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: 10389828
• 负责人: Gillian Fitz
• 金额: $ 3.2万
• 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 2025-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10389828
• 关键词: Actins; Agammaglobulinaemia tyrosine kinase; Binding; Biochemical; Biological; Biophysics; C-terminal; Cell membrane; Cell physiology; Charge; Crohn' s disease; Cues; 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; Proteins; Research; Role; Structure; Surface; System; Tail; Testing; Transmembrane Domain; Transmission Electron Microscopy; arm; base; 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 在尤卡里亚，以肌动蛋白为基础的突起，如丝状足、立体纤毛和微绒毛，支持各种各样的 包括运动、机械感觉和营养吸收在内的细胞功能。曲面的形成 突起涉及质膜的变形，先前的研究表明肌动蛋白聚合 产生机械力使薄膜向前移动。除了肌动蛋白产生的力之外 聚合、突起的形成也可能受到其他因素的推动。事实上，基于肌动蛋白的力量 肌球蛋白是以肌动蛋白为基础的突起中数量最多的一种。以前的研究 已经表明，这些马达起到了货运载体的作用，将组装所需的关键部件转移到 远端尖端。然而，只有突起中的一些肌球蛋白已知携带货物，而所有的都相互作用 直接或间接地与质膜结合。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 能够施加力量，我们假设肌球蛋白可能具有对 膜的力学性能。事实上，根据我们的初步数据，我们发现了一个 通过对接产生肌球蛋白的力量来促进突起生长的第二条不依赖于货物的途径- 10马达在质膜上。我们假设肌球蛋白对血浆施加倾向力 膜能促进肌动蛋白的组装和突起伸长。为了检验这一假设，我们将确定 肌球蛋白马达是如何通过控制肌球蛋白-10的机械性能来驱动突起伸长的 作为在质膜上对接额外类别的运动域。接下来，我们将定义哪些模式 通过将肌球蛋白-10运动域对接到三个不同的位置来延长膜附着冲击突起 膜附着构造物(即跨膜区、磷脂酰肌醇或负性 膜的内部小叶的电荷)。最后，我们将确定膜相互作用的肌球蛋白是如何 利用传输电子影响丝状肌动蛋白(F-肌动蛋白)组装的生长的带刺末端 显微镜下观察我们系统中形成的突起远端的细胞质隔间。如果 如果成功，这项研究将扩大肌球蛋白作为货物帮助伸长的教条 承运人。这个项目试图了解膜结合的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，这对理解克罗恩病等疾病具有广泛的意义 疾病、耳聋和癌症。