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.

第18类肌球蛋白与传统的2类非肌肉肌蛋白（NM2）最密切相关。令人惊讶的是，果蝇，小鼠和人肌球蛋白18a（M18a）的纯化头域缺乏肌动蛋白激活的ATPase活性和易位分配肌动蛋白丝的能力，认为M18A INVIVO的功能不取决于固有运动活性。 M18a具有2级肌球蛋白以外的任何肌球蛋白的第二最长的盘绕线圈，这表明它可能形成类似于传统肌球蛋白的双极丝。为了解决这种可能性，我们使用Baculovirus/SF9系统表达并纯化了全长小鼠M18A。在测试的任何条件下，M18A都不形成大型双极丝。 取而代之的是，M18A形成了一个65 nm长的双极结构，两端有两个头。重要的是，当NM2在M18A存在下聚合时，这两种肌动物形成混合的双极丝，如引起，电子显微镜和单分子成像所证明的那样。此外，使用荧光标记的蛋白质和内源性蛋白的免疫染色的NM2和M18A的超分辨率成像显示，NM2和M18A在活细胞内的各个细丝中都存在。我们的体外和活细胞成像数据共同表明，M18A将NM2与混合的双极丝结合在一起。 M18a可以调节这些丝的生物物理特性，并根据其额外的N和C末端结构域确定细丝的定位和/或分子相互作用。鉴于归因于NM2的无数细胞和发育作用，我们的结果具有生物学上的意义。 18A类肌球蛋白是一个知名的肌球蛋白的子类，其领域架构类似于II类肌球蛋白。 与II类肌球蛋白相反，肌球蛋白18a没有ATPase活动，因此似乎不是真正的肌球蛋白运动。 值得注意的是，通过其扩展的盘绕螺旋型域在体外和体内共聚成双极细丝，这表明肌球蛋白18a在调节细丝周转中的潜在作用，或者作为对不同的细胞结构或信号分子的不同无需与NMII Interferting Intnection nmii Interferting Nimii链接到不同的细胞结构或信号分子。哺乳动物肌球蛋白18a基因的替代剪接至少导致2种同工型（肌球蛋白18a和）。 肌球蛋白18A和肌球蛋白18A A均具有运动结构域的介绍，其次是短颈部区域，延伸的盘绕螺旋域和C末端的非螺旋尾部，该尾部具有SH3和PDZ结构域蛋白质的结合位点。肌球蛋白18A具有N末端扩展，其中包含一个KE富含ATP的肌动蛋白结合域和PDZ域。肌球蛋白18a的敲除导致小鼠和苍蝇的胚胎致死性，这表明在发育中起着基本作用。肌球蛋白18a在某些细胞类型中的表达升高和同工型特异性表达升高的哺乳动物组织中似乎无处不在。这些研究的目的是研究上皮细胞和上皮组织中的M18A。我们使用肌球蛋白18a特异性抗体分析了肌球蛋白18a在极化MDCK细胞表中的定位以及各种含有小鼠上皮的组织的冷冻截面。 我们显示了肌球蛋白18a在培养物中极化MDCK细胞的顶端表面上的细胞细胞连接的优先定位，其中NMII对于维持上皮完整性至关重要。在组织切片（例如肾脏和肠道）中，肌球蛋白18a富含近端小管和微绒毛。两种组织也表达NMII。此外，在分泌组织（例如胰腺和唾液腺）中，M18A在分泌之前就位于分泌颗粒的外表面。 这类似于NMII在这些结构上的定位动力学，在这些结构中，NMII对于维持适当的静水压力以进行分泌至关重要。 唾液腺中的初步实验表明，M18A可能与NMII一起招募到颗粒。 总之，这些数据表明，M18A可以调节NMII，因为它可以保持经典的上皮完整性及其在更专业的过程中（例如胰腺和唾液分泌）中起作用。