Quantitative live cell imaging of vimentin network assembly and regulation

摘要--项目3 III型中间丝蛋白Vimentin与细胞定向运动性增强有关。不管有多少 至于Vimentin在细胞定向迁移中发挥的具体功能，目前仍不清楚。我们过去的数据 资助期证明Vimentin中间丝(VIF)可以与其他细胞骨架相互作用 用于稳定微管(MT)调节的细胞极化的组件和细胞-基质黏附机械 和牵引定向，增强了迁移方向性的持久性。分子因素和 在迁移细胞的背景下，调节这些相互作用的机械机制尚未被揭示。 为了揭示VIF稳定MT极化的分子机制，我们将进一步开发我们的 定量成像方法及分析VIF-MT相互作用和交叉的可能机制 链接器(目标1)。我们还将分析VIF对肌动蛋白收缩性能、黏附分布和 使用高分辨率牵引力显微镜产生牵引力(目标2)。VIF的建议功能在 稳定MT极化和组织细胞牵引预测VIF周转设定时间尺度 坚持细胞的极性和定向牵引力。因此，在方向线索变化较快的环境中 相比VIF周转的时间尺度，VIF网络可能会产生潜在的不利迁移惯性。 几种蛋白水解酶，如PKC和PKA，可以磷酸化波形蛋白，而这种磷酸化增加了 波形蛋白的溶解度。我们假设，这些激酶在前沿的激活是为了响应 改变方向线索可局部分解VIF，促进细胞骨架重组和突起 在新的方向上形成。为了验证这一假设，我们将把局部PKC和PKA的激活与 通过突变Vimentin的PKC和PKA来分解和扰乱VIF对引导信号的反应 磷酸化位点。我们还将量化野生型VS网络中的重组动态 趋化试验中诱变的VIF，其中细胞外部变异的大小和时间尺度 引导线索可以通过实验进行控制。这项拟议的研究计划将增进我们对 Vimentin在定向迁移中的作用并产生创新的成像方法，对 细胞骨架领域。

ABSTRACT-PROJECT 3 The type III intermediate filament protein, vimentin is relevant to enhanced directed cell motility. However many of the specific functions vimentin plays in directed cell migration have remained elusive. Our data of the past funding period demonstrate that Vimentin Intermediate Filaments (VIF) can interact with the other cytoskeleton components and the cell-substrate adhesion machinery to stabilize microtubule (MT)-regulated cell polarization and traction orientation, which enhances the persistence in migration directionality. The molecular factors and mechanical mechanisms that mediate these interactions in the context of migrating cells have yet to be revealed. To uncover the molecular mechanism of VIF’s function in stabilizing MT polarization we will further develop our quantitative imaging approaches and analyze possible mechanisms of VIF-MT interaction and VIF-MT cross- linkers (Aim 1). We will also analyze the effects of VIF on actomyosin contractility, adhesion distribution and traction generation using high-resolution traction-force microscopy (Aim 2). The proposed functions of VIF in stabilizing MT polarization and organizing cell traction predict that the turnover of VIF sets the time scale of persistence in cell polarity and directed traction. Hence, in environments where directional cues change faster than the time scale of VIF turnover, the VIF network may generate potentially unfavorable migration inertia. Several kinases, such as PKC and PKA, can phosphorylate vimentin and the phosphorylation increases the solubility of vimentin. We hypothesize that the activation of these kinases at the leading edge in response to changing directional cues disassembles VIF locally to facilitate cytoskeleton reorganization and protrusion forming in the new direction. To test this hypothesis we will correlate local PKC and PKA activation with the rate of VIF disassembly and perturb the VIF response to guidance cues by mutagenizing vimentin’s PKC and PKA phosphorylation sites. We will also quantify the dynamics of reorganization in networks of wildtype vs mutagenized VIF in chemotaxis assays, where the magnitude and time scale of variations in cell external guidance cues can be experimentally controlled. This proposed research plan will enhance our understanding of vimentin’s function in directed migration and produce innovative imaging methods that impact on the cytoskeleton field.