Non-muscle myosin II (NM II) powers myriad developmental and cellular processes, including embryogenesis, cell migration, and cytokinesis. To exert its functions, monomers of NM II assemble into bipolar filaments that produce a contractile force on the actin cytoskeleton. Mammalian cells express up to three isoforms of NM II (NM IIA, IIB and IIC), each of which possesses distinct biophysical properties and supports unique, as well as redundant, cellular functions. Despite previous efforts, it remains unclear if NM II isoforms assemble in living cells to produce mixed (heterotypic) bipolar filaments, or if filaments consist entirely of a single isoform (homotypic). We addressed this question using fluorescently-tagged versions of NM IIA, IIB and IIC, isoform-specific immunostaining of the endogenous proteins, and two-color total internal reflection fluorescence structured-illumination microscopy, or TIRF-SIM, to visualize individual myosin II bipolar filaments inside cells. We show that NM II isoforms co-assemble into heterotypic filaments in a variety of settings, including various types of stress fibers, individual filaments throughout the cell, and the contractile ring. We also show that the differential distribution of NM IIA and NM IIB typically seen in confocal micrographs of well-polarized cells is reflected in the composition of individual bipolar filaments. Interestingly, this differential distribution is less pronounced in freshly-spread cells, arguing for the existence of sorting mechanism acting over time. Together, our work argues that individual NM II isoforms are potentially performing both isoform-specific and isoform-redundant functions while co-assembled with other NM II isoforms. Class 18 myosins are most closely related to conventional class 2 nonmuscle myosins (NM2). Surprisingly, the purified head domains of Drosophila, mouse and human myosin 18A (M18A) lack actin-activated ATPase activity and the ability to translocate actin filaments, arguing that the functions of M18A in vivo do not depend on intrinsic motor activity. M18A has the second longest coiled-coil of any myosin outside of the class 2 myosins, suggesting that it might form bipolar filaments similar to conventional myosins. To address this possibility, we expressed and purified full-length mouse M18A using the baculovirus/Sf9 system. M18A did not form large bipolar filaments under any conditions tested. Instead, M18A formed a 65 nm-long bipolar structure with two heads at each end. Importantly, when NM2 was polymerized in the presence of M18A, the two myosins formed mixed bipolar filaments, as evidenced by cosedimentation, electron microscopy, and single-molecule imaging. Moreover, super-resolution imaging of NM2 and M18A using fluorescently tagged proteins and immunostaining of endogenous proteins showed that NM2 and M18A are present together within individual filaments inside living cells. Together, our in vitro and live-cell imaging data argue strongly that M18A coassembles with NM2 into mixed bipolar filaments. M18A could regulate the biophysical properties of these filaments, and, by virtue of its extra N- and C-terminal domains, determine the localization and/or molecular interactions of the filaments. Given the myriad cellular and developmental roles attributed to NM2, our results have far reaching biological implications.

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Project End
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Budget End
Support Year
31
Fiscal Year
2014
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U.S. National Heart Lung and Blood Inst
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Hammer, John A (2018) Myosin goes for blood. Proc Natl Acad Sci U S A 115:4813-4815
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Heimsath Jr, Ernest G; Yim, Yang-In; Mustapha, Mirna et al. (2017) Myosin-X knockout is semi-lethal and demonstrates that myosin-X functions in neural tube closure, pigmentation, hyaloid vasculature regression, and filopodia formation. Sci Rep 7:17354
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Varadarajan, Ramya; Hammer, John A; Rusan, Nasser M (2017) A centrosomal scaffold shows some self-control. J Biol Chem 292:20410-20411
Beach, Jordan R; Bruun, Kyle S; Shao, Lin et al. (2017) Actin dynamics and competition for myosin monomer govern the sequential amplification of myosin filaments. Nat Cell Biol 19:85-93
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Billington, Neil; Beach, Jordan R; Heissler, Sarah M et al. (2015) Myosin 18A coassembles with nonmuscle myosin 2 to form mixed bipolar filaments. Curr Biol 25:942-8

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