Methods and Procedures

Immunofluorescent Staining Surface Protein Biotinylation

Western BlotFigure 3: The steps and protocol taken to biotinylate the surface protein from the plasma membrane.

Cell lysates and biotinylated proteins were prepared as shownabove. Protein samples were resolved using 4-12% Bis-Tris gels,transferred to nitrocellulose membranes and blocked with 5% milksolution in TBS containing 0.05% Tween-20. The membranes wereincubated with primary antibodies overnight at 4°C and withhorseradish peroxidase conjugated secondary antibodies for 30minutes at room temperature. Enhanced chemiluminescence wasused to detect the primary antibody through a Western blotdetection system.

Plate: Plate approximately hundredthousand cells onto coverslipsFix: Fix cells in 4% PFA for 18minutesPermeabilize: Permeabilize the cellsby adding 0.15% Surfact-Amps inPBS for 4 minutesBlock: Block in Normal Goat SerumPrimary Antibody: Incubate inprimary antibody overnight at 4°CSecondary Antibody: Incubate insecondary antibody for 1 hour at4°CNuclear stain: Stain DNA withHoechst Stain for 2 minutesAffix: Dry and affix coverslips withInvitrogen Slow-Fade onto slides

The transcription factor NF-kB plays an essential role inimmunity. Under basal conditions, NF-kB is sequestered inthe cytosol by IkB proteins, which prevent its nuclearaccumulation. IkB degradation requires the sequential actionof a kinase and ubiquitin ligase for NF-kB activation. Ourgroup has found that deficiency in components of theCOMMD/CCDC22/CCDC93 or CCC complex impairsdegradation of particular isoforms of IkB. The CCC complex isfound in early endosomes and plays a required role in therecycling of cell surface proteins that have been internalizedinto the endosomal compartment. The CCC complex isrecruited to endosomal membranes by FAM21, a proteinwithout which the CCC complex cannot localize toendosomes or perform its function in protein recycling. Inthe studies we have performed, we explored the hypothesisthat the CCC complex may regulate the localization ofsurface receptors involved in NF-kB activation and thatlocalization of the receptors influences the signaling output,meaning how the IkB proteins are degraded. I investigatedwhether CCC deficiency results in endosomal accumulationof two receptors, IL1R1 and TNFR1. These receptors bind toIL1b and TNFa, respectively, resulting in IkB degradation.Loss of CCC leads to altered patterns of IkB degradation.

The Role of Endosomal Recycling in NF-kB Pathway Activation

Riya Mohan1, Lindsey Morris1, Rebecca Faulkner1, Ezra Burstein1,2

Department of Internal Medicine1, Department of Molecular Biology2, UT Southwestern Medical

Center, Dallas, TX

Conclusion

Future Direction

Acknowledgements

Introduction

Thank you to the Burstein Lab for being so inviting and willing to help me throughout my time in their lab. I would also like to thank Dr. Ezra Burstein for allowing me to be apart of his lab and for his expertise, guidance, and advice during my project. Next, I owe thanks to my mentor, Dr. Lindsey Morris, for taking the time to teach me in depth andfor helping advance my passion for science, biology, and scientific research. Lastly, I would like to thank the entire STARS team for creating a program that changed my

perception of science and gave me more reasons to pursue research.

Objective: Identify if internalization and recycling of cell surface receptors occur in the absence of the CCC complex.

Figure 1: The normal process ofinternalization and recycling of surfaceproteins in a cell. Ligand binding, receptorinternalization, endosomal localization, andCCC dependent recycling of receptors backto the plasma membrane are shown.

1. Traffic. 2018 Aug;19(8):578-590.2. Mol Biol Cell. 2015 Jan 1;26(1):91-103.3. J Clin Invest. 2013 May;123(5):2244-56.4. Nat Cell Biol. 2017 Oct;19(10):1214-1225.

Our plan in the future is to further examine and investigatewhether receptor localization affects degradation of IkBisoforms. In order to do this, we will test additionalantibodies, and also utilize dynamin inhibitors such asDynasore or MiTMAB, to achieve plasma membraneenrichment of TNFR1 and IL1R. This will allow us to assesswhether plasma membrane relocalization of the receptoraffects the pattern of IkB degradation.

Results

ParentalCCC deficient

(CCDC93 KO)

CCDC93

IkBα

IkBβ

IkBε

Actin

TNF (0-60 min)

A549

Huh7

TNF-R1 / Fam21

Figure 4: Loss of the CCC subunit CCDC93results in impaired degradation of the IkBsubunits -b and -e.

Figure 5: Loss of the FAM21, which is requiredfor CCC complex recruitment to endosomes alsoresults in impaired degradation of the IkBsubunits -b and -e.

Figure 6: Biotinylation experiments to detect the plasmamembrane fraction of TNFR1 and IL1R1. The experimentsincluded the lung adenocarcinoma cell line A549, which has highlevel of TNFR1 expression. Under the conditions used, the plasmamembrane fraction is only easily detectable in this cell line.Optimization of the experiment is ongoing.

Huh7 hepatocellular cancer cells

Figure 7: Immunofluorescence staining for TNFR1 and theendosomal marker FAM21. While TNFR1 signal is stronger inA549 cells compared to Huh7 cells, the pattern of staining did notshow any membranous component, but only endosomal andnuclear staining. New antibodies against TNFR1 are being tested.

Throughout our experimentation, we have been able toconclude that IkB-b and -e degradation are affected by theCCC complex and its endosomal recruitment. However, atthis point in time, it is too early for us to accuratelydetermine whether TNFR1 or IL1R1 recycling is affectedspecifically by CCC deficiency through the cell lines andantibodies currently in use.

Figure 2: The cell on the left depicts normalendosomal recycling of cell surface receptors,where plasma membrane localization is greaterthan endosomes. The cell on the right depicts aCCC-deficient cell where endosomal localization isgreater than plasma membrane.

Literature Cited

IkBα

IkBβ

Actin

IkBε

siControl

FAM21

TNF (0-120 min)

CCC deficient(Fam21 KO)

TNFR1

IL1R1

Integrin b1

A549

Hu

h7

CC

DC

93KO

A549

Hu

h7

CC

DC

93KO

WCL

Biotinylated

proteins

293T Human embryonic kidney cells