MOLECULARANDCELLULARBIOLOGY,July1987,p.2457-2466Vol.7,No.70270-7306/87/072457-10$02.00/0CopyrightC1987,AmericanSocietyforMicrobiologyExpressionofTransfectedMutant,-ActinGenes:AlterationsofCellMorphologyandEvidenceforAutoregulationinActinPoolsJOHNLEAVITh,l*SUN-YUNG,'UELIAEBI,2'3MADHUVARMA,'GERALDLATTER,'STEPHENBURBECK,lLARRYKEDES,4ANDPETERGUNNINGt4ArmandHammerCancerResearchCenter,LinusPaulingInstituteofScienceandMedicine,PaloAlto,California943061;DepartmentofCellBiologyandAnatomy,TheJohnsHopkinsUniversitySchoolofMedicine,Baltimore,Maryland212052;M.E.MullerInstituteforHighResolutionElectronMicroscopy,Biozentrum,UniversityofBasel,CH4056Basel,Switzerland3;andMedigenProject,DepartmentofMedicine,StanfordUniversitySchoolofMedicineandVeteransAdministrationMedicalCenter,PaloAlto,California943044Received10November1986/Accepted6April19%!Twodifferentmutanthumanj-actingeneshavebeenintroducedintonormaldiploidhuman(KD)fibroblastsandtheirimmortalizedderivativecellline,HuT-12,toassesstheimpactofanabnormalcytoskeletalproteinoncellularphenotypessuchasmorphology,growthcharacteristics,andpropertiesrelatingtotheneoplasticphenotype.Amutant,-actincontainingasinglemutation(Gly-244--Asp-244)wasstableandwasincorporatedintocytoskeletalstressfibers.TransfectedKDcellswhichexpressedthestablemutantI(-actininexcessofnormal,-actinweremorphologicallyaltered.Incontrast,asecondmutant,-actingenecontainingtwoadditionalmutations(Gly-36-*Glu-36andGlu-83-*Asp-83,aswellasGly-244-*Asp-244)didnotaltercellmorphologywhenexpressedathighlevelsintransfectedcells,buttheproteinwaslabileanddidnotaccumulateinstressfibers.InbothKDandHuT-12cells,endogenous,-andy-actindecreasedinresponsetohigh-levelexpressionofthestablemutantI-actin,inamannerconsistentwithautoregulatoryfeedbackofactinconcentrations.SincethepercentdecreasesintheendogenousI8-andy-actinswereequal, theratioofnet0-actin(mutantplusnormal)toy-actinwassignificantlyincreasedinthetransfectedcells.Antiseracapableofdistinguishingthemutantfromthenormalepitoperevealedthatthemutantj-actinaccumulatedinstressfibersbutdidnotparticipateintheformationoftheactinfilament-richperinuclearnetwork.Theseobservationssuggestthatdifferentintracellularlocationsdifferentiallyincorporateactinintocytoskeletalmicrofilaments.ThedramaticimpactoncellmorphologyandonP-actin/y-actinratiosinthetransfecteddiploidKDcellsmayberelatedtotheacquisitionofsomeofthecharacteristicsofcellsthatunderwenttheneoplastictransformationeventthatoriginallyledtotheappearanceofthej-actinmutations.Actinisaubiquitous,highlyabundantproteinthatisresponsibleforavarietyofcellularactivities,includingmotilityandthestructuralpropertiesofthecytoplasm.Thisprotein,highlyconservedinevolution,formscomplexstruc-turesinalleucaryoticcellsbyacombinationofself-polymerizationandinteractionswithahostofbindingpro-teins(reviewedinreference37).Thesecomplexinteractionshavebeenthesubjectofintensebiochemicalandstructuralstudybuthavenotyetbeenamenabletogeneticanalysis.Fewstructuralmutationsinactinareknown,asmightbeexpectedforaproteinwhoseactionsaresocentraltonormalcellmorphologyandfunction.Therecessiveflightlessmu-tantsofDrosophilamelanogastershowadisorderedstruc-tureofthesarcomeres(18,31)andinductionofstressproteins(15,18,31)intheindirectflightmusclesofhomozygotesinwhichmutantallelesofatissue-specificactinareexpressed(18,31).HumanfibroblastswhichexpressmutantP-actinshavebeengeneratedbytheneoplastictransformationofdiploidKDfibroblastsinvitroandtheisolationofstablefocus-derived,neoplasticstrains(16,25).TheKDfibroblastsarephenotypicallynormaldiploidcellswithafinitelifespaninculture(2,16,20,22,25,26,40,42);HuT-11,HuT-12,andHuT-13areneoplasticallytransformed"immortalized"strainsderivedfromKDcellsthatexpresstransformation-*Correspondingauthor.tPresentaddress:Children'sMedicalResearchFoundation,Camperdown,Australia2050.specificproteinmarkers(3,10,11,20-22,25,26,40)andanchorage-independentgrowth(16,17),butrarelyproducetumorsinnudemice(20,22).HuT-14isahighlytumorigenicstrainthatexhibitsadditionaldifferencesincellularmorphol-ogyandgeneexpression(19-22,25,
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