Microtubule Actin Interactions In Cell Motility

细胞运动中微管肌动蛋白的相互作用

• 批准号: 8557948
• 负责人: Clare Michal Waterman
• 金额: $ 52.23万
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8557948
• 关键词: 3-Dimensional; ATP phosphohydrolase; Actins; Actomyosin; Affect; Affinity; Area; Automobile Driving; Binding Proteins; Biological Assay; Biological Sciences; Caenorhabditis elegans; Categories; Cell Polarity; Cells; Cellular Morphology; Centromere; Complex; Computer software; Cytoskeleton; Data; Defect; Down-Regulation; Endothelial Cells; Ensure; Event; Exhibits; Extracellular Matrix; F-Actin; Family; Feedback; Frequencies; Goals; Growth; Guanosine Triphosphate Phosphohydrolases; Human; Image; Interphase; Kinesin; Manuscripts; Mediating; Methods; Microtubule Depolymerization; Microtubule Polymerization; Microtubule-Associated Proteins; Microtubules; Mitosis; Mitotic; Molecular; Monomeric GTP-Binding Proteins; Morphogenesis; Motion; Occupations; Pathway interactions; Philadelphia; Phospho-Specific Antibodies; Phosphorylation; Phosphotransferases; Plus End of the Microtubule; Polymers; Positioning Attribute; Process; Proteins; Publications; Publishing; RNA Interference; Regulation; Research Personnel; Role; Science; Signal Pathway; Signal Transduction; Singapore; Small Interfering RNA; Speed; Stimulus; System; Testing; Time; Tissues; Tubulin; United States National Institutes of Health; Universities; Variant; angiogenesis; aurora kinase; aurora-A kinase; base; cell behavior; cell motility; computer program; crosslink; directional cell; extracellular; genetic regulatory protein; human STK6 protein; migration; movie; mutant; neuronal cell body; new technology; novel; osteosarcoma; polarized cell; polymerization; research study; response; rho; rho GTP-Binding Proteins; two-dimensional; volunteer

Project 1: An RNAi screen of microtubule-regulatory proteins identifies MARK2/Par1 as an effector of Rac1-mediated microtubule growth. Yukako Nishimura, Kathryn Applegate, Gaudenz Danuser, Clare Waterman Proper regulation of microtubule (MT) assembly dynamics is essential for directed cell migration. Microtubule dynamics in migrating cells are spatially regulated by Rho GTPases. We have previously shown that activated Rac1 induces MT net growth by suppressing catastrophe and increasing growth velocity, and that Rac1 activity is required for polarized MT growth in the leading edge of migrating cells. We identified a necessary, but not sufficient PAK kinase-mediated pathway downstream of Rac1 that promoted MT growth. Therefore, we hypothesized that additional factors promote MT net growth downstream of Rac1. To find these factors, we performed a RNAi screen in human U2OS osteosarcoma cells to determine if known MT-regulatory proteins were required for constitutively activated Rac1 promotion of MT growth. To analyze MT dynamics, we imaged fluorescent-tagged EB3, a MT plus-end binding protein that serves as a probe for the position of MT ends, and tracked the motion of EB3 comets in time-lapse movies using an automated computer program. Our results indicate that depletions of several MT-binding proteins change the growth rate of MT in activated Rac1-expressing cells. We have focused on MARK2, a microtubule affinity-regulating kinase homologous to the C. elegans polarity protein Par1, whose depletion reduces the number of elongated MTs in the leading edge of Rac1-activated cells. We are currently testing how MARK2 is involved in promoting MT growth downstream of Rac1 and it requirement in cell migration. A manuscript describing these results was published in Plos One in 2012.Dr Nishimura moved on to a position at the National University of Singapore Project 2: MCAK Activity Controls Interphase Microtubule Dynamics and Directed Cell Migration. Myers, K.A.; Applegate, K; Danuser G.; and Waterman, C.M. Directional cell migration is initiated through extracellular stimuli that coordinate changes in the cytoskeleton to establish a polarized cellular morphology. Cell