE cells was suppressed by 2'-FL. Compared to 5-FU treated mice, 2'-FL pretreatment restrained bodyweight loss, and ameliorated inflammation scores, proinflammatory cytokine production, shortening of villi, apoptosis, and loss of goblet cells in the small intestine. Concurrent treatment with 2'-FL had less effects on intestinal mucositis as compared to 2'-FL pretreatment. Interestingly, no effect of 2'-FL on 5-FU-induced S-phase arrest was identified in MSIE, AGS and HT29. 2'-FL pretreatment or concurrent treatment did not affect inhibition of proliferation by 5-FU in small intestinal epithelial cells in mice. Conclusions. This study reveals a novel direct impact of 2'-FL on protecting against 5-FU-induced apoptosis in small intestinal epithelial cells. This effect may contribute to prevention of intestinal mucositis by 2'-FL.

P60

Nanoscale organization and molecular assemblies of transcellular nanocolumns in chemical synapses

Sun, R; Wilson, L; Haider, M; Wang, X; Alten, Baris; Zhou, Q

ABSTRACT As key functional units in neural circuits, neuronal synapses play important roles in brain information processing, learning and memory. Synaptic abnormalities are believed to underlie various neurological and psychiatric disorders. In our lab, we use an innovative, interdisciplinary approach that combines cutting-edge structural techniques including cryogenic electron tomography (cryo-ET), biochemical methods, electrophysiological recording and RNA sequencing to reveal the molecular assembly and dynamics at the nanoscale level in the synapses, and to bridge structural biology with cellular and molecular physiology. We visualized the in situ three-dimensional organization of synaptic organelles and macromolecules in their native state by cryo-ET. Analyses of the synaptic tomograms of cultured neurons revealed high density protein clusters which are aligned with presynaptic vesicles. High-resolution tomograms obtained from the unique synaptosomes sample with PSD allows us to detect similar protein clusters in the PSD, as well as glutamate receptor-like and adhesion molecules. Our lab also studies the role of aberrant spontaneous neurotransmission in SNAP25-associated encephalopathies by electrophysiology and RNA sequencing techniques.

P62

Metabolic reprogramming on the development and progression of metaplasia induced by activated Ras expression in gastric chief cells

Yoonkyung Won, Brianna Caldwell, Eunyoung Choi

ABSTRACT Gastric cancer (GC) is the third leading cause of cancer-related death worldwide. Intestinal-type gastric cancer, which is the most common form of GC, usually develops within a carcinogenic cascade of pre-cancerous metaplasias, such as spasmolytic polypeptide-expressing metaplasia (SPEM), intestinal metaplasia (IM), to dysplasia and adenocarcinoma. Gastric chief cells are a mature gastric cell type that secretes digestive enzymes and have been considered the origin of metaplasia through transdifferentiation process. Up to 40% of human gastric cancer have signatures of activated Ras protein, suggesting that Ras activation may promote the progression of metaplasia to cancer during gastric carcinogenesis. We h ave recently established a gastric chief cell-specific driver using GIF gene locus and utilized to express the active Kras in GIF-expressing chief cells. Recent studies have suggested that metabolic reprogramming has crucial roles for supporting the requirements of exponential growth and proliferation in cancer development and progression. However, it remains undefined how metabolism regulates the transition state between pre-cancer and cancer. In this study, we have focused on the metabolic changes in a cascade of metaplasia to dysplasia to determine which metabolic pathway associates the progression of metaplasia to dysplasia. We first crossed the GIF-rtTA mice with TetO-Cre and LSL-K-Ras(G12D) mice and generated the GIF-rtTA;TetO-Cre;LSL-K-Ras(G12D) (GCK) mouse allele to continuously express active Kras in chief cells. Histological analysis of GCK mouse stomachs demonstrated that the active Kras expression in chief cells developed SPEM within 1 month and IM/dysplasia within 3-4 months after doxycycline treatment. Further understanding the mechanistic insights on the metabolism, we analyzed the metabolite changes in the GCK mouse stomach tissue by imaging mass spectrometry (IMS) for mapping the spatial distribution of biomolecules across the tissue of interest. We observed numerous metabolites associated with glucose, fatty acid, and phosphatidic acid metabolism changed in GCK mouse stomach. In particular, we examined unique regional patterns of distribution for hexose bisphosphate which is an intermediate metabolite in glycolysis highly accumulated in the base of the metaplastic gland in the GCK mouse stomach. Taken together, our findings indicate that expression of active form of Kras in GIF-expressing chief cell leads to the development and progression of precancerous metaplasia in stomach corpus. Moreover, glycolytic pathway is highly activated in the development of metaplasia in the stomach corpus. Future studies will examine the functional roles of glycolytic pathway in the progression of metaplasia to dysplasia and even cancer during gastric carcinogenesis.

