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类肌球蛋白与传统的2类非肌肉肌球蛋白(NM2)关系最为密切。令人惊讶的是，果蝇、小鼠和人类肌球蛋白18A(M18A)的纯化头域缺乏肌动蛋白激活的ATPase活性和肌动蛋白细丝移位的能力，认为M18A在体内的功能不依赖于内在的运动活性。在2类肌球蛋白之外，M18A的卷曲卷曲长度在所有肌球蛋白中排名第二，这表明它可能形成类似于传统肌球蛋白的双极细丝。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丝。相反，M18A形成了一个65纳米长的双极结构，两端各有两个头。重要的是，当NM2在M18A存在下聚合时，两个肌球蛋白形成了混合的双极细丝，共沉淀、电子显微镜和单分子成像证明了这一点。此外，使用荧光标记蛋白对NM2和M18A进行的超分辨率成像和内源性蛋白的免疫染色表明，NM2和M18A一起存在于活细胞内的单个细丝中。综上所述，我们的体外和活细胞成像数据有力地证明了M18A与NM2共同组装成混合的双极细丝。M18A可以调节这些细丝的生物物理性质，并通过其额外的N-末端和C-末端结构域来决定这些细丝的定位和/或分子相互作用。鉴于NM2具有众多的细胞和发育作用，我们的研究结果具有深远的生物学意义。 18A类肌球蛋白是肌球蛋白中一个知之甚少的亚类，其结构域类似于II类肌球蛋白。然而，与II类肌球蛋白相比，肌球蛋白18A没有ATPase活性，因此，似乎不是真正的肌球蛋白马达。值得注意的是，18A类肌球蛋白和II类肌球蛋白在体外和体内通过其扩展的卷曲结构域共聚合成双极细丝，这表明肌球蛋白18A在调节细丝周转或作为适配器将细丝连接到不同的细胞结构或信号分子而不干扰NMII运动活动方面具有潜在的作用。哺乳动物肌球蛋白18A基因的选择性剪接导致至少两种亚型(肌球蛋白18A和#61538；)。肌球蛋白18A&61537和肌球蛋白18A#61472都是由一个运动区和一个短颈区、一个延伸的螺旋线圈结构域和一个含有SH3和PDZ结构域结合位点的C-末端非螺旋尾段组成。肌球蛋白有一个N-末端延伸，它包含一个富含KE的区域、一个三磷酸腺苷不敏感的肌动蛋白结合域和一个PDZ结构域。敲除肌球蛋白18A会导致小鼠和果蝇的胚胎死亡，这表明在发育过程中起着重要作用。肌球蛋白18A在哺乳动物组织中普遍表达，在某些类型的细胞中表达上调，并具有异构体特异性表达。这些研究的目标是研究上皮来源的普通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在极化的MDCK细胞片和不同小鼠含上皮组织的冰冻切片中的定位。我们发现肌球蛋白18A在培养的极化MDCK细胞的顶端表面优先定位于细胞-细胞连接，在那里NMII被认为是维持上皮完整性的关键。在肾、肠等组织切片中，肌球蛋白18A主要分布在近端小管和微绒毛中。这两种组织也都表达NMII。此外，在分泌组织中，如胰腺和唾液腺，M18A定位于分泌颗粒分泌前的外表面。这类似于NMII在这些结构上的定位动力学，在这些结构上，NMII被认为是维持适当的静水压力以发生分泌所必需的。唾液腺的初步实验表明，M18A可能与NMII一起被招募为颗粒。综上所述，这些数据表明，M18A可能调节NMII，因为它的功能是维持经典的上皮细胞的完整性，以及它在更专门的过程中发挥作用，如胰腺和唾液分泌。