polarity can be achieved through regional regulation of microtubule (MT) dynamics, including MT growth toward the leading edge and MT shortening in the cell rear. Mitotic Centromere Associated Kinesin (MCAK) is a MT depolymerase that is down-regulated in mitosis by Aurora kinase phosphorylation. While its mitotic functions have been well-characterized, whether MCAK regulates MT dynamics during cell migration is not known. We hypothesize that MCAK is down-regulated locally via a Rac1/Pak1/Aurora-A kinase signaling pathway to establish preferential MT growth toward the leading edge and to promote MT shortening within the cell rear. To test this hypothesis, we performed time-lapse imaging of fluorescently tagged EB3 as a marker of MT plus end growth in HUVEC cells and analyzed MT dynamics and cell behavior under different manipulations of the proposed signaling cascade. We find that MCAK knockdown (KD) produces expected effects on the MT cytoskeleton, including increased levels of tubulin polymer and decreased MT catastrophe frequency. MCAK-KD cells show a reduction in MT polymerization speeds and exhibit a mal-oriented MT array, as well as a statistically significant reduction in cell migration velocity, directional persistence, and distance to origin, indicating a defect in cell migration and/or polarization. These effects are rescued through expression of exogenous wild-type-MCAK, but not by expression of either an inactive (ATPase-dead) MCAK mutant or an MCAK mutant that is incapable of phospho-regulation by Aurora-A kinase. Immunolabeling of cells expressing either constitutively active-Rac1 or constitutively active-Pak1 suggests that Rac1 and Pak1 activities correlate with increased Aurora-A activity, as assayed with a phospho-specific antibody, and also correlate with decreased levels of MCAK expression. These data suggest that interphase regulation of MCAK is achieved downstream of a Rac1/Pak1/Aurora-A signaling pathway in order to locally coordinate MCAK-mediated MT depolymerization as a method to ensure proper cell polarization and motility. A manuscript describing these studies is being prepared for publication. Dr Myers received a job as a tenure-track investigator at the University of the Sciences in Philadelphia and will finis this manuscrips as an NIH special volunteer. Project 3: Regulation of microtubules in migrating endothelial cells in 3D ECMs Ken Myers, Kathryn Applegate, Gaudenz Danuser, Robert Fischer, Clare Waterman Cytoskeletal dynamics driving endothelial cell (EC) branching morphogenesis during angiogenesis are thought to be regulated in part by cellular signals elicited in response to compliance and topology of the extracellular matrix (ECM) via a process termed mechanosensing. We hypothesized that ECM mechanosensing of compliance or topology (2 dimensional vs 3 dimensional ECMs, i.e. ECM dimensionality) could elicit different responses of the microtubule (MT) cytoskeleton to mediate EC branching morphogenesis. To test this, we used novel MT end-tracking software to analyze spatial variations in MT dynamics in ECs in 2D and 3D compliance-controlled ECMs during branching morphogenesis. Pharmacological inhibition showed that MT dynamics negatively regulate EC branching, independent of ECM compliance or dimensionality. Analyzing MT dynamics incompliant 2D and 3D ECMs with or without myosinII inhibition indicated that myosinII down-regulation by compliance mechanosensing promotes fast MT assembly, but we found dimensionality-specific effects on MT growth persistence. Comparing MT dynamics in cell bodies versus cell branches in in 2D and 3D ECMs of varying stiffness revealed faster, more dynamically unstable MT growth in EC bodies and slower, more persistent MT growth in EC branches. Thus, distinct compliance and dimensionality ECM mechanosensing pathways regionally regulate MT dynamics in ECs to guide branching morphogenesis in physically complex ECMs. A manuscript describing these results was published in the JCB.

