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.

项目概要/摘要 在真核生物中，基于肌动蛋白的突起，如丝状伪足、静纤毛和微绒毛，支持多种 细胞功能，包括运动、机械感觉和营养吸收的形成。 突出涉及质膜的变形，之前的研究表明肌动蛋白聚合 除了肌动蛋白产生的力外，还产生使膜向前移动的机械力。 事实上，基于肌动蛋白的力也可能由其他因素驱动。 肌球蛋白是肌动蛋白突起中最丰富的居民之一。 已经表明，这些电机可以充当货物运输工具，将组装所需的关键部件移动到 然而，仅已知突起中的一些肌球蛋白携带货物，而所有肌球蛋白都相互作用。 因为在基于肌动蛋白的突起中发现的所有肌球蛋白马达也直接或间接地与质膜相关。 能够施加力量，我们认为肌球蛋白可能具有对 事实上，根据我们的初步数据，我们发现了膜的机械特性。 通过对接产生肌球蛋白的力来促进突起生长的次要的、与货物无关的途径 10 质膜上的马达 我们勇敢地承认肌球蛋白对质膜施加了倾斜的力。 为了测试这是否有帮助，我们将确定膜促进肌动蛋白组装和突出伸长。 肌球蛋白马达如何通过操纵肌球蛋白-10 的机械特性来驱动突出伸长 作为在质膜上对接其他类别的运动域，接下来我们将定义哪些模式。 通过将肌球蛋白 10 运动域对接至三个不同的膜附着物影响突起伸长 膜附着结构（即跨膜结构域、磷脂酰肌醇或负 最后，我们将确定膜如何与肌球蛋白相互作用。 使用传输电子影响丝状肌动蛋白 (F-肌动蛋白) 组装的生长、带刺末端 显微镜观察我们系统中形成的突起远端的细胞质区室。 成功后，这项研究将扩展肌球蛋白通过充当货物来协助突出伸长的教条 该项目旨在了解膜结合肌球蛋白如何驱动基于肌动蛋白的突起。 在不同的生物环境中形成，这对于理解克罗恩病等疾病具有广泛的意义 疾病、耳聋和癌症。