Structure And Function of Convential and Unconventional Myosins

常规和非常规肌球蛋白的结构和功能

• 批准号: 9354304
• 负责人: JOHN A HAMMER
• 金额: $ 59.61万
• 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/9354304
• 关键词: Actins; Actomyosin; Address; Adherens Junction; Affect; Alternative Splicing; Amoeba genus; Antibodies; Apical; Architecture; Binding; Binding Sites; Biochemistry; Biological; Biological Models; Brush Border; C-terminal; Cell physiology; Cells; Cellular Structures; Clustered Regularly Interspaced Short Palindromic Repeats; Coiled-Coil Domain; Color; Cytoplasm; Data; Development; Dictyostelium; Dimerization; Embryo; Epithelial; Epithelial Cell Proliferation; Epithelial Cells; Epithelial Physiology; Epithelium; Event; Fiber; Fibroblasts; Filament; Frequencies; Goals; Growth; Homeostasis; Image; In Vitro; Individual; Intercellular Junctions; Intestines; Knock-in; Knock-out; Life; Link; Lymphocyte; MDCK cell; MYLK gene; Microfilaments; Modeling; Motor; Motor Activity; Movement; Mus; Myosin ATPase; N-terminal; Neck; Neurons; Nuclear; Null Lymphocytes; Pancreas; Process; Protein Isoforms; Proteins; Proximal Kidney Tubules; Regulation; Resistance; Resolution; Rho-associated kinase; Role; Salivary Glands; Secretory Vesicles; Signal Pathway; Signaling Molecule; Site; Structure; Surface; System; Testing; Tissues; Work; cell growth regulation; cell type; fly; imaging modality; in vivo; insight; melanocyte; migration; monolayer; monomer; mutant; non-muscle myosin; single molecule; structural biology

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.

非肌肉肌球蛋白2（NM 2）细丝组装在活细胞中的空间和时间控制机制在很大程度上是未知的。使用EGFP-NM 2A敲入成纤维细胞和多种超分辨率成像模式，我们描述了一种连续扩增机制，用于板层内的NM 2细丝组装，其中从单个成核事件发出的单个细丝连续分区以形成细丝簇，然后在细胞更深处填充大规模肌动球蛋白结构。活的，双色成像表明，个别分区事件在空间和时间上与底层肌动蛋白纤维的运动相一致，肌动蛋白动力学抑制分区。这些和其他数据表明，NM 2A丝被它们所结合的肌动蛋白纤维的动态运动所分割。分配频率和丝状体生长速率都依赖于MLCK活性。重要的是，我们提供了直接的证据，即可用于细丝组装的匪2A单体库是有限的，使得MLCK与Rho激酶竞争在细胞质中更深处作用的单体以驱动层状细丝组装。总之，我们的研究结果为细胞中NM 2细丝组装的机制和调控提供了新的见解。 18 A类肌球蛋白（M18 A）是一类了解甚少的肌球蛋白，其结构域结构与肌球蛋白2（M2）相似。具体而言，M18 A和M18 A（通过选择性剪接产生的两种M18 A同种型）均由马达结构域、随后的短颈区和驱动二聚化的延伸卷曲螺旋结构域组成。然而，与M2不同的是，它们具有C末端非螺旋尾部，其中含有SH 3和含PDZ结构域的蛋白质的结合位点。此外，M18 A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。敲除M18 A导致小鼠和苍蝇的胚胎死亡，表明在发育中的基本作用。尽管它们与M2的整体结构相似，但M18 A亚型没有肌动蛋白激活的ATP酶活性，并且在体外不移位肌动蛋白丝，这表明它们的功能不需要运动活性。此外，M18 A同种型本身不会组装成细丝。 然而，M18 A同种型确实在体外和体内与M2共组装以形成混合双极细丝（Billington和海滩等人，Curr. 2015）。这一关键发现表明，M18 A亚型可能用于调节M2细丝周转和/或作为衔接子，通过其额外的N-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运动活性。M18 A在哺乳动物组织中广泛表达，在许多细胞类型（包括上皮细胞）中表达升高和亚型特异性表达。在这项研究中，我们确定了亚细胞定位M18 A在极化MDCK细胞片和冷冻切片的各种小鼠上皮细胞使用M18 A特异性抗体。我们发现M18 A集中在极化MDCK细胞顶端表面附近的细胞：细胞连接处，已知M2对维持粘附连接的完整性至关重要。使用CRISPR方法，我们生成了M18 A无效MDCKII系，并测试了成熟单层的屏障功能。在M18 A无效细胞中，跨上皮电阻和FLUX均受到影响。我们还发现，M18 A是丰富的肾脏近端小管和M2定位在分泌组织，如胰腺和唾液腺的分泌颗粒。最后，我们发现，M18 A定位沿着与M2的细胞：细胞连接在肠道刷状缘上皮。为了研究M18 A在肠道中的功能，我们建立了一个长期的肠上皮细胞培养系统，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M18 A如何与M2在上皮生理学和稳态中的两个保守过程中一起工作：顶端细胞挤出和核迁移。