项目1：微管调节蛋白的RNAi屏幕将Mark2/PAR1识别为Rac1介导的微管生长的效应子。 Yukako Nishimura，Kathryn Applegate，Gaudenz Danuser，Clare Waterman 适当调节微管（MT）组装动力学对于定向细胞迁移至关重要。迁移细胞中的微管动力学在空间上由Rho GTPase进行空间调节。我们先前已经表明，激活的Rac1通过抑制灾难并增加生长速度来诱导MT净生长，并且Rac1活性是迁移细胞前缘的极化MT生长所必需的。我们确定了促进MT生长的Rac1下游的必要但没有足够的PAK激酶介导的途径。因此，我们假设其他因素促进了Rac1下游MT净生长。为了找到这些因素，我们在人U2OS骨肉瘤细胞中进行了RNAi筛选，以确定是否需要已知的MT调节蛋白来组成型激活的Rac1促进MT生长。为了分析MT动力学，我们成像了荧光标记的EB3，这是一种MT加末端结合蛋白，可作为MT末端位置的探针，并使用自动化计算机程序在延时电影中跟踪EB3彗星的运动。 我们的结果表明，几种MT结合蛋白的耗竭改变了激活Rac1的细胞中MT的生长速率。我们专注于Mark2，Mark2是一种与秀丽隐杆线虫极性蛋白PAR1同源的微管亲和力调节酶，其耗竭减少了Rac1激活细胞前缘的细长MT数量。我们目前正在测试Mark2如何参与Rac1下游促进MT增长及其在细胞迁移中的需求。 一份描述这些结果的手稿于2012年在PLOS One发表。 项目2：MCAK活动控制相间微管动力学和定向细胞迁移。 迈尔斯，K.A。； Applegate，K； Danuser G。；和沃特曼（C.M.） 定向细胞的迁移是通过细胞外刺激启动的，该刺激坐落在细胞骨架中的变化以建立极化的细胞形态。细胞极性可以通过微管（MT）动力学的区域调节，包括MT向前缘的MT生长和细胞后部的MT缩短。有丝分裂的丝粒相关驱动蛋白（MCAK）是一种MT解聚酶，在Aurora激酶磷酸化中被丝分裂下调。尽管它的有丝分裂功能已经很好地表征，但MCAK是否在细胞迁移过程中调节MT动力学。我们假设MCAK通过RAC1/PAK1/Aurora-A激酶信号传导途径在本地下调，以建立向前沿的优先MT生长，并促进细胞后部内MT缩短。为了检验这一假设，我们对荧光标记的EB3进行了延时成像，作为HUVEC细胞中MT Plus最终生长的标记，并在所提出的信号级联的不同操纵下分析了MT动力学和细胞行为。我们发现MCAK敲低（KD）会对MT细胞骨架产生预期影响，包括升高小管蛋白聚合物和MT灾难频率降低。 MCAK-KD细胞显示出MT聚合速度的降低，表现出面向MAL的MT阵列，以及细胞迁移速度，方向持久性和起源距离的统计学显着降低，表明细胞迁移和/或偏置的缺陷。这些作用是通过表达外源野生型-MCAK的表达来挽救的，而不是通过表达非活性（ATPASE DEAD）MCAK突变体或MCAK突变体的表达，而MCAK突变体无法通过Aurora-A激酶进行磷酸化调节。表达组成性活性-RAC1或组成性活性PAK1的细胞的免疫标记表明，RAC1和PAK1活性与Aurora-A活性的增加相关，并与磷酸化特异性抗体进行了测定，并且与MCAK表达水平降低相关。这些数据表明，在Rac1/Pak1/Aurora-A信号通路的下游实现MCAK的相间调节，以便局部协调MCAK介导的MT脱聚合，以确保正确的细胞极化和运动性。描述这些研究的手稿正在准备出版。 迈尔斯博士在费城科学大学担任终身调查员的工作，并将作为NIH特别志愿者罚款。 项目3：在3D ECM中迁移的内皮细胞中微管的调节 Ken Myers，Kathryn Applegate，Gaudenz Danuser，Robert Fischer，Clare Waterman 驱动内皮细胞（EC）在血管生成过程中驱动内皮细胞（EC）分支形态发生的细胞骨架动力学被认为部分受到细胞信号的响应和细胞外基质（ECM）拓扑的响应，并通过称为机械感应的过程。 我们假设依从性或拓扑的ECM机械连敏（2维vs 3维ECM，即ECM维度）可能会引起微管（MT）细胞骨架的不同反应以介导EC分支分支形态发生。为了测试这一点，我们使用了新型MT端跟踪软件来分析分支形态发生过程中2D和3D合规性控制的ECM的ECS中MT动力学的空间变化。药理学抑制作用表明，MT动态对EC分支负面调节，而与ECM依从性或维度无关。分析有或没有肌性抑制的2D和3D ECM的MT动力学表明，通过合规机械效率通过合规性机械效率下调肌动菌会促进快速MT组装，但我们发现特定于MT生长持久性的维度特异性影响。 比较细胞体的MT动态与2D和3D ECM中的细胞分支的变化刚度的速度更快，在EC体中动态不稳定的MT生长更加动态不稳定，并且EC分支中的MT增长更加持久。因此，不同的合规性和维度ECM机械感应途径在区域调节EC中的MT动力学，以指导物理复杂的ECM中的形态发生。 描述这些结果的手稿发表在JCB中。