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: 7969103
• 负责人: Clare Michal Waterman
• 金额: $ 74.12万
• 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/7969103
• 关键词: 3-Dimensional; ATP phosphohydrolase; Actins; Actomyosin; Affect; Affinity; Area; Binding Proteins; Biological Assay; Biological Sciences; Caenorhabditis elegans; Categories; Cell Adhesion; Cell Polarity; Cells; Cellular Morphology; Centromere; Cytoskeleton; Data; Defect; Endothelial Cells; Ensure; Event; Exhibits; Extracellular Matrix; F-Actin; Family; Feedback; Frequencies; Goals; Growth; Guanosine Triphosphate Phosphohydrolases; Human; Image; Interphase; Kinesin; Mediating; Methods; Microtubule Depolymerization; Microtubule Polymerization; Microtubule Stabilization; Microtubule-Associated Proteins; Microtubules; Mitosis; Mitotic; Molecular; Monomeric GTP-Binding Proteins; Morphogenesis; Motion; Myosin Type II; Nocodazole; Paclitaxel; Pathway interactions; Phospho-Specific Antibodies; Phosphorylation; Phosphotransferases; Plus End of the Microtubule; Polymers; Positioning Attribute; Property; Proteins; RNA Interference; Regulation; Role; Scheme; Signal Pathway; Signal Transduction; Small Interfering RNA; Speed; Stimulus; Stress Fibers; System; Testing; Time; Tissues; Tubulin; adhesion receptor; aurora kinase; aurora-A kinase; base; blebbistatin; cell behavior; cell motility; computer program; crosslink; directional cell; extracellular; genetic regulatory protein; human STK6 protein; migration; movie; mutant; new technology; novel; osteosarcoma; physical property; polarized cell; polymerization; research study; response; rho; rho GTP-Binding Proteins

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. 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. Project 3: Regulation of microtubules in migrating endothelial cells in 3D ECMs Ken Myers, Kathryn Applegate, Gaudenz Danuser, Clare Waterman ECM dimensionality and stiffness regulate endothelial cell branching morphogenesis via mechanosensitive cell adhesion receptors that elicit bidirectional signals to and from the actomyosin cytoskeleton. Stiff ECMs promote myosin II activity that inhibits cell branching, while compliant ECMs reduce myosin II activity and promote branching. The MT cytoskeleton controls cell morphology through its structural and regulatory interactions with actomyosin. However the role of MTs in cellular responses to ECM properties, and the effects of ECM properties on MT organization and dynamics are unknown. To explore the role of MTs in cell responses to ECM dimensionality and compliance, we analyzed MT and actin organization in HUVEC cells on two different stiffnesses of 2D and 3D ECMs. This showed that stiffer substrates enhanced MT presence in cell branches. Depolymerization of MTs with nocodazole induced actin stress fibers and inhibited cell branching independent of substrate stiffness or dimensionality, showing that MT depolymerization-induced activation of contractility is not regulated by physical properties of ECM. In contrast, stabilization of MTs with taxol inhibited MT presence in branches. To determine how ECM stiffness and dimensionality affect MTs, we analyzed MT dynamics by tracking fluorescently-tagged MT plus ends. This revealed that on soft substrates, MT growth rate was independent of ECM dimensionality, while on stiff substrates, MT growth was faster in 3D compared to 2D ECMs. In 2D, MT growth rate was independent of stiffness, while in 3D, stiffer ECM promoted faster MT growth than soft ECM. Thus, the fastest MT growth occurred in stiff 3D ECMs, and the slowest occurred in 2D ECMs, independent of stiffness. Inhibition of myosin II with blebbistatin increased MT growth rate under all conditions of ECM dimensionality and stiffness, indicating that myosin II regulates MT growth independent of ECM physical properties. Furthermore, blebbistatin did not abrogate the effects of ECM stiffness on MT growth. Thus, we find a bidirectional regulation scheme where MTs mediate cellular responses to ECM, and ECM regulates MTs by both contractility-dependent and independent mechanisms.

