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 微管（MT）的正确组织对于神经回路的发育和功能至关重要。MT表格 分子马达驱动蛋白和动力蛋白将货物运输到特定细胞位置的轨道。的 MT的固有正端/负端极性指导马达运输。在轴突中，MT与它们的正 末端远离细胞体。MT极性必须在最初的轴突形成过程中建立， 分支事件调节轴突和分支中MT极性的机制知之甚少，但 大量的证据表明，分子马达是至关重要的MT极性。驱动蛋白和动力蛋白有助于 MT极性，并假设通过MT的运输发挥作用。然而，可视化MT聚合物转运是 具有挑战性，并且仅在体外或培养的神经元中进行。 我们的实验室以前表明，Clstn 1，驱动蛋白-1适配器，调节感觉神经元中的轴突分支。我们 初步数据显示，Clstn 1也是轴突MT极性所需的。一个已知的驱动蛋白适配器 主要介导货物运输，也调节MT极性是未知的。我的目标是揭示 机制等已知Clstn 1结合并激活KLC，KLC激活KHC和驱动蛋白运动活性， 抑制KHC尾部与MT结合。我假设Clstn 1激活KLC阻止了过度的MT 通过KHC尾-MT结合进行交联。在缺乏Clstn 1的情况下，这种交联可能会对抗动力蛋白的能力， 从轴突上去除定向错误的MT，导致错误极化的MT增加。我将使用高速在 体内4D成像方法来测试这些假设。 在目标1中，我将首先测试使用Clstn 1 W-酸性结构域肽激活KLC是否足以拯救MT Clstn 1-/-突变体的极性。其次，我将使用两种方法来破坏KHC尾与MT的结合， 减少KHC与MT的交联，并测试MT极性Clstn 1-/-突变体的拯救。最后，我将测试角色 KLC磷酸化和Clstn 1蛋白水解裂解的MT极性。 在目标2中，我建议与光学和光学实验室合作使用先进的成像技术。 计算仪器成像MT动态在体内。我将标记MT聚合物和正/负末端 与多个荧光团，并使用光漂白后的荧光恢复，以确定是否MT 转运有助于体内的极性。我将测试Clstn 1丢失是否会影响MT传输，从而 影响极性。我还将检验Clstn 1在轴突生长过程中组织MT极性的假设。 通过在分支点成像MT动力学来分支。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