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Coiciv & Igiv Biology 4:5, 612-615; Spb/Ocob 2011; 2011 Ls Bioscic

612 Coiciv & Igiv Biology Vol 4 Iss 5

Addendum to: Garca Fernndez B, Gaspar P,

Brs-Pereira C, Jezowska B, Rebelo SR, Janody F.

Actin-Capping Protein and the Hippo pathway

regulate actin accumulation and tissue growth

in Drosophila. Development 2011; 138:233746.

PMID: 21525075; DOI: 10.1242/dev.063545

Key words: capping protein,Hippo signaling pathway, yorkie, at,expanded, actin cytoskeleton, Drosophilaadult retina, neuronal degeneration

Submitted: 06/08/11

Accepted: 06/08/11

DOI: 10.4161/cib.4.5.16853

*Correspondence to: Florence Janody;

Email: [email protected]

The conserved Hippo signalingpathway regulates multiple cellu-lar events, including tissue growth, cell

ate decision and neuronal homeostasis. While the core Hippo kinase moduleappears to mediate all the eects o thepathway, various upstream inputs havebeen identifed depending on tissue con-text. We have recently shown that, in theDrosophila wing imaginal disc, actin-Capping Protein and Hippo pathwayactivities inhibit F-actin accumulation.In turn, the reduction in F-actin sus-tains Hippo pathway activity, prevent-ing Yorkie nuclear translocation and the

upregulation o prolieration and sur-vival genes. Here, we investigate the roleo Capping Protein in growth-unrelatedevents controlled by the Hippo pathway.

We provide evidence that loss o CappingProtein induces degeneration o the adultDrosophila retina through misregula-tion o the Hippo pathway. We propose amodel by which F-actin dynamics mightbe involved in all processes that requirethe activity o the core Hippo kinasemodule.

The conserved Hippo (Hpo) signalingpathway has emerged as a critical regulatoro tissue growth both in Drosophila andin mammals. At the center o the path-way are the two Ser/Thr kinases Hpo and Warts (Wts), and their adaptor proteinsSalvador (Sav) and dMob as a tumour sup-pressor (Mats).1-6 This core kinase modulerepresses tissue growth by sequestering thetranscriptional co-activator Yorkie (Yki)in the cytosol through phosphorylation

Homeostasis o the Drosophila adult retina by actin-capping proteinand the Hippo pathway

Catarina Brs-Pereira,1 Tianyi Zhang,2 Francesca Pignoni2, and Florence Janody1,*1Instituto Gulbenkian de Cincia; Oeiras, Portugal; 2Department o Ophthalmology; Harvard Medical School and the Massachusetts Eye and Ear Inrmary;

Boston, MA USACurrent address: Center or Vision Research; Upstate Medical University; Syracuse, NY USA

and direct binding.7-9 Sequestration o Ykiprevents ormation o complexes between Yki and DNA-binding transcription co-

actors and activation o target genes thatregulate cell growth, survival and proli-eration.10 In addition to controlling tis-sue growth, the core kinase module alsoregulates growth-unrelated events, such asstress-induced apoptosis, cell ate decisionand neuronal homeostasis.11-13 Most othese outputs rely on Yki activity, includ-ing the control o tissue growth and themaintenance o neuronal homeostasis othe Drosophila adult retina,11 while othersare Yki-independent.13

Multiple upstream inputs have alsobeen shown to regulate the core Hpokinase module at various levels. Amongthose, the atypical Cadherin Fat wasproposed to transduce signals rom theatypical Cadherin Dachsous (Ds) andFour-jointed (Fj), while the two Ezrin/Radixin/Moesin (ERM) amily mem-bers, Expanded (Ex) and Merlin (Mer) arebelieved to exert their growth suppressiveactivity by activating the Hpo kinase.14,15These inputs can act in both a coordinatedand independent ashion on Hpo pathway

activity depending on the tissue context.In the wing imaginal disc, Fat and Exare major regulators o the pathway.16,17In contrast, in the pupal Drosophila eye,Fat and Ex play only minor roles,18 whilein the adult retina, Fat, but not Ex, isabsolutely critical to prevent neuronaldegeneration.11

Despite much progress in understand-ing the molecular regulation betweenthe core components o this module, key


