Using Quantitative Proteomics to Elucidate the Signaling Consequences of Microtubule Disruption

 描述(申请人提供)：微管是大型细胞结构，在几秒钟到几分钟的数量级上生长和收缩。微管动力学的化学破坏通常会导致周期细胞和非周期细胞的死亡，尽管导致这一现象的机制尚未得到很好的描述。有趣的是，微管药物刺激激酶- 在细胞死亡之前介导的应激信号，这表明细胞可以检测到微管破坏并对其做出反应。我建议解决围绕这一耐人寻味的压力现象的问题。首先，我想了解微管扰动对细胞信号的总体影响。鉴于微管破裂后磷酸化应激信号的先例，我建议对微管药物治疗后立即发生的蛋白质磷酸化进行高度定量的质谱学测量。其次，我想确定不同的微管扰动(例如，稳定和不稳定)是否对细胞信号有不同的影响。为了做到这一点，我将使用不同的药物来扰乱微管，并比较对蛋白质磷酸化的影响。最后，关于微管如何直接调节信号，特别是对化学侮辱的反应，目前还知之甚少。我希望阐明信号蛋白(如激酶、磷酸酶)受微管动力学调节并在微管破裂时被激活的机制。为此，我将使用免疫荧光成像、传统的转基因技术和统计推断方法。

 DESCRIPTION (provided by applicant): Microtubules are large cellular structures that grow and shrink on the order of seconds to minutes. Chemical disruption of microtubule dynamics typically leads to death in both cycling and non-cycling cells, though the mechanisms responsible for this are not well characterized. Interestingly, microtubule drugs stimulate kinase- mediated stress signaling prior to cell death, which suggests that cells can detect and respond to microtubule disruption. I propose to address the questions surrounding this intriguing stress phenomenon. First, I would like to understand the general impact that microtubule perturbations have on cellular signaling. Given the precedent of phosphorylation stress signals in response to microtubule disruption, I propose to generate highly quantitative mass spectrometry measurements of protein phosphorylation that occurs immediately following microtubule drug treatment. Second, I want to determine whether different microtubule perturbations (e.g. stabilization and destabilization) have different effects on cellular signaling. To do this, I willuse different drugs to perturb microtubules and compare effects on protein phosphorylation. Finally, there is currently very little knowledge regarding how microtubules might directly regulate signaling, especially in response to chemical insults. I hope to elucidate mechanisms by which signaling proteins (e.g. kinases, phosphatases) are regulated by microtubule dynamics and activated in response to microtubule disruption. To this end, I will use immunofluorescence imaging, conventional transfection techniques, and statistical inference methods.