Investigating the role of myosin-based force generation in protrusion formation.

研究基于肌球蛋白的力产生在突起形成中的作用。

• 批准号: 10609802
• 负责人: Gillian Fitz
• 金额: $ 3.07万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-01-01 至 2023-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10609802
• 关键词: Acceleration; Actins; Agammaglobulinaemia tyrosine kinase; Binding; Biochemical; Biological; Biophysics; C-terminal; Cell membrane; Cell physiology; Charge; Crohn' s disease; Cues; Cytoplasm; Data; Disease; Distal; Docking; Electrostatics; Elongation Factor; Exhibits; F-Actin; Filopodia; Fingers; Generations; Growth; Hearing; Immunofluorescence Immunologic; Integral Membrane Protein; Length; Lipid Binding; Maintenance; Malignant Neoplasms; Measures; Membrane; Modeling; Motor; Myosin ATPase; N-terminal; Nature; Nonmuscle Myosin Type IIA; PH Domain; Pathway interactions; Peripheral; Phosphatidylinositols; Plus End of the Actin Filament; Polymers; Proteins; Research; Role; Structure; Surface; System; Tail; Testing; Transmembrane Domain; Transmission Electron Microscopy; Visualization; arm; cell motility; cellular microvillus; deafness; ezrin; in vivo; live cell imaging; mechanical force; mechanical properties; moesin; novel; nutrient absorption; phosphatidylinositol 3,4,5-triphosphate; phosphoinositide-3,4,5-triphosphate; platelet protein P47; polymerization; radixin protein; response; vasodilator-stimulated phosphoprotein

PROJECT SUMMARY/ABSTRACT In Eukarya, actin-based protrusions such as filopodia, stereocilia, and microvilli, support a wide variety of cellular functions including motility, mechanosensation, and nutrient absorption. Formation of a surface protrusion involves deformation of the plasma membrane, and previous studies indicate that actin polymerization produces the mechanical force to displace the membrane forward. In addition to the force generated by actin polymerization, protrusion formation may also be powered by other factors. Indeed, the actin-based force generators, myosins, are some of the most abundant residents of actin-based protrusions. Previous research has shown that these motors function as cargo carriers to move critical components needed for assembly to the distal tips. However, only some of the myosins in protrusions are known to carry cargo, while all interact either directly or indirectly with the plasma membrane. As all myosin motors found in actin-based protrusions are also able to exert force, we hypothesize that myosins likely hold the potential to apply a significant impact on the mechanical properties of the membrane. Indeed, based on our preliminary data we have discovered a secondary, cargo-independent pathway for promoting protrusion growth by docking the force generating myosin- 10 motor on the plasma membrane. We hypothesize that myosins exert tipward forces on the plasma membrane to promote actin assembly and protrusion elongation. To test this hypothesize, we will determine how myosin motors drive protrusion elongation by manipulating the mechanical properties of myosin-10, as well as docking additional classes of motor domains on the plasma membrane. Next we will define which modes of membrane attachment impact protrusions elongation by docking the myosin-10 motor domain to three different membrane attachment constructs (i.e. a transmembrane domain, a phosphatidylinositol, or to the negative charge of the inner leaflet of the membrane). Lastly, we will determine how membrane-interacting myosins impact the growing, barbed-ends of filamentous actin (F-actin) assembly by using transmission electron microscopy to visualize the cytoplasmic compartment of the distal tips of protrusions formed in our system. If successful, this research will expand the dogma that myosins assist in protrusion elongation by acting as cargo carriers. This project seeks to understand how membrane-bound myosins drive actin-based protrusions formation in diverse biological settings, which has broad implications for understanding diseases such as Crohn’s disease, deafness, and cancer.

项目摘要/摘要 在Eukarya，基于肌动蛋白的突起，例如丝状，立体尾肌和微绒毛，支持多种多样 细胞功能，包括运动性，机械性和营养滥用。表面的形成 突出涉及质膜的变形，以前的研究表明肌动蛋白聚合 产生机械力来置换膜的前部。除了肌动蛋白产生的力 聚合，蛋白质形成也可能由其他因素提供动力。确实，基于肌动蛋白的力量 发电机，肌球蛋白是基于肌动蛋白的突起的一些最丰富的居民。先前的研究 已经表明，这些电动机是将组装所需的关键组件移动到的货物载体 远端技巧。但是，众所周知，仅突起的某些肌动物可以携带货物，而全部相互作用 直接或间接使用质膜。正如所有在基于肌动蛋白的蛋白中发现的肌球蛋白电动机一样 能够发挥力，我们假设肌球蛋白可能有可能对 膜的机械性能。确实，根据我们的初步数据，我们发现了一个 次要的，无货物的途径，用于通过对接产生肌球蛋白的力来促进突出生长 质膜上的10马达。我们假设肌动物在等离子体上施加尖端力 促进肌动蛋白组装和突出伸长的膜。为了测试这一假设，我们将确定 肌球蛋白电动机如何通过操纵肌球蛋白10的机械性能来驱动蛋白质伸长 随着质膜上的其他类别的电机域对接。接下来我们将定义哪些模式 通过将肌球蛋白10电机域停靠到三个不同的 膜附着构建体（即跨膜结构域，磷脂酰肌醇或阴性 膜内部小叶的电荷）。最后，我们将确定膜相互作用的肌球蛋白 通过使用传输电子来影响丝状肌动蛋白（F-肌动蛋白）组装的刺骨末端 显微镜以可视化系统中形成的蛋白质远端尖端的细胞质室。如果 成功的这项研究将扩大肌动物的教条 载体。该项目旨在了解膜结合的肌球蛋白如何驱动基于肌动蛋白的蛋白 潜水员生物环境中的形成，这对理解克罗恩等疾病具有广泛的影响 疾病，死亡和癌症。