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artICLe addendum

Capping Proteinand Neuronal

Degeneration through the HippoSignaling Pathway

Independently o its role in growth control,the Hpo signaling pathway prevents neu-ronal degeneration o the adult Drosophila

retina.11 CP has also been shown to preventretinal degeneration.20,21 Thereore, CPmight have a general requirement in con-trolling the dierent outputs o Hpo sig-naling activity. To test whether loss o CPtriggers neuronal degeneration throughinhibition o Hpo pathway activity, wecarried out genetic interactions betweencpa or cpb and components o the hpo sig-naling pathway. To perorm this study, weexpressed independent double-strandedRNAs to knock down Cpa (UAS-cpa-IR)or Cpb (UAS-cpb-IR) using the ato348 or

GMR-Gal4 drivers, which drive expres-sion in committed G

1-arrested cells prior

to photoreceptor dierentiation in thedierentiating retina respectively. Whileexpressing cpb-IR45668 under ato348-Gal4control did not induce morphologicaldeects (Fig. 1A), driving cpa-IRC10 withato348-Gal4 (Fig. 1D and J) orGMR-Gal4(Fig. 1G) or cpb-IR45668 with GMR-Gal4(data not shown) triggered the appearanceo black omatidial clusters, which revealsretinal degeneration. Reducing one copy

ohpo strongly enhanced neuronal degen-eration o Cpa (Fig. 1B) or Cpa-depletedphotoreceptor cells using either ato348 orGMR-Gal4 (data not shown). While het-erozygote mutant animals or hpo, carry-ing either ato348-(Fig. 1C) or GMR-Gal4(data not shown) showed no visible deectso the adult retina. This indicates that CPand hpo genetically interact to maintainneuronal homeostasis.

The neuroprotective unction oHpo signaling required Yki activity.11Expressing an RNAi construct against

Yki with ato348

-Gal4 had no visible eecton eye morphogenesis (Fig. 1F) but sup-pressed the neurodegenerative pheno-type due to Cpa depletion (Fig. 1E). Weobserved a similar suppression with GMR-Gal4 (data not shown). This indicates that Yki activity is part o the signaling cas-cade, which trigger neuronal degenerationo Cpa-depleted photoreceptors cells.

We then investigated the epistatic rela-tionship between CPand components o

interplay between Hpo pathway activityand F-actin dynamics, in which regula-tion o an apical F-actin network by Hposignaling activity and CP sustains Hpopathway activity, thereby limiting Ykinuclear import and the activation o pro-lieration and survival genes.19 Here, weinvestigate whether CP is also requiredor growth-unrelated events controlled by

the Hpo signaling pathway. We provideevidence that loss o CP induces neuro-nal degeneration o the adult Drosophilaretina through the control o Hpo path-way activity. Based on our work and theincreasing number o reports, implicat-ing F-actin in Hpo signaling, we proposethat F-actin might be a central player othe pathway, integrating signals rom vari-ous inputs and mediating tissue-specicoutputs.

unanswered questions remain, such ashow the dierent inputs are activated andintegrated by the core kinase module invarious tissues and how this triggers spe-cic developmental outputs.

The Interplay betweenHpo Signaling Activity

and F-Actin Dynamics

We have recently shown that the actin-Capping protein (CP) heterodimer, com-posed o an (Cpa) and (Cpb) subunits,which regulates actin polymerization, alsounctions to suppress inappropriate tis-sue growth by inhibiting Yki activity.Interestingly, Hpo signaling activity, likeCP, limits actin lament (F-actin) accu-mulation at apical sites, independently o Yki. Thus, our ndings indicate a novel

Figure 1. Kockig ow Cppig Poi iggs il gio hogh ihibiio o

Hippo siglig civiy. all pls show l dosophil i. th goyps o h ils

iic bov h pls. ato348 o GMR s o ato348-Gl4 o GMR-Gl4 ivig xpssio

o h iic sgs (uaS-cpb45668

, uaS-cpa-IRC10

, uaS-yki4005R-2

, uaS-hpoM11.1

o uaS-ex) ih wil-yp o h hpo g (a) o hozygo o h hpo42-47 lll (B C).


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www.lsbioscic.co Coiciv & Igiv Biology 614

(National Institute o Genetics, NIG);

UAS-cpa-IR

7009

and UAS-cpb-IR

45668

(Vienna Drosophila Research Center,VDRC); UAS-hpoM11.1;2 UAS-ex;4GMR-Gal4;26 FRT42D, hpo42-48, a git romD. Pan.1 Crosses with the ato348-Gal4 orGMR-Gal4 drivers were maintained at25C or 18C respectively. Each cross wasperormed in parallel with the appropri-ate controls. Adult fies were collected andphotographed 24 to 48 h a ter hatching. Togenerate ato348-Gal4 transgenic lines thatexpress Gal4 within, and anterior to, themorphogenetic urrow in the eye disc, the

minimal3'-ato eye enhancer27

was clonedin the pChs-Gal4 vector. Transgenic fieswere generated by standard methods.

