Microinjection offluorescently labeled a-actinin into living fibroblasts

a-Actinin fromchicken gizzard labeled with tetramethylrhodamine isothiocyanate hasbeenincorporated into living fibroblast cells bymicroinjection. Fluorescent la- beling ofa-actinin wascarried outsuchthat theconjugated protein wasfunctional invitro asshownbyits ability tobind toF-actin. Within 1-2hrafter injection, diffuse fluorescence wasobserved throughout thecytoplasm andonly faint fluores- cence wasapparently associated with thestress fibers. During theensuing 2-15hr, however, mostofthefluorescence wasseen asperiodicities along thestress fibers andasfoci ofthemicro- filament polygonal networks. This distribution ofa-actinin in theliving cells wasstrikingly similar tothat found byindirect immunofluorescence localization ofendogenous a-actinin in fixed samples ofthesamecell type. Control studies inwhich heat-treated (100'C, 2min)fluorescent a-actinin ortetrameth- ylrhodamine isothiocyanate alone wasinjected into thecells indicated that thestress fiber andpolygonal network labeling wasspecific for "native" fluorescently labeled a-actinin. These results suggest that thedynamic properties ofproteins and structures incultured mammalian cells canbestudied with the useofmicroinjection andfluorescence microscopic tech- niques. During thepast several years, muchinformation hasbeen ob- tained intheidentification andlocalization ofcontractile and cytoskeletal proteins innonmuscle cells (for review, seeref1). Forexample, byelectron microscopy andimmunofluorescence microscopy, thepresence ofactin filaments (e.g., refs 2and3), myosin (4, 5), tropomyosin (6), a-actinin (7), andfilamin (8) inthestress fibers ofnonmuscle cells hasbeendemonstrated. Though itisnotyetproven that thestress fibers ofnonmuscle cells function ascontractile organelles, thepresence ofcon- tractile proteins inthe stress fibers, thealternating "sarcomeric" arrangement ofmyosin anda-actinin along thefibers (9), and theobservation that stress fibers contract inresponse toionic conditions that induce muscle contraction (10) suggest that the stress fibers mayfunction ascontractile structures andcontract inamanner analogous tomuscle sarcomeres (i.e., viaaslid- ing-filament mechanism). However, inorder toobserve the dynamic process ofcell movement atthemolecular level, techniques other than microscopic inspection offixed cells must beutilized. Anovel approach totheproblem ofstudying adynamic in- tracellular process hasrecently beendescribed inwhich fluo- rescently labeled actin wasmicroinjected into thecytoplasm oftheprotozoans Physarum polycephalum andChaos caroli- nens*s (11). Inthis manner thedistribution offluorescent actin intheliving organisms wasobtained. Because oftherelatively large size andthickness ofthese protozoans, though, itisdifficult toexamine thecells with fluorescence microscopy todetermine thelocation ofthefluorescent proteins injected orthelocation oftheendogenous proteins byimmunofluorescence. Main- malian cells such asfibroblasts areofthesize that makes them especially suitable forfluorescence microscopy, butatthesame time areofasufficiently small size that microinjection ofthem isnotaseasily done asitiswith thelarger protozoans. However, with thedevelopment oftheelegant techniques forthemi- croinjection ofmammalian cells byGraessmann andGraess- mann(12), theproblems ofinjecting small cells havebeen solved. Inthepresent paper Idescribe theincorporation of fluorescently labeled a-actinin, aprotein component ofthe Z-lines ofmuscle (13), into thefunctional pools ofca-actinin in living fibroblasts after microinjection ofthefluorescently la- beled protein. Analysis oftheinjected cells byfluorescence microscopy shows theincorporation ofthefluorescent protein into periodicities along thestress fibers aswell asinto thefoci ofthepolygonal microfilament networks, types ofstructures that byimmunofluorescent