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.
Aim1:DefinethefunctionofGGAinrhodopsintransportinvivo.
• DoesGGAknockoutaffectrhodopsinimport?‐ RaisetheflyindarktostopRh1turnoverandmeasureRh1clearanceaftermovingtolight.
GGAisessential:examinemosaiceyefromFM7ywaB;P{donor}XP{70‐ey4x‐FLP.Exel}1,y1w1118;P{70I‐SceI}ninaE‐
Gal4‐UAS‐Rh1::GFP.
Aim1:DefinethefunctionofGGAinrhodopsintransportinvivo.
Aim2:Createageneticsystemforinvivostructure‐functionanalysisofGGA.
• WhichdomainisessentialforsurvivalorRh1transport?• Domainanalysisbysitedirectedmutagensisusing
QuikchangeTMSite‐DirectedMutagenesiskit.
Aim2:Createageneticsystemforinvivostructure‐functionanalysisofGGA.
• Control:RescueGGAknockoutmutant
GGA+
• Alternatives:ConstructmutantformGGAwithGSTtagandcarryoutpulldownassaytostudyinteractions.
Aim2:Createageneticsystemforinvivostructure‐functionanalysisofGGA.
WT
GGAK/DRNAi
PreliminarydataofGGAknockdownusingdsRNAshoweddelayedinrhodopsintransportintotherhabdomerescomparetowild‐type.
Aim3:StudypathwaythatGGAinvolvedinmaintainingretinalhomeostasis.
Aim3:StudypathwaythatGGAinvolvedinmaintainingretinalhomeostasis.
• InvestigatetheunderlyingmechanismofrhodopsintraffickingonseveralcandidateproteinsknowntointeractwithGGA.
(Hirstetal2001)
GGA1 Clathrin Merge
Aim3:StudypathwaythatGGAinvolvedinmaintainingretinalhomeostasis.
• InvestigatetheunderlyingmechanismofrhodopsintraffickingonseveralcandidateproteinsknowntointeractwithGGA.
1.testroleofclathrininrhodopsintransport.CHCandCLCRNAi.
UsevitaminAreplacementtherapy,observeaccumulationofrhodopsininrhabdomere.
2.Arf,Rab6willalsobetested.3.MicroarrayonGGAknockoutflies
Thankyou!