P64

Compartment-specific role of retinoic acid receptor activation in AKI Yang M, Lopez N L, Delgado R, Brewer M, Gewin S. L, Yang H, de Caestecker P. M

ABSTRACT Background: Retinoic acid receptors (RARs) are activated in proximal tubules (PT), collecting duct (CD), and renal macrophages (Møs) after ischemia reperfusion AKI (IR-AKI), and systemic RAR inactivation increases Mø-dependent injury after IR-AKI. However, the functional roles of RAR activation in different cellular compartments are unknown. Methods: RARE-LacZ (RAR reporter); PEPCK-CRE; R26R-Dominant Negative RAR (PT-DNRAR); AQP2-CRE; DNRAR (CD-DNRAR); LysM-CRE; DN-RAR (Mø-DNRAR) underwent bilateral IR- and/or rhabdomyolysis-AKI (rhabdo-AKI). Injury and RARE-LacZ localization were evaluated by BUN, LacZ staining and IF. Renal Mø activation determined by FACS; primary PTEC proliferation and metabolic activity using Seahorse. Results: RARs are more widely activated after rhabdo- vs. IR-AKI: ~90% in LTL or Kim1+ PTECs; ~5% in AQP2+ CD; ~2-3% in F4/80+ Møs; and <2% in THP1+ thick ascending limb. To evaluate RAR function, we performed IR- and rhabdo-AKI in PT-, CD- and Mø-DNRAR mice. AKI was less severe in PT-DNRAR mice: day 3 BUN in CRE- vs. +; rhabdo-AKI: 52.9 (11.2) vs. 29.1 (1.8); IR-AKI: 71.7 (8.8) vs. 38.4 (8.3) mg/dl, p<0.005. In contrast, Mø- DNRAR had more severe injury: IR-AKI, day 3 BUN CRE- vs. + 37.0 (2.9) vs. 63.1 (10.6), p<0.05. There was no difference in IR- or rhabdo-AKI severity in CD-DNRAR mice. Despite decreased injury, there was increased Kim1 and F4/80+ Møs after AKI, associated with decreased MLKL (necrosis) and increased Sox9 and Ki67 (de-differentiation and repair) in PT-DNRAR mice. FACS also showed decreased Ly6C inflammatory renal Møs after AKI. Uninjured PT-DN-RAR mice also had patchy increase in Kim1/Sox9+ PTECs; increased F4/80+ CD206+ reparative Møs; and PTECs from PT-DNRAR CRE+ mice were more metabolically active and proliferative than CRE- mice. Conclusion: Inhibition of RAR in PTs protects against AKI by increasing reparative, metabolically active PTECs, and suppresses Mø activation, while inhibition of RAR in Møs exacerbates AKI. In contrast, inhibition of RARs in CDs does not affect the severity of injury. These findings indicate that RAR activation in different cellular compartments exert opposing effects on the severity AKI through distinct mechanisms, and provides the first evidence that dedifferentiated and inflammatory PTECs, recently described molecular signatures of failed repair, may be protective in AKI.

P66

Using Expansion Microscopy to Unveil Cardiomyocyte Organelle Contact Sites

Zachary Sanchez & Dylan T. Burnette

ABSTRACT A limitation of modern microscopy is the resolution limit, which prevents imaging small structures with enough spatial resolution to be able to identify separate structures or proteins. Super-resolution microscopy techniques such as PALM or SIM are used to image proteins or structures of interest that are unable to be resolved normally due to the diffraction limit of conventional microscopy techniques. However, these techniques often require costly software or equipment to minimize focal drift and prove to be a barrier for some labs. Expansion Microscopy (ExM) addresses this problem by augmenting the size of the sample rather than increasing the resolving power of the microscope. By embedding a tissue or cell sample in a hydrogel, I am able to swell and isometrically space out cellular structures with the addition of water. This expansion allows for imaging of samples exogenously labeled with fluorophores. I have been able to successfully utilize human derived iPSC cardiomyocytes ( as a model to test the limitations and hallmarks of this technique. These cells contain sarcomeres which are easily recognizable structures with predictable spacing allowing for conducive interpretation of expansion results. Preliminary data shows that at least 4X expansion of the original sample can be achieved. Furthermore, this technique is being applied to tissue sections to see if similar results can be achieved thereby allowing for a comparison with adult structures due to the neonatal-like nature of the iPSC-derived cardiomyocytes. I hypothesize that this technique can reveal previously unresolved contact sites between the organelles of cardiomyocytes and their sarcomeres. If this is true, then new hypotheses regarding the influence of organelles on sarcomere assembly can be generated.

