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.

项目总结/摘要 在真核生物中，以肌动蛋白为基础的突起，如丝状伪足、静纤毛和微绒毛， 包括运动、机械感觉和营养吸收的细胞功能。形成表面 突起涉及质膜的变形，先前的研究表明，肌动蛋白聚合 产生机械力使膜向前移动。除了肌动蛋白产生的力之外 除了聚合之外，突起形成还可以由其他因素驱动。事实上， 肌球蛋白是肌动蛋白基突起的最丰富的居民。以前的研究 已经表明，这些发动机的功能是作为货物载体，将组装所需的关键部件移动到 远端尖端。然而，只有一些突起中的肌球蛋白已知携带货物，而所有的相互作用， 直接或间接与质膜结合。由于肌动蛋白为基础的突起中发现的所有肌球蛋白马达也 能够施加力，我们假设肌球蛋白可能具有对肌球蛋白产生重大影响的潜力。 膜的机械性能。事实上，根据我们的初步数据，我们发现了一个 第二，货物独立的途径，促进突起的生长，通过对接的力量产生肌球蛋白， 10马达在质膜上。我们假设肌球蛋白对血浆施加尖端力 膜，以促进肌动蛋白组装和突起伸长。为了验证这一假设，我们将确定 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延长，以及 在质膜上对接其他类型的马达结构域。接下来，我们将定义哪些模式 膜附着通过对接肌球蛋白-10马达结构域与三种不同的 膜附着构建体（即，跨膜结构域、磷脂酰肌醇，或负性的 膜的内小叶的电荷）。最后，我们将确定如何膜相互作用肌球蛋白 利用透射电子轰击丝状肌动蛋白（F-actin）组装体不断生长的倒刺末端， 在显微镜下观察在我们的系统中形成的突起的远端尖端的细胞质隔室。如果 这项研究的成功，将扩大肌球蛋白作为货物协助突出伸长的教条 载波这个项目旨在了解膜结合肌球蛋白如何驱动肌动蛋白为基础的突起 在不同的生物环境中形成，这对理解克罗恩病等疾病具有广泛的意义， 疾病耳聋和癌症