M&M

MicroscopicMthodsforStudyingCllWatrandVolumRgulationinApoptoticCllsBrandonAGibbons1,NathanJMudrak1,MichalAModl11DpartmntofBiologicalScincs,KntStatUnirsity,Knt,OhioCllshrinkagandwatrlossarhallmarksofapoptoticclldathDspitsdcadsofrsarch,thirsignificancandmchanismofdlopmntarpoorlyundrstood[1]Progrssinthisaradpndsonthabilitytomasurcllolum,cllwatrcontntandintracllularconcntrationsofmonoalntionsCllolumTransmissionthroughdy(TTD)imagingachisaccuratandrobustolummasurmntsofliingcllsusingastandardbrightfildmicroscop[2]ThmthodisbasdonxclusionofastronglyabsorbingxtracllulardybyintactcllsCllsarplacdinashallowchambr(i,canbgrownonacorslip,whichisthnmountdonaslidwithcllsfacingthslid)Cllsarkptinthirnormalmdium,towhichanontoxicandmmbranimprmantdyAcidBlu9isadddWhndryingisprntd,cllsrmainiablinsuchannironmntandxhibitnormalbhaiorforatlast24hoursThsamplisimagdintransmittdlightatawalngthofmaximaldyabsorption(630nm)Bcauscllsxcludthdy,thdpthofthabsorbinglayriscomplmntarytocllthicknss,andtransmissionimagquantitatilyrflctscllthicknssandolum(Fig1A);importantly,thrsultsarnotaffctdbypossiblinstabilityinthlightsourcTTDproidsaccuratmasurmntsnonsrlydformdclls(whicharcommonduringapoptosis)andiscompatiblwithfluorscncCllwatrApoptoticshrinkagcanoccurbytworydiffrntprocsss:bywatrxpulsionorbycllfragmntationTodistinguishbtwnthstwopossibilitis,dirctwatrmasurmntsmustcomplmntmasurmntsofthcllolumIntracllularwatrisdirctlyrlatdtotharagrfractiindx;thlattrcanbobtaindfromtholum(whichcanbdtrmindbyTTD)andfromthphasdlayofalightwacrossingthsamplAsimplandffctiwaytomasurthphasisbasdonthtransportofintnsityquation(TIE)[3,4]Twotransmissionbrightfildimagsarcollctdatdiffrntdistancstothsampl,andthphasisfoundbysolingthTIEInhalthycllsthrisgoodcorrlationbtwnthphasandthhightmapobtaindbyTTD(Fig1B)Thrisnosuchcorrlationinapoptoticclls,andthmismatchbtwnTTDandTIEcanbusdtoquantifycllwatrcontnt[4]CalibrationofionprobsIntracllularionsmakamajorcontributiontothosmolarityofthcytosol,whichdtrminsthamountofintracllularwatrWhilthrisxtnsilitraturonionmasurmnts,thproblmofcalibrationinfluorscncbasdassayshasbninsufficintlyaddrssdItiswllknownthationindicatorsaraffctdbythirnironmnt,andthrforthyndtobcalibratdinsituCalibrationistypicallyachidbyapplyingionophors,withthaimtoqualizthionconcntrationsinsidIiandoutsidIoHowr,suchqualizationisonlypossiblwhnthmmbranpotntialisdstroyd(=0),andthatrquirsadditionalharshtratmntofcllswithpumpinhibitorsandmtabolicpoisonsQuitoftn,thisstpisaoiddaltogthr,andthatprcludsrliablcalibrationWproposadiffrnt,twostpapproachtocalibration(1)Instadoftryingtoliminatthmmbranpotntial,thactualismasurdfromthquilibriumdistributionofapotntialsnsitiprob[5],whichcanbaccomplishdwithaconfocalmicroscop(2)FluorscntsignalFfromanionprobismasurdinthprsncofanionophoratariablxtrnalionconcntrationsFromthknowldgofandIo,IicanbcalculatdandrlatdtoF;thusacalibrationcurF(Ii)canbconstructd(Fig1A)(3)Finally,IicanbobtaindinintactcllsloaddwithafluorscntprobandapotntiomtricdyThismthodhasbndmonstratdforintracllularK+masurmnts,butitshouldbquallyapplicabltoNa+,Ca2+andothrionsFigur1(A)ThprinciplofTTD(B)ComparisonofTIEandTTDimagsThphasTIEimagwasconstructdfromthtwobrightfildimags,BF1andBF2,sparatdrticallyby5mThlngthofthscalbaris25m(C)Thstpsinoldinionprobcalibration[1]MAModl,AmJPhysiol306(2014),C417[2]MAModl,CurrProtocCytom72(2015),12391[3]KIshizukaandBAllman,MicroscopyToday13(2005),22[4]MAModlandESchonbrun,JPhysiol591(2013),5843[5]BEhrnbrgtal,BiophysJ53(1988),785[6]ThworkwassupportdbyNIHgrant1R15GM186816,bythOhioBoardofRgntsandbythKntStatUnirsityRsarchCouncil