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类非肌球蛋白（NM 2）最密切相关。令人惊讶的是，果蝇，小鼠和人类肌球蛋白18 A（M18 A）的纯化头部结构域缺乏肌动蛋白激活的ATP酶活性和移位肌动蛋白丝的能力，认为M18 A在体内的功能不依赖于内在的运动活性。M18 A在2类肌球蛋白之外具有第二长的卷曲螺旋，这表明它可能形成类似于常规肌球蛋白的双极丝。为了解决这种可能性，我们使用杆状病毒/Sf 9系统表达和纯化全长小鼠M18 A。在任何测试条件下，M18 A均未形成大的双极细丝。 相反，M18 A形成了一个65 nm长的双极结构，两端各有两个头。重要的是，当NM 2在M18 A存在下聚合时，两种肌球蛋白形成混合的双极丝，如共沉降、电子显微镜和单分子成像所证明的。此外，使用荧光标记的蛋白质和内源性蛋白质的免疫染色对NM 2和M18 A进行的超分辨率成像表明，NM 2和M18 A一起存在于活细胞内的单个细丝中。总之，我们的体外和活细胞成像数据有力地证明了M18 A与NM 2共同组装成混合双极丝。M18 A可以调节这些纤维的生物物理性质，并且，凭借其额外的N-和C-末端结构域，确定纤维的定位和/或分子相互作用。鉴于NM 2在细胞和发育中的无数作用，我们的研究结果具有深远的生物学意义。 18 A类肌球蛋白是肌球蛋白的一个知之甚少的亚类，具有与II类肌球蛋白相似的结构域结构。 与II类肌球蛋白相反，肌球蛋白18 A没有ATP酶活性，因此似乎不是真正的肌球蛋白马达。 值得注意的是，18 A类肌球蛋白和II类肌球蛋白在体外和体内通过其延伸的卷曲螺旋结构域共聚成双极肌丝，这表明肌球蛋白18 A在调节肌丝周转或作为衔接子将肌丝连接到不同的细胞结构或信号分子而不干扰匪II运动活性中的潜在作用。哺乳动物肌球蛋白18 A基因的选择性剪接产生至少2种同种型（肌球蛋白18 A和）。 肌球蛋白18 A和肌球蛋白18 A都由一个马达结构域、一个短颈区、一个延伸的卷曲螺旋结构域和一个C-末端非螺旋尾片段组成，该尾片段含有SH 3和PDZ结构域蛋白的结合位点。肌球蛋白18 A的N-末端延伸区含有一个KE富集区、一个ATP不敏感的肌动蛋白结合区和一个PDZ区。敲除肌球蛋白18 A会导致小鼠和苍蝇的胚胎死亡，这表明肌球蛋白18 A在发育中起着重要作用。肌球蛋白18 A似乎在哺乳动物组织中普遍表达，在某些细胞类型中具有升高的表达和同种型特异性表达。这些研究的目的是研究上皮来源的一般细胞和上皮组织中的M18 A。我们使用肌球蛋白18 A特异性抗体分析了肌球蛋白18 A在极化MDCK细胞片和各种小鼠上皮组织的冷冻切片中的定位。 我们显示肌球蛋白18 A优先定位于极化MDCK细胞培养中的顶端表面的细胞-细胞连接处，其中NMII已知对于维持上皮完整性至关重要。在组织切片中，如肾和肠，肌球蛋白18 A在近端小管和微绒毛中富集。这两种组织也表达NMII。此外，在分泌组织中，如胰腺和唾液腺，M18 A定位于分泌颗粒的外表面，就在它们分泌之前。 这类似于匪II在这些结构上的定位动力学，其中已知匪II对于维持分泌发生的适当流体静压是必需的。 在唾液腺中的初步实验表明，M18 A可能与NMII一起被募集到颗粒中。 总之，这些数据表明M18 A可能正在调节NMII，因为它的功能是维持经典的上皮完整性，并且在更专门的过程中发挥作用，例如胰腺和唾液分泌。