ACTIN-BASED MOTILITY BY CLAMPED-FILAMENT MOTORS

钳位灯丝电机基于肌动蛋白的运动

• 批准号: 6731384
• 负责人: Daniel Lee Purich
• 金额: $ 24.18万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-04-01 至 2007-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6731384
• 关键词: actins; adenosine triphosphate; adenosinetriphosphatase; biomechanics; cell motility; enzyme mechanism; enzyme model; fluorescent dye /probe; hydrolysis; microfilaments; nucleotide analog; protein protein interaction; protein structure function

DESCRIPTION (provided by applicant): Our recently introduced actoclampin motor model describes how force is generated in actin-based motility through clamped-filament elongation. We proposed a three-step mechanoenzymatic process (called the "Lock, Load & Fire" or "LLF" mechanism): during locking, an affinity-modulated clamp protein (also tethered to the surface of a motile object) binds onto ATP-containing actin monomers situated at a filament's barbed-end; in the loading step, new actin ATP monomers bind onto the barbed end of a clamped-filament, an event that triggers the firing step, whereto ATP hydrolysis on the clamped actin subunit greatly attenuates the clamp's affinity for the filament. ATP hydrolysis initiates clamp translocation and re-locking to the new ATP-containing terminus, starting another LLF cycle. This model explains how surface-tethered filaments can grow while exerting flexural or tensile force on the motile surface, and stochastic simulations reproduce the signature motions of motile Listeria. This elongation motor exploits actin's intrinsic ATPase activity to provide a simple, high fidelity enzymatic reaction cycle for force production that does not require elongating filaments to dissociate from the motile surface. Our research proposal addresses model-based hypotheses designed to test specific features of the LLF mechanism of actin polymerization motors. Specific Aim-1 includes experiments aimed at differentiating the LLF model from Brownian Ratchet-type mechanisms (a) by conducting motility studies near and below the (+)-end critical concentration, and (b) by using covalently cross-linked profilin-actin that cannot release profilin after each LLF cycle. Specific Aim-2 focuses on (a) the role of ATP hydrolysis in motility using slowly hydrolyzing ATP analogues pp(NH)pA and ATPgammaS to identify the likely force-producing step(s) in the LLF mechanism. Specific Aim-3 deals with profilin's potential role in a kinetic proofreading pathway to suppress loading of actin-ADP into clamped-filament motors. In Specific Aim-4, we will apply sedimentation and fluorescence anisotropy measurements to learn if and how ATP hydrolysis modulates the strength of binding interactions of VASP's clamping domain with the ends of actin filaments. Together, these investigations promise to shed new light on actin-based motility by testing fundamental mechanistic properties.

描述（由申请人提供）：我们最近引入的actoclampin运动模型描述了如何通过夹紧丝伸长在基于肌动蛋白的运动中产生力。我们提出了一个三步机械酶过程（称为“锁定、加载和激发”或“LLF”机制）：在锁定过程中，亲和力调节的钳蛋白（也束缚在运动物体的表面）结合到含有 ATP 的物质上。肌动蛋白单体位于细丝的倒刺末端；在加载步骤中，新的肌动蛋白 ATP 单体结合到夹紧丝的有刺末端，这一事件触发发射步骤，其中夹紧肌动蛋白亚基上的 ATP 水解大大减弱了夹具对丝的亲和力。 ATP 水解引发钳位易位并重新锁定到新的含 ATP 末端，开始另一个 LLF 循环。该模型解释了表面系留的细丝如何在对运动表面施加弯曲或拉伸力的同时生长，并且随机模拟再现了运动李斯特菌的特征运动。这种伸长马达利用肌动蛋白固有的 ATP 酶活性，为产生力提供简单、高保真度的酶反应循环，不需要伸长丝从运动表面解离。我们的研究提案提出了基于模型的假设，旨在测试肌动蛋白聚合马达 LLF 机制的特定特征。具体的 Aim-1 包括旨在区分 LLF 模型与布朗棘轮型机制的实验 (a) 通过在 (+) 端临界浓度附近和以下进行运动研究，以及 (b) 通过使用共价交联的肌动蛋白-肌动蛋白在每个 LLF 周期后不能释放 profilin。具体目标 2 侧重于 (a) ATP 水解在运动中的作用，使用缓慢水解 ATP 类似物 pp(NH)pA 和 ATPgammaS 来识别 LLF 机制中可能的力产生步骤。具体的 Aim-3 涉及 profilin 在动力学校对途径中的潜在作用，以抑制肌动蛋白-ADP 加载到夹紧丝马达中。在 Specific Aim-4 中，我们将应用沉降和荧光各向异性测量来了解 ATP 水解是否以及如何调节 VASP 夹紧结构域与肌动蛋白丝末端的结合相互作用的强度。总之，这些研究有望通过测试基本的机械特性，为基于肌动蛋白的运动提供新的线索。