Formation and Toxicity of Soluble Polyglutamine Oligomers in Living Cells 英文参考文献.docVIP

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Formation and Toxicity of Soluble Polyglutamine Oligomers in Living Cells 英文参考文献.doc

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Formation and Toxicity of Soluble Polyglutamine Oligomers in Living Cells 英文参考文献

FormationandToxicityofSolublePolyglutamine OligomersinLivingCells PatrickLajoie,ErikLeeSnapp* DepartmentofAnatomyandStructuralBiology,AlbertEinsteinCollegeofMedicine,Bronx,NewYork,UnitedStatesofAmerica Abstract Background:Aggregationandcytotoxicityofmutantproteinscontaininganexpandednumberofpolyglutamine(polyQ) repeatsisahallmarkofseveraldiseases,includingHuntington’sdisease(HD).Withincells,mutantHuntingtin(mHtt)and otherpolyglutamineexpansionmutantproteinsexistasmonomers,solubleoligomers,andinsolubleinclusionbodies(IBs). Determiningwhichoftheseformsconstituteatoxicspecieshasprovendifficult.RecentstudiessupportaroleforIBsasa cellularcopingmechanismtosequesterlevelsofpotentiallytoxicsolublemonomericandoligomericspeciesofmHtt. Methodology/PrincipalFindings:Whenfusedtoafluorescentreporter(GFP)andexpressedincells,thesolublemonomeric and oligomeric polyglutamine species are visually indistinguishable. Here, we describe two complementary biophysical fluorescence microscopy techniques to directly detect soluble polyglutamine oligomers (using Htt exon 1 or Httex1 ) and monitortheirfatesinlivecells.PhotobleachinganalysesrevealedasignificantreductioninthemobilitiesofmHttex1 variants consistent with their incorporation into soluble microcomplexes. Similarly, when fused to split-GFP constructs, both wildtypeandmHttex1formedoligomers,asevidencedbytheformationofafluorescentreporter.OnlythemHttex1split-GFP oligomersassembledintoIBs.BothFRAPandsplit-GFPapproachesconfirmedtheabilityofmHttex1tobindandincorporate wildtypeHttintosolubleoligomers.Weexploitedtheirreversiblebindingofsplit-GFPfragmentstoforciblyincreaselevels ofsolubleoligomericmHttex1.AcorrespondingincreaseintherateofIBsformationandthenumberformedwasobserved. Importantly, higher levels of soluble mHttex1 oligomers significantly correlated with increased mutant cytotoxicity, independentofthepresenceofIBs. Conclusions/Significance: Ourstudydescribespowerfulandsensitivetoolsforinvestigatingsolubleoligomericformsof expanded