Sites of Glucose Transporter-4 Vesicle Fusion with the Plasma Membrane Correlate Spatially with Microtubules 英文参考文献.docVIP

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Sites of Glucose Transporter-4 Vesicle Fusion with the Plasma Membrane Correlate Spatially with Microtubules 英文参考文献.doc

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Sites of Glucose Transporter-4 Vesicle Fusion with the Plasma Membrane Correlate Spatially with Microtubules 英文参考文献

SitesofGlucoseTransporter-4VesicleFusionwiththe PlasmaMembraneCorrelateSpatiallywithMicrotubules JennineM.Dawicki-McKenna1,2,YaleE.Goldman1,2,E.MichaelOstap1,2 * 1PennsylvaniaMuscleInstitute,PerelmanSchoolofMedicineattheUniversityofPennsylvania,Philadelphia,Pennsylvania,UnitedStatesofAmerica,2Departmentof Physiology,PerelmanSchoolofMedicineattheUniversityofPennsylvania,Philadelphia,Pennsylvania,UnitedStatesofAmerica Abstract Inadipocytes,vesiclescontainingglucosetransporter-4(GLUT4)redistributefromintracellularstorestothecellperipheryin responsetoinsulinstimulation.Vesiclesthenfusewiththeplasmamembrane,facilitatingglucosetransportintothecell.To gain insight into the details of microtubule involvement, we examined the spatial organization and dynamics of microtubulesinrelationtoGLUT4vesicletraffickinginliving3T3-L1adipocytesusingtotalinternalreflectionfluorescence (TIRF)microscopy.InsulinstimulatedanincreaseinmicrotubuledensityandcurvaturewithintheTIRF-illuminatedregionof the cell. The high degree of curvature and abrupt displacements of microtubules indicate that substantial forces act on microtubules.Thetimecourseofthemicrotubuledensityincreaseprecedesthatoftheincreaseinintensityoffluorescently- taggedGLUT4inthissameregionofthecell.Inaddition,portionsofthemicrotubulesarehighlycurvedandarepulled closertothecellcortex,asconfirmedbyParallaxmicroscopy.MicrotubuledisruptiondelayedandmodestlyreducedGLUT4 accumulationattheplasmamembrane.QuantitativeanalysisrevealedthatfusionsofGLUT4-containingvesicleswiththe plasmamembrane,detectedusinginsulin-regulatedaminopeptidasewithapH-sensitiveGFPtag(pHluorin),preferentially occurnearmicrotubules.Interestingly,long-distancevesiclemovementalongmicrotubulesvisibleatthecellsurfaceprior to fusion does not appear toaccount for this proximity. We conclude that microtubules may be important in providing spatialinformationforGLUT4vesiclefusion. Citation:Dawicki-McKennaJM,GoldmanYE,OstapEM(2012)SitesofGlucoseTransporter-4VesicleFusionwitht