Nonmuscle Myosin II Isoforms Coassemble in Living Cells

Abstract

Janelia Farm Research Campus, Howard Hughes MedicalInstitute, Ashburn, VA 20147, USASummaryNonmuscle myosin II (NM II) powers myriad developmentaland cellular processes, including embryogenesis, cellmigration, and cytokinesis [1]. To exert its functions, mono-mers of NM II assemble into bipolar filaments that produce acontractile force on the actin cytoskeleton. Mammalian cellsexpress up to three isoforms of NM II (NM IIA, IIB, and IIC),each of which possesses distinct biophysical propertiesand supports unique as well as redundant cellular functions[2–8]. Despite previous efforts [9–13], it remains unclearwhether NM II isoforms assemble in living cells to producemixed (heterotypic) bipolar filaments or whether filamentsconsist entirely of a single isoform (homotypic). We ad-dressed this question using fluorescently tagged versionsof NM IIA, IIB, and IIC, isoform-specific immunostaining oftheendogenousproteins,andtwo-color totalinternalreflec-tion fluorescence structured-illumination microscopy, orTIRF-SIM, to visualize individual myosin II bipolar filamentsinside cells. We show that NM II isoforms coassemble intoheterotypic filaments in a variety of settings, includingvarious types of stress fibers, individual filamentsthroughout the cell, and the contractile ring. We also showthatthedifferentialdistributionofNMIIAandNMIIBtypicallyseen in confocal micrographs of well-polarized cells is re-flected in the composition of individual bipolar filaments.Interestingly, this differential distribution is less pro-nounced in freshly spread cells, arguing for the existenceof a sorting mechanism acting over time. Together, ourwork argues that individual NM II isoforms are potentiallyperforming both isoform-specific and isoform-redundantfunctions while coassembled with other NM II isoforms.Results and DiscussionTIRF-SIM Reveals Individual NM II Bipolar FilamentsIn an initial effort to identify individual nonmuscle myosin II(NMII)bipolarfilaments,weexpressedNMIIAwithanN-termi-nalEGFPtag(EGFP-NMIIA;notethatalltagswerefusedtotheNM II heavy chain; Figure 1A) in U2OS cells and imaged thecells by total internal reflection fluorescence structured-illumi-nation microscopy (TIRF-SIM) (Figure 1C), a TIRF version of apreviously established SIM technique [14] that provides two-color imaging capability and achieves a lateral resolution ofw100 nm. This resolution should allow unequivocal identifica-tionofw300nmNMIIbipolarfilaments[15,16],unlikeimagingperformed by conventional microscopy, where the lateralresolution is w250 nm (although see [17]). In TIRF-accessibleregions of the lamellar extensions and the cell interior, imagesrevealed what appeared to be individual NM IIA filamentspossessing two puncta spaced w300 nm apart (Figure 1Cand insets C1 and C2). These putative filaments were usuallyembedded in actin networks (Figure 1D and insets D1 andD2) or aligned with linear actin filaments/bundles (Figure 1EandinsetsE1andE2)labeledwithF-tractin,anF-actinreporter[18, 19]. In regions rich in transverse arcs and ventral stress fi-bers, however, EGFP-NM IIA puncta were too numerous andclose together to identify individual filaments unequivocally(Figure1CandinsetC3).Toconfirmthatthese300nm-spacedpuncta correspond to individual NM IIA bipolar filaments andto resolve these structures in filament-rich regions of the cell,weusedNMIIAwithaC-terminalmAppletag(NMIIA-mApple;Figure 1A). Because NM II filaments are bipolar, coexpressionof NM IIA-mApple with EGFP-NM IIA should result in filamentswith EGFP puncta at both ends of the filament (correspondingto the N termini of the head domains) bifurcated by a singlemApple punctum (corresponding to C termini of the taildomains)(Figure1B).Consistently,whenthesetwoconstructswere coexpressed in U2OS cells, we observed two greenpuncta w300 nm apart that were bifurcated by a single redpunctum (Figure 1F and inset F1). Moreover, these two-colorstructures were readily resolvable in filament-rich regions ofthecell(Figure1FandinsetsF2andF3).Importantly,thelocal-izationofC-terminallytaggedNMIIAwasqualitativelyindistin-guishable from N-terminally tagged NM IIA, arguing that theC-terminal tag has no obvious deleterious effects on bipolarfilament structure (although minor effects cannot be entirelyruledout).WenotethatasimilarapproachemployinganN-ter-minalantibodyandaC-terminalfluorophorewasusedrecentlyto identify bipolar filaments containing NM IIC in epithelia [17].Exogenous NM II Isoforms Form Heterotypic FilamentsTo determine whether NM II isoforms form heterotypicfilaments, we coexpressed NM IIA-mApple and EGFP-NMIIB in U2OS cells. Although both isoforms are overexpressed,the ratio of NM IIA to NM IIB in transfected cells remainsessentiallythesameasinuntransfectedcells(w25:1)becausethe fold increase over endogenous protein levels is approxi-mately the same for both isoforms (w2.5- to 3-fold) (see Fig-ure S1 and Table S1 available online). Confocal microscopydemonstrated that these two isoforms ‘‘colocalize’’ to somedegree throughout most of the cell (Figure 2A), although NMIIAis enrichedinperipherallamellarelative toNMIIB,whereasNM IIB is enriched in central and posterior regions relative toNM IIA, as reported by others [8, 20, 21] (see also below). Todetermine whether this ‘‘colocalization’’ corresponds at leastin part to heterotypic filaments of NM IIA and NM IIB, weimaged using TIRF-SIM. Figure 2B shows that the green-red-green signal expected for heterotypic filaments is indeedvery common in TIRF-accessible interior regions of cells (seealso insets B1 and B2), although not all puncta conform to thispattern (presumably due to the presence of homotypic fila-ments, unipolar filaments [22], filaments that are partially outof the TIRF zone, bipolar filaments with a unipolar distributionof one isoform, and/or heterotypic filaments where the signalfor one isoform is below the level of detection; see Experi-mental Procedures for further discussion). Heterotypic