P68

Investigating the Gene Regulatory Network and Epigenetic-Guidance Patterns of Mnx1 During β-cell Development

Ziehm, E., Ray, M., Jarvis, B., Booth, D., Ursu, V., Wright, C.

ABSTRACT The homeodomain-protein-encoding gene Motor Neuron Homeobox 1 (Mnx1, a.k.a. Hb9 or Hlxb9) encodes a poorly studied pancreatic β-cell (insulin-producing cell) transcription factor (TFs). Mnx1 is also expressed early in tissues sending intercellular signals to induce formation of the adjacent pancreatic anlagen. Later, Mnx1 is produced in pancreatic cells after endocrine-lineage commitment, beginning at the Pax6+ post-mitotic stage. Previous studies clearly indicate Mnx1 as a core TF regulating β-cell specific differentiation and function, but its lack of molecular analysis is especially surprising considering the profound phenotypes accompanying mouse or human mutations. When Mnx1 is inactivated early in mouse β-cell development, DMnx1 cells still have “momentum” to follow the β-cell developmental track, but in the early postnatal period (P5-10), all transform into somatostatin-producing δ-like cells. We are testing if DMnx1 cells undergo a progressive cumulative build-up of cryptic deflections from the normal differentiation-guidance program, leading to an inability to maintain the b-cell GRN (gene-regulatory network) when faced with “checkpoint control” at the early postnatal stage. An alternate model is that the GRN and epigenetic control mechanisms undergo sudden paroxysmal alterations only postnatally, triggering rapid β-to-δ transdifferentiation. Human Mnx1 point mutations cause severe permanent neonatal diabetes mellitus (PNDM). Our new mouse models mimic human Mnx1 PNDM (F272L in helix 2 of the DNA-binding homeodomain), prospectively allowing complete study of the abnormal developmental program. The profound phenotype, including postnatal death, suggests an extreme effect on the pancreas and its endocrine-cell population. We are investigating the Mnx1 GRN and epigenetic-guidance patterns at various stages of β-cell development. Defining Mnx1 target genes, and activating or repressing effects, will be paramount in understanding how Mnx1 drives and maintains cells in the β-cell fate. Because experiments such as ChIP-Seq, CUT&Tag, scRNA-seq will unveil key chromatin-Mnx1 interactions, we need effective and high signal:noise antibodies. Currently, we have rabbit, goat, and alpaca polyclonal affinity-purified antibodies, with alpaca nanobodies being made. As triangulation methods, we created novel epitope-tagged mouse strains, genetically engineered via CRISPR-Cas9 with a knock-in of either 3x[FLAG] or the new ALFA-tag at the C-terminus of Mnx1. Both strains seemingly have normal Mnx1 function. The ALFA tag is an innovative, rationally designed a-helical tag and extremely high-affinity fluorescent-nanobody detection. The nanobodies should maneuver better through often compact molecular complexes such as found in chromatin, to find and tightly bind the ALFA-tagged Mnx1 in situ.

P70

EP4 Perturbation Resulting in Mouse Patent Ductus Arteriosus may be Dependent on Developmental Timing

Michael T. Yarboro, Naoko Brown, Chris W. Hooper, Ting Wong, Stan D. Poole, Elaine L. Shelton, Jeff Reese

ABSTRACT The ductus arteriosus (DA) is a vascular shunt which allows oxygenated blood to bypass the developing lungs in utero. After birth, normal DA closure may be disrupted, leading to persistent patency of the DA (PDA). COX-derived PGE2 signaling via the prostanoid receptor EP4 appears to be critical for the normal closure process, with neonatal lethality in EP4 KO and COX double KO mice due to PDA. Previous studies have examined the EP4 KO phenotype, but questions of developmental timing and vascular function remain. We hypothesize that EP4 signaling in late gestation is critical for establishing the contractile properties of the mature DA, including its myogenic tone, biomechanical properties, and responsiveness to key molecular signals. For developmental timing, a gavage model using a selective EP4 antagonist (ONO-AE3-208) was employed. Mice treated from D15-D19 showed partially open DAs at 4hrs of age. Contractile properties of EP4 KO mice were examined through pressurized vessel myography. KO vessels lacked the myogenic response to pressure increase typical of the WT. KO vessels also showed a decreased dynamic range compared to WT. Mathematically this translated to a decrease in compliance of the KO vessel. In addition, term gestation KO vessels showed vasomotion characteristic of the preterm DA. Mounted DAs were exposed to signaling compounds of various pathways. Significant differences were seen in responsiveness to KCl, PGE2, and SNP. Together, these data indicate that EP4 signaling is required during the D15-D19 window for proper DA development, and that EP4 signaling is required for establishing the myogenic tone, compliance, and various signaling properties of the mature DA.

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