Quantitative live cell imaging of vimentin network assembly and regulation

项目3 III型中间丝蛋白，波形蛋白与增强的定向细胞运动有关。然而许多 波形蛋白在定向细胞迁移中所起的具体作用仍然是难以捉摸的。我们过去的数据 一项研究表明，波形蛋白中间丝（VIF）可以与其他细胞骨架相互作用， 组分和细胞基质粘附机制，以稳定微管（MT）调节的细胞极化 和牵引方向，这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介导这些相互作用的机械机制还有待揭示。 为了揭示VIF稳定MT极化的分子机制，我们将进一步发展我们的 定量成像方法，并分析可能的机制VIF-MT相互作用和VIF-MT交叉， 接头（目标1）。我们还将分析VIF对肌动球蛋白收缩性、粘附分布和 使用高分辨率牵引力显微镜产生牵引力（目标2）。VIF的拟议功能 稳定MT极化和组织细胞牵引预测VIF的周转设定了 细胞极性和定向牵引的持久性。因此，在方向线索变化较快的环境中， 与VIF周转的时间尺度相比，VIF网络可能会产生潜在的不利迁移惯性。 几种激酶，如PKC和PKA，可以磷酸化波形蛋白，磷酸化增加波形蛋白的表达。 波形蛋白的溶解度。我们假设，这些激酶在前沿的激活是对 改变方向性线索可局部分解VIF，促进细胞骨架重组和突起 形成新的方向。为了验证这一假设，我们将局部PKC和PKA激活与 VIF的解体和干扰VIF响应的指导线索，通过诱变波形蛋白的PKC和PKA 磷酸化位点。我们还将量化野生型与非野生型之间的网络重组动力学。 在趋化性测定中，诱变的VIF的细胞外基质中的变化的幅度和时间尺度可以在趋化性测定中确定。 可以通过实验来控制引导提示。这项拟议的研究计划将增进我们对 波形蛋白在定向迁移中的功能，并产生创新的成像方法， 细胞骨架场

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.