title: Myosin 18A coassembles with nonmuscle myosin 2 to form mixed bipolar filaments.
abstract: 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, suggesting that the functions of M18A in vivo do not depend on intrinsic motor activity. M18A has the 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 of the conditions tested. Instead, M18A formed an ∼ 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 numerous, fundamental cellular and developmental roles attributed to NM2, our results have far-reaching biological implications.
authors.affiliation: Laboratory of Molecular Physiology, National Heart, Lung, and Blood Institute, NIH, Bethesda, MD 20892-8015, USA.
authors.forename: Neil
authors.initials: N
authors.lastname: Billington
authors.affiliation: Cell Biology and Physiology Center, National Heart, Lung, and Blood Institute, NIH, Bethesda, MD 20892-8015, USA.
authors.forename: Jordan R
authors.initials: JR
authors.lastname: Beach
authors.affiliation: Laboratory of Molecular Physiology, National Heart, Lung, and Blood Institute, NIH, Bethesda, MD 20892-8015, USA.
authors.forename: Sarah M
authors.initials: SM
authors.lastname: Heissler
authors.affiliation: Cell Biology and Physiology Center, National Heart, Lung, and Blood Institute, NIH, Bethesda, MD 20892-8015, USA.
authors.forename: Kirsten
authors.initials: K
authors.lastname: Remmert
authors.affiliation: Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY 12180, USA.
authors.forename: Stephanie
authors.initials: S
authors.lastname: Guzik-Lendrum
authors.affiliation: Laboratory of Molecular Physiology, National Heart, Lung, and Blood Institute, NIH, Bethesda, MD 20892-8015, USA.
authors.forename: Attila
authors.initials: A
authors.lastname: Nagy
authors.affiliation: Laboratory of Molecular Physiology, National Heart, Lung, and Blood Institute, NIH, Bethesda, MD 20892-8015, USA.
authors.forename: Yasuharu
authors.initials: Y
authors.lastname: Takagi
authors.affiliation: Janelia Farm Research Campus, Howard Hughes Medical Institute, Ashburn, VA 20147, USA.
authors.forename: Lin
authors.initials: L
authors.lastname: Shao
authors.affiliation: Janelia Farm Research Campus, Howard Hughes Medical Institute, Ashburn, VA 20147, USA.
authors.forename: Dong
authors.initials: D
authors.lastname: Li
authors.affiliation: Laboratory of Functional Proteomics, College of Veterinary Medicine, Hunan Agricultural University, Changsha, Hunan 410128, People's Republic of China.
authors.forename: Yi
authors.initials: Y
authors.lastname: Yang
authors.affiliation: Laboratory of Molecular Cardiology, National Heart, Lung and Blood Institute, NIH, Bethesda, MD 20892, USA.
authors.forename: Yingfan
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Laboratory of Molecular Genetics, National Institute on Deafness and Other Communication Disorders, NIH, Bethesda, MD 20892, USA.
authors.forename: Melanie
authors.initials: M
authors.lastname: Barzik
authors.affiliation: Janelia Farm Research Campus, Howard Hughes Medical Institute, Ashburn, VA 20147, USA.
authors.forename: Eric
authors.initials: E
authors.lastname: Betzig
authors.affiliation: Cell Biology and Physiology Center, National Heart, Lung, and Blood Institute, NIH, Bethesda, MD 20892-8015, USA. Electronic address: hammerj@nhlbi.nih.gov.
authors.forename: John A
authors.initials: JA
authors.lastname: Hammer
authors.affiliation: Laboratory of Molecular Physiology, National Heart, Lung, and Blood Institute, NIH, Bethesda, MD 20892-8015, USA. Electronic address: sellersj@nhlbi.nih.gov.
authors.forename: James R
authors.initials: JR
authors.lastname: Sellers
mesh_terms: D000818:Animals; D003599:Cytoskeleton / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure; D051379:Mice; D008854:Microscopy, Electron / Q000379:methods; D008856:Microscopy, Fluorescence / Q000379:methods; D057054:Molecular Imaging / Q000379:methods; D024462:Myosin Type II / Q000378:metabolism; D009218:Myosins / Q000378:metabolism*; D020033:Protein Isoforms / Q000378:metabolism
chemical_list: C407101:Myo18a protein, mouse; D020033:Protein Isoforms; D024462:Myosin Type II; D009218:Myosins