investigate molecules required for rhodopsin trafficking...

InvestigatemoleculesrequiredforrhodopsintraffickinginDrosophila

photoreceptors

ShanLuan’sOralexam16Dec2010

Overview

Details

From: Boman, A.L. (2001) J. Cell Sci. 114: 3413-3418

p56p200

ubiquitin

ModularorganizationofGGAfamilyproteins

Mutantsgeneratebytransposableelement

GenerateGGAknockoutflies

RescueGGAknockoutmutant

GGA+

Overview

Details

Whydowestudyflyeyes?

•  Wearesimilar!50%offlyproteinsequenceshavemammaliananalogues.61%ofknownhumandiseasegeneshavearecognizablematchinthegeneticcodeoffruitflies.

•  Eyeisanon‐essentialorgan.Knockdownofanessentialgeneineyewillgiveusasurvivalanimalwithamutanteyephenotype.

•  Mutationscausingimpairedrhodopsintargetingwillleadtoretinaldegenerationinbothhumanandflies.

Wolff,T.andReady,D.F.(1993).PatternformationintheDrosophilaretina.In:TheDevelopmentofDrosophilamelanogaster.ColdSpringHarborLaboratoryPress.Vol.2Pp.1277‐1325

http://stke.sciencemag.org/content/vol2006/issue366/cover.dtl

Rhodopsin•  belongtotheG‐proteincoupled

receptorfamilyandareextremelysensitivetolight.

•  Rhodopsin=opsin+retinal

•  rhodopsin(Rh1),isglycosylatedintheERbymolecularchaperone,NinaA.

•  VariousRabproteinfacilitateRh1exitfromtheERandtransportthroughthesecretorypathwaytorhabdomerebase

Rh1signalingcascade

Rhodopsinturnover?

•  Initiatedbyarrestinbindingtometarhodopsin

•  TGNproteinsorting:‐Adaptorprotein1‐4mammals

‐Clathrin

ButK/DofallAP1‐4doesnotinhibittransportfromTGN.Otheradaptorproteins…?

•Golgi‐localized,ϒ‐ear‐containing,arf‐bindingprotein•monomericclathrinadaptors•UniqueGGAfamilyproteininflies•implicatedincargoselectionandinvesicleformationandtrafficking

GGA

From: Boman, A.L. (2001) J. Cell Sci. 114: 3413-3418

p56p200

ubiquitin

ModularorganizationofGGAfamilyproteins

5xUAS GAL4

Actin5C-GAL4 driver transgeneUAS RNAi transgene

X

Any non-Curly adults?

inverted repeat/Curly wings

RNAi knockdown assay for essential genes

GGA is essential in Drosophila

GGAknockdown(14days)ommatidiahaveirregular,displacedandfragmentedrhabdomeres

GMRDriver‐onlyGGAknockdown

Mutantsgeneratebytransposableelement

But…

RescueLethalityComplementationtestwithfliescarryinterchromosomalduplications

fromBloomingtonStockCenter

lethalbetween8C3and8C12

WTmale GGAP1malesrescuedbyduplication

Hypothesis

GGAisrequiredintheadultretinafortrafficking of rhodopsin fromTGN torhabdomeres.

Aim1:DefinethefunctionofGGAinrhodopsintransportinvivo.

•  GenerateGGAknockoutflies

Select red eye fly from white eye background: yw;P(Donor)*/+

Balancing: : ✕♂ FM7 ywa B Mapping: ✕ ☿yw; Sco/CyO; TM3y+/Sb

After 2 generations

FM7 ywa B; P(Donor)/P(Donor) Able to map p-element insertion

GGA-

FM7 ywa B;

FM7 ywa B× FM7 ywa B ♂

FM7 ywa B;P(70FLP)

Possibleoutcomes:Red,SingleBareyefemalewillbeheterozygousforGGA‐(Balancedstock).

IfthereisnoRed,non‐Bareyemale,GGAK/Oislethal.ResultswillbeconfirmedbyPCR.

GGA-FM7 ywa B

•  DoesGGAknockoutaffectrhodopsinexport?‐  UseVitaminAreplacementtherapytosynchronizesynthesisanddeploymentofRh1.

GGAisessential:examinemosaiceyefromFM7ywaB;P{donor}XP{70‐ey4x‐FLP.Exel}1,y1w1118;P{70I‐SceI}ninaE‐

Gal4‐UAS‐Rh1::GFP.


•  DoesGGAknockoutaffectrhodopsinimport?‐  RaisetheflyindarktostopRh1turnoverandmeasureRh1clearanceaftermovingtolight.

GGAisessential:examinemosaiceyefromFM7ywaB;P{donor}XP{70‐ey4x‐FLP.Exel}1,y1w1118;P{70I‐SceI}ninaE‐

Gal4‐UAS‐Rh1::GFP.


Aim2:Createageneticsystemforinvivostructure‐functionanalysisofGGA.

•  WhichdomainisessentialforsurvivalorRh1transport?•  Domainanalysisbysitedirectedmutagensisusing

QuikchangeTMSite‐DirectedMutagenesiskit.


•  Control:RescueGGAknockoutmutant

GGA+

•  Alternatives:ConstructmutantformGGAwithGSTtagandcarryoutpulldownassaytostudyinteractions.


WT

GGAK/DRNAi

PreliminarydataofGGAknockdownusingdsRNAshoweddelayedinrhodopsintransportintotherhabdomerescomparetowild‐type.

Aim3:StudypathwaythatGGAinvolvedinmaintainingretinalhomeostasis.


•  InvestigatetheunderlyingmechanismofrhodopsintraffickingonseveralcandidateproteinsknowntointeractwithGGA.

(Hirstetal2001)

GGA1 Clathrin Merge


•  InvestigatetheunderlyingmechanismofrhodopsintraffickingonseveralcandidateproteinsknowntointeractwithGGA.

1.testroleofclathrininrhodopsintransport.CHCandCLCRNAi.

UsevitaminAreplacementtherapy,observeaccumulationofrhodopsininrhabdomere.

2.Arf,Rab6willalsobetested.3.MicroarrayonGGAknockoutflies

Thankyou!

investigate molecules required for rhodopsin trafficking...

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