项目 1：微管调节蛋白的 RNAi 筛选将 MARK2/Par1 鉴定为 Rac1 介导的微管生长的效应子。 西村由花子、凯瑟琳·艾伯盖特、高登兹·丹努瑟、克莱尔·沃特曼 微管 (MT) 组装动力学的正确调节对于定向细胞迁移至关重要。迁移细胞中的微管动态受 Rho GTPases 的空间调节。我们之前已经证明，激活的 Rac1 通过抑制突变和提高生长速度来诱导 MT 净生长，并且 Rac1 活性是迁移细胞前缘极化 MT 生长所必需的。我们确定了 Rac1 下游必要但不充分的 PAK 激酶介导途径，可促进 MT 生长。因此，我们假设其他因素促进 Rac1 下游 MT 净增长。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，以确定组成型激活 Rac1 促进 MT 生长是否需要已知的 MT 调节蛋白。为了分析 MT 动力学，我们对荧光标记的 EB3（一种 MT 加端结合蛋白，用作 MT 末端位置的探针）进行成像，并使用自动化计算机程序跟踪延时电影中 EB3 彗星的运动。 我们的结果表明，几种 MT 结合蛋白的消耗改变了活化的 Rac1 表达细胞中 MT 的生长速率。我们重点关注 MARK2，一种与线虫极性蛋白 Par1 同源的微管亲和力调节激酶，其缺失会减少 Rac1 激活细胞前缘中伸长 MT 的数量。我们目前正在测试 MARK2 如何参与促进 Rac1 下游 MT 的生长及其在细胞迁移中的要求。 项目 2：MCAK 活性控制间期微管动力学和定向细胞迁移。 迈尔斯，K.A.；阿普尔盖特，K；丹努瑟·G.；和沃特曼，C.M. 定向细胞迁移是通过细胞外刺激启动的，细胞外刺激协调细胞骨架的变化以建立极化的细胞形态。细胞极性可以通过微管 (MT) 动力学的区域调节来实现，包括 MT 向前缘生长和 MT 在细胞后部缩短。有丝分裂着丝粒相关驱动蛋白 (MCAK) 是一种 MT 解聚酶，在有丝分裂中通过 Aurora 激酶磷酸化下调。虽然 MCAK 的有丝分裂功能已得到充分表征，但 MCAK 是否在细胞迁移过程中调节 MT 动态尚不清楚。我们假设 MCAK 通过 Rac1/Pak1/Aurora-A 激酶信号通路局部下调，以建立朝向前缘的 MT 优先生长，并促进细胞后部内的 MT 缩短。为了检验这一假设，我们对荧光标记的 EB3 作为 HUVEC 细胞中 MT 加末端生长的标记物进行了延时成像，并分析了在所提出的信号级联的不同操作下的 MT 动态和细胞行为。我们发现 MCAK 敲低 (KD) 对 MT 细胞骨架产生预期的影响，包括微管蛋白聚合物水平增加和 MT 灾难频率降低。 MCAK-KD 细胞表现出 MT 聚合速度降低并表现出方向错误的 MT 阵列，并且细胞迁移速度、方向持久性和到原点的距离在统计上显着降低，表明细胞迁移和/或极化存在缺陷。这些效应可以通过外源野生型 MCAK 的表达来挽救，但不能通过表达无活性（ATP 酶死亡）的 MCAK 突变体或无法通过 Aurora-A 激酶进行磷酸调节的 MCAK 突变体来挽救。对表达组成型活性 Rac1 或组成型活性 Pak1 的细胞进行免疫标记表明，Rac1 和 Pak1 活性与磷酸特异性抗体测定的 Aurora-A 活性增加相关，并且还与 MCAK 表达水平降低相关。这些数据表明，MCAK 的间期调节是在 Rac1/Pak1/Aurora-A 信号通路下游实现的，以便局部协调 MCAK 介导的 MT 解聚，作为确保适当的细胞极化和运动的方法。 项目 3：3D ECM 中迁移内皮细胞微管的调节 肯·迈尔斯、凯瑟琳·艾伯盖特、高登兹·丹努瑟、克莱尔·沃特曼 ECM 维度和刚度通过机械敏感细胞粘附受体调节内皮细胞分支形态发生，这些受体引发往返于肌动球蛋白细胞骨架的双向信号。僵硬的 ECM 会促进抑制细胞分支的肌球蛋白 II 活性，而顺应性的 ECM 则会降低肌球蛋白 II 的活性并促进分支。 MT 细胞骨架通过其与肌动球蛋白的结构和调节相互作用来控制细胞形态。然而，MT 在细胞对 ECM 特性的反应中的作用以及 ECM 特性对 MT 组织和动力学的影响尚不清楚。为了探索 MT 在细胞对 ECM 维度和顺应性的反应中的作用，我们分析了 HUVEC 细胞在两种不同硬度的 2D 和 3D ECM 上的 MT 和肌动蛋白组织。 这表明较硬的基质增强了细胞分支中 MT 的存在。 MT 与诺考达唑的解聚诱导肌动蛋白应力纤维并抑制细胞分支，与基质硬度或维度无关，表明 MT 解聚诱导的收缩性激活不受 ECM 物理性质的调节。相反，用紫杉醇稳定 MT 会抑制分支中 MT 的存在。为了确定 ECM 刚度和维度如何影响 MT，我们通过跟踪荧光标记的 MT 加末端来分析 MT 动力学。这表明，在软基材上，MT 生长速率与 ECM 维度无关，而在硬基材上，与 2D ECM 相比，3D 中 MT 生长更快。在 2D 中，MT 增长率与刚度无关，而在 3D 中，较硬的 ECM 比软 ECM 促进更快的 MT 增长。因此，最快的 MT 增长发生在刚性 3D ECM 中，最慢的发生在 2D ECM 中，与刚度无关。在所有 ECM 维度和硬度条件下，用肌球蛋白 II 进行抑制可增加 MT 生长速率，表明肌球蛋白 II 调节 MT 生长与 ECM 物理性质无关。此外，blebbistatin 并没有消除 ECM 硬度对 MT 生长的影响。因此，我们发现了一种双向调节方案，其中 MT 介导细胞对 ECM 的反应，而 ECM 通过收缩性依赖和独立机制调节 MT。