The Role of EMT Signaling and ECM Stiffness in Cytokinetic Abscission

项目摘要： 细胞分裂的最后阶段，细胞动力学分裂是一个复杂的过程， 成功地切断了连接两个女儿的细胞间桥梁 细胞脱落失败产生多核细胞，其在正常组织中观察到，例如人的组织。 表皮、成肌细胞和泌乳乳腺的末端上皮，以及在几个人中， 肿瘤，包括乳腺癌。多核细胞继续分裂会导致非整倍体， 染色体的不稳定性，强调了解微环境条件如何 导致分裂失败和多核化。我们最近发现，乳腺上皮细胞诱导， 在僵硬的微环境中经历上皮-间充质转化（EMT），类似于在 正常乳腺或乳腺肿瘤的末端，不能完成胞质分裂，导致 多核细胞通过Snail的EMT信号传导导致septin-6表达增加，septin-6是一种丝蛋白， 形成在胞质分裂期间被募集到细胞间桥，特别是中间体的GT3。细胞 在软基质上培养不上调septin-6表达，不