Structure And Function of Convential and Unconventional Myosins

活细胞中非肌肉肌球蛋白2(NM2)微丝组装的空间和时间控制机制在很大程度上尚不清楚。使用EGFP-NM2A敲入成纤维细胞和多种超分辨率成像方式，我们描述了NM2细丝在板层内组装的顺序放大机制，其中来自单个成核事件的单个细丝连续分裂形成丝状簇，然后填充到细胞更深处的大规模肌动球蛋白结构中。实时双色成像显示，单个分割事件在空间和时间上与潜在肌动蛋白纤维的运动一致，抑制肌动蛋白动力学抑制分割。这些和其他数据表明，NM2A细丝被它们结合的肌动蛋白纤维的动态运动所分割。MLCK的活性决定了膜片的分割频率和纤丝生长速度。重要的是，我们提供的直接证据表明，可用于细丝组装的NM2A单体池是有限的，因此MLCK与Rho Kinase竞争，在细胞质中更深地作用于单体以驱动板层细丝组装。总之，我们的结果为细胞内NM2丝组装的机制和调控提供了新的见解。 18A类肌球蛋白(M18A)是一类鲜为人知的肌球蛋白，其结构域结构类似于肌球蛋白2(M2)。具体地说，M18A和M18A都是通过交替剪接产生的两种M18A亚型，都由一个马达结构域和一个紧随其后的短颈区和一个驱动二聚的扩展盘绕线圈结构域组成。然而，与M2不同的是，它们拥有一个C末端的非螺旋尾段，其中含有SH3和PDZ结构域蛋白的结合位点。此外，M18A还具有一个N-末端延伸，包含一个富含KE的区域、一个ATP不敏感的肌动蛋白结合位点和一个PDZ结构域。M18A基因的敲除会导致小鼠和果蝇的胚胎死亡，这表明它在发育中发挥了重要作用。尽管M18A亚型在结构上与M2相似，但它们在体外没有肌动蛋白激活的ATPase活性，也不转移肌动蛋白细丝，这表明它们的功能不需要运动活性。此外，M18A亚型本身不能组装成细丝。然而，M18A亚型在体外和体内确实与M2共同组装形成混合的双极细丝(Billington和比奇等人，Curr.比奥尔。2015年)。这一重要发现表明，M18A亚型可能起到调节M2细丝周转的作用，和/或作为适配器，通过额外的N-末端和C-末端结构域将M2细丝连接到不同的细胞结构/信号分子，所有这些都不会干扰M2的运动活动。M18A在哺乳动物组织中普遍表达，在包括上皮细胞在内的多种细胞类型中均有高表达和异构体特异性表达。在这项研究中，我们使用M18A特异性抗体确定了M18A在极化的MDCK细胞片和各种小鼠上皮冷冻切片中的亚细胞定位。我们发现M18A集中在细胞：极化的MDCK细胞顶面附近的细胞连接，在这个位置，M2被认为是维持粘连连接完整性的关键。使用CRISPR方法，我们生成了M18A空MDCKII线，并测试了成熟的单层屏障功能。M18A缺失细胞的跨上皮阻力和通透性均受到影响。我们还发现，M18A在肾脏近端小管中丰富，并与M2定位于胰腺和唾液腺等分泌组织中的分泌颗粒上。最后，我们发现M18A和M2一起定位于细胞：细胞连接位于肠刷状缘上皮。为了研究M18A在肠道中的功能，我们建立了一个长期的肠道肠样培养系统，这是一个研究上皮细胞增殖、迁移和分化的相关模型。我们现在的重点是M18A如何与M2一起在上皮生理学和动态平衡中的两个保守过程中发挥作用：顶端细胞挤出和动间核迁移。

The mechanisms governing the spatial and temporal control of non-muscle myosin 2 (NM2) filament assembly in living cells are largely unknown. Using EGFP-NM2A knock-in fibroblasts and multiple super-resolution imaging modalities, we describe a sequential amplification mechanism for NM2 filament assembly within lamella wherein individual filaments emanating from single nucleation events continuously partition to form filament clusters that then populate large scale actomyosin structures deeper in the cell. Live, two-color imaging demonstrates that individual partitioning events coincide spatially and temporally with the movements of underlying actin fibers, and inhibition of actin dynamics suppresses partitioning. These and other data indicate that NM2A filaments are partitioned by the dynamic movements of actin fibers to which they are bound. Both partition frequency and the rate of filament growth in the lamella are dependent on MLCK activity. Importantly, we provide direct evidence that the pool of NM2A monomer available for filament assembly is limiting, such that MLCK competes with Rho Kinase acting deeper in the cytoplasm for monomer to drive lamellar filament assembly. Together, our results provide new insights into the mechanism and regulation of NM2 filament assembly in cells. Class 18A myosins (M18A) are a poorly understood class of myosin with domain architecture similar to that of myosin 2 (M2). Specifically, both M18A and M18A, two M18A isoforms generated by alternative splicing, consist of a motor domain followed by a short neck region and an extended coiled-coil domain that drives dimerization. Unlike M2, however, they possess a C-terminal non-helical tailpiece that harbors binding sites for SH3 and PDZ domain-containing proteins. Moreover, M18A also possesses an N-terminal extension containing a KE-rich region, an ATP-insensitive actin-binding site, and a PDZ domain. Knockout of M18A results in embryonic lethality in both mice and flies, suggesting a fundamental role in development. Despite their overall structural similarity to M2, M18A isoforms have no actin-activated ATPase activity and do not translocate actin filaments in vitro, suggesting that their functions do not require motor activity. Moreover, M18A isoforms do not assemble into filaments on their own. M18A isoforms do, however, co-assemble with M2 both in vitro and in vivo to form mixed bipolar filaments (Billington and Beach et al, Curr. Biol. 2015). This critical finding suggests that M18A isoforms may serve to regulate M2 filament turnover and/or act as adaptors to link M2 filaments to different cellular structures/signaling molecules via their extra N- and C-terminal domains, all without interfering with M2 motor activity. M18A is ubiquitously expressed across mammalian tissues, with elevated expression and isoform-specific expression in numerous cell types, including epithelia. In this study we determined the subcellular localization of M18A in polarized MDCK cell sheets and in cryo-sections of various mouse epithelia using an M18A-specific antibody. We find M18A concentrated at cell: cell junctions near the apical surface of polarized MDCK cells, a site where M2 is known to be critical for maintaining the integrity of adherens junctions. Using a CRISPR approach, we generated M18A null MDCKII lines and tested mature monolayers for barrier function. Both trans-epithelial resistance and FLUX were affected in M18A null cells. We also find that M18A is enriched in kidney proximal tubules and localizes with M2 on secretory granules in secretory tissues such as the pancreas and salivary gland. Finally, we find that M18A localizes along with M2 to cell: cell junctions in intestinal brush border epithelium. To investigate M18A function in the gut, we established a long-term intestinal enteroid culture system, a pertinent model to study epithelial cell proliferation, migration, and differentiation. Our focus now is on how M18A may be working together with M2 in two conserved processes in epithelial physiology and homeostasis: apical cell extrusion and interkinetic nuclear migration.