Acknowledgments

We thank D.J. Pan, R.G. Fehon, theBloomington Drosophila Stock Center,the Drosophila Genomics ResourceCenter, the Vienna Drosophila ResearchCenter (VDRC) and the NationalInstitute o Genetics (NIG) or fy stocks.

neuronal homeostasis.11 Moreover, the role

o F-actin may be independent o tissue-specic inputs since F-actin seems to beinvolved in all processes that require theactivity o the core kinase module, whereasEx, which has a critical role in regulatingHpo-dependent tissue growth,16,17 is notinvolved in neuronal homeostasis.11

In conclusion, the interplay betweenHpo signaling activity and F-actindynamics may be a general requirementin Hpo signaling. Consistent with this, inthe wing disc tissue, the core kinase mod-ule inhibits F-actin accumulation, pre-

vents excess F-actin o CP-depleted cells19

and we observed similar epistatic relation-ships between CP, exor hpo in the adultretina. Nevertheless, urther studies willbe required to validate the central role oF-actin in the Hpo pathway.

Materials and Methods

Fly stocks used were UAS-cpa-IRC10,19UAS-cpa-IR10540R-2; UAS-yki-IR4005R-2

the Hpo pathway. Adult eye overexpress-ing hpo (Fig. 1I) or expressing both hpoand cpa-IR (Fig. 1H) under GMR-Gal4control displayed identical morphologicaldeects. This indicates that Hpo activityovercomes the eect o CP loss. Moreover,ex overexpression, which had no visible

eects in the adult eye (Fig. 1L) suppressedthe neuronal degeneration o Cpa-depletedtissues (Fig. 1K). Taken together, we con-clude that retinal degeneration as a resulto CP loss is mediated, at least in part, byinhibiting Hpo pathway activity.

The Actin Cytoskeleton: A CentralRole in Hippo Signaling Activity

All outputs o the Hpo signaling pathwayseem to be dependent on the activity o thecore kinase module, whereas the strength

and identity o the upstream inputs dependon tissue context.14,15 The core kinasemodule is thereore central to the pathway, integrating multiple signals that aretranslated into tissue-specic responses. We propose that F-actin dynamics mayalso be a central player o the Hpo sig-naling pathway (Fig. 2). CP restricts theaccessibility o the lament barbed end,inhibiting addition or loss o actin mono-mers.22 In addition to CP, other actinregulators have also been shown to regu-

late Hpo signaling activity. Thus, overex-pression o a constitutive active orm othe actin-nucleator Diaphanous inhibitsthe pathway activity upstream o Wts butin parallel to Hpo.23 The MST1/2 Hpoorthologs also co-localize with F-actinstructures and are activated upon F-actindepolymerization; 24 and, Ajuba, whichnegatively regulates Hpo signaling activ-ity downstream o Hpo and upstream o Wts, belongs to an actin-associated am-ily o LIM-domain-containing proteins.25In addition, CP appears to be required

not only to control Hpo-dependent tissuegrowth but also Hpo-dependent growth-unrelated events, such as neuronal homeo-stasis. Thus, CP and consequently F-actin,may mediate all unctions o Hpo signal-ing. Consistent with this, in addition toacilitate Yki phosphorylation by the Hpokinase cassette in the wing disc tissue,19CP may also regulate non-phophorylatedYki since Wts-dependent phosphorylationo Yki does not appear to be involved in

Figure 2. mol o h ol o F-ci i Hippo siglig civiy. th co kis ol o h

Hpo phwy gls lipl ops, iclig iss gowh, ol hoossis -

ophgy. Hpo-p iss gowh is i hogh ihibiio o Yki by phosphoyl-

io-p ip chiss. th opociv fc o Hpo siglig lso

qi Yki ihibiio, ip o is phosphoylio by Ws. I cos, Yki is o ivolv

i Hpo-p ophgy. th co kis coplx is gl by lipl ips, iclig

F ex. th iply bw Hpo siglig civiy F-ci yics y b gl

qi i Hpo siglig.


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