Structure And Function Of Unconventional Myosins

非常规肌球蛋白的结构和功能

• 批准号: 9157305
• 负责人: JOHN A HAMMER
• 金额: $ 52.5万
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/9157305
• 关键词: ATP phosphohydrolase; Actins; Address; Alternative Splicing; Amoeba genus; Antibodies; Apical; Architecture; Baculoviruses; Binding; Binding Sites; Biochemistry; Biological; Biological Models; C-terminal; Cell physiology; Cells; Cellular Structures; Coiled-Coil Domain; Cytoplasmic Granules; Data; Development; Dictyostelium; Drosophila genus; Electron Microscopy; Embryo; Epithelial; Epithelial Cells; Epithelium; Filament; Generic Drugs; Genes; Goals; Head; Human; Hydrostatic Pressure; Image; In Vitro; Individual; Intercellular Junctions; Intestines; Kidney; Kinetics; Knock-out; Length; Life; Link; Lymphocyte; MDCK cell; Microfilaments; Motor; Motor Activity; Mus; Myosin ATPase; Myosin Type II; N-terminal; Neck; Neurons; Pancreas; Process; Protein Isoforms; Proteins; Recruitment Activity; Regulation; Resolution; Role; Salivary Glands; Secretory Vesicles; Signal Pathway; Signaling Molecule; Structure; Surface; System; Testing; Tissues; Work; biophysical properties; cell growth regulation; cell type; cellular imaging; cellular microvillus; fly; in vivo; melanocyte; mutant; non-muscle myosin; pancreatic juice; research study; saliva secretion; single molecule; structural biology

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. Class-18A myosins are a poorly understood subclass of myosins with a domain architecture similar to that of class II myosins. In contrast to class II myosins though, myosin 18A has no ATPase activity and therefore, does not appear to be a true myosin motor. Notably, class-18A myosins and class II myosins copolymerize in vitro and in vivo into bipolar filaments via their extended coiled-coil domains, suggesting a potential role for myosin 18A in the regulation of filament turnover or as an adaptors to link the filaments to different cellular structures or signaling molecules without interfering with NMII motor activity. Alternative splicing of the mammalian myosin 18A gene results in at least 2 isoforms (myosin 18A and ). Both myosin 18A and myosin 18Aconsist of a motor domain followed by a short neck region, an extended coiled-coil domain, and a C-terminal non-helical tailpiece harboring binding sites for SH3 and PDZ domain-containing proteins. Myosin 18A has an N-terminal extension that contains a KE-rich region, an ATP-insensitive actin-binding domain, and a PDZ domain. Knockout of myosin 18A results in embryonic lethality in both mice and flies, suggesting a fundamental role in development. Myosin 18A appears ubiquitously expressed across mammalian tissues with elevated expression and isoform-specific expression in certain cell types. The goal of these studies was to investigate M18A in epithelia-derived generic cells and epithelial tissues. We analyzed the localization of myosin 18A in both polarized MDCK cell sheets and in cryo-sections of various mouse epithelia-containing tissues using a myosin 18A-specific antibody. We show preferential localization of myosin 18A to cell-cell junctions at the apical surface of polarized MDCK cells in culture, where NMII is known to be critical for maintaining epithelial integrity. In tissue sections, such as kidney and intestine, myosin 18A is enriched in proximal tubules and microvilli. Both tissues are also expressing NMII. Additionally, in secretory tissues, such as the pancreas and salivary gland, M18A localizes to the outer surface of secretory granules immediately prior to their secretion. This is similar to the localization kinetics of NMII on these structures, where NMII is known to be essential for maintaining proper hydrostatic pressure for secretion to occur. Preliminary experiments in the salivary gland suggest that M18A might be recruited to granules together with NMII. Together, these data argue that M18A may be regulating NMII as it functions to maintain classic epithelia integrity and as it functions in more specialized processes, such as pancreatic and salivary secretion.

18类肌凝蛋白与常规的2类非肌凝蛋白（NM2）关系最为密切。令人惊讶的是，纯化的果蝇、小鼠和人肌球蛋白18A （M18A）头部结构域缺乏肌动蛋白激活的atp酶活性和肌动蛋白丝转运的能力，这表明M18A在体内的功能并不依赖于内在的运动活性。M18A是除2类肌凝蛋白外所有肌凝蛋白中第二长的盘绕线圈，这表明它可能形成与传统肌凝蛋白类似的双极细丝。为了解决这种可能性，我们使用杆状病毒/Sf9系统表达和纯化了全长小鼠M18A。在任何测试条件下，M18A都没有形成大的双极细丝。相反，M18A形成了65 nm长的两极结构，两端各有两个头。重要的是，当NM2在M18A存在下聚合时，两种肌球蛋白形成混合的双极性细丝，共沉淀、电子显微镜和单分子成像证明了这一点。此外，利用荧光标记蛋白和内源性蛋白的免疫染色对NM2和M18A进行超分辨率成像显示，NM2和M18A同时存在于活细胞内的单个细丝内。总之，我们的体外和活细胞成像数据有力地证明M18A与NM2结合成混合双极细丝。M18A可以调节这些细丝的生物物理特性，并通过其额外的N端和c端结构域决定细丝的定位和/或分子相互作用。考虑到NM2在细胞和发育中的作用，我们的研究结果具有深远的生物学意义。