Organization of Microtubule Polarity During Neuronal Axon Development in vivo

体内神经元轴突发育过程中微管极性的组织

• 批准号: 10267658
• 负责人: Elizabeth Haynes
• 金额: $ 2.08万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-06-01 至 2020-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10267658
• 关键词: 4D Imaging; Adaptor Signaling Protein; Address; Affect; Afferent Neurons; Axon; Binding; Biological; Brain; Cells; Collaborations; Data; Defect; Dendrites; Development; Distal; Dynein ATPase; Embryo; Event; Fluorescence Recovery After Photobleaching; Goals; Human; Image; Imaging technology; In Vitro; Kinesin; Label; Laboratories; Light; Location; Mediating; Membrane; Microtubules; Molecular; Molecular Motors; Motor; Motor Activity; Movement; Nature; Neurons; Neurophysiology - biologic function; Optics; Phosphorylation; Photobleaching; Plus End of the Microtubule; Polymers; Regulation; Resolution; Role; Sensory; Site; Speed; System; Tail; Tertiary Protein Structure; Testing; Tubulin; Work; Zebrafish; axon growth; brain health; crosslink; developmental disease; experimental study; fluorophore; imaging approach; in vivo; in vivo imaging; insight; instrumentation; mutant; nervous system disorder; neural circuit; neurite growth; neuron development; novel; prevent; spectrograph; trafficking

PROJECT SUMMARY Proper organization of microtubules (MTs) is critical for development and function of neural circuits. MTs form the tracks on which the molecular motors kinesin and dynein transport cargo to specific cell locations. The inherent plus end/minus end polarity of MTs directs motor transport. In axons MTs are arranged with their plus ends distal to the cell body. MT polarity must be established during initial axon formation, and also during axon branching events. The mechanisms regulating MT polarity in axons and branches are poorly understood, but a body of evidence suggests that molecular motors are crucial to MT polarity. Kinesin and dynein contribute to MT polarity and are hypothesized to act by transport of MTs. However, visualizing MT polymer transport is challenging and has only been done in vitro or cultured neurons. Our lab showed previously that Clstn1, a kinesin-1 adaptor, regulates axon branching in sensory neurons. Our preliminary data show that Clstn1 is also required for MT polarity in axons. How a kinesin adaptor, known primarily to mediate cargo transport, also regulates MT polarity is unknown. My goal is to reveal the underlying mechanisms. Clstn1 is known to bind and activate KLC, which activates KHC and kinesin motor activity, and inhibits KHC tail binding to MTs. I hypothesize that Clstn1 activation of KLC prevents excessive MT crosslinking by KHC tail-MT binding. In the absence of Clstn1, this crosslinking may oppose dynein’s ability to remove misoriented MTs from the axon, leading to an increase in mispolarized MTs. I will use high-speed in vivo 4D imaging approaches to test these hypotheses. In Aim 1, I will first test if activation of KLC using a Clstn1 W-acidic domain peptide is sufficient to rescue MT polarity in Clstn1-/- mutants. Second, I will use two approaches to disrupt the binding of the KHC tail to MTs to reduce KHC crosslinking to MTs, and test for rescue of MT polarity Clstn1-/- mutants. Finally, I will test the roles of KLC phosphorylation and proteolytic cleavage of Clstn1 in MT polarity. In Aim 2 I propose to use advanced imaging technologies in collaboration with the Laboratory of Optical and Computational Instrumentation to image MT dynamics in vivo. I will label MT polymers and plus/minus ends with multiple fluorophores, and use fluorescence recovery after photobleaching to determine whether MT transport contributes to polarity in vivo. I will test whether Clstn1 loss affects MT transport, and thereby influences polarity. I will also test the hypothesis that Clstn1 functions to organize MT polarity during axon branching by imaging MT dynamics at branch points. These experiments will enhance our understanding of the cellular and molecular mechanisms that establish axon MT polarity.

项目摘要 正确组织微管（MTS）对于神经回路的发展和功能至关重要。 MTS形式 分子电动机动力蛋白和动力蛋白传输到特定细胞位置的轨道。这 MTS的固有加上末端/减去末端极性指导电动机传输。在轴突中，MT与他们的加 末端到细胞体远端。 MT极性必须在初始轴突形成期间以及轴突期间建立 分支事件。轴突和分支中调节MT极性的机制知之甚少，但是 证据表明，分子电动机对MT极性至关重要。动力蛋白和动力蛋白有助于 MT极性，并假设通过MT的运输来起作用。但是，可视化MT聚合物传输是 具有挑战性，仅在体外或培养的神经元中完成。 我们的实验室以前表明，旋转蛋白-1衔接子ClstN1调节感觉神经元中的轴突分支。我们的 初步数据表明，轴突中MT极性也需要CLSTN1。风机适配器如何已知 主要到介导货物运输，也调节MT极性尚不清楚。我的目标是揭示基础 机制。众所周知，CLSTN1会结合和激活KLC，该KLC激活KHC和动力蛋白运动活性，并且 抑制KHC尾巴与MTS结合。我假设KLC激活CLSTN1可以防止多余的MT 通过KHC Tail-MT结合进行交联。在没有Clstn1的情况下，这种交联可能反对Dynein的能力 从轴突中去除不良导向的MT，导致MT偏振的增加。我将使用高速 Vivo 4D成像方法检验这些假设。 在AIM 1中，我将首先测试如果使用Clstn1 W-酸性结构型肽的KLC激活足以营救MT Clstn1 - / - 突变体中的极性。其次，我将使用两种方法破坏KHC尾巴与MTS的结合 将KHC的交联降低到MTS，并测试MT极性Clstn1 - / - 突变体。最后，我将测试角色 MT极性中CLSTN1的KLC磷酸化和蛋白水解裂解 在AIM 2中，我建议使用高级成像技术与光学实验室合作 计算仪器在体内图像MT动力学。我将标记MT聚合物和加/减 使用多个荧光团，并在光漂白后使用荧光回收来确定MT是否 运输有助于体内极性。我将测试CLSTN1损失是否影响MT运输，从而 影响极性。我还将检验以下假设：Clstn1在轴突期间起作用MT极性 通过成像分支点的分支在分支点。这些实验将增强我们对 建立轴突MT极性的细胞和分子机制。

项目成果

KLC4 shapes axon arbors during development and mediates adult behavior.

• DOI: 10.7554/elife.74270
• 发表时间: 2022-10-12
• 期刊: eLife
• 影响因子: 7.7
• 作者: Haynes EM;Burnett KH;He J;Jean-Pierre MW;Jarzyna M;Eliceiri KW;Huisken J;Halloran MC
• 通讯作者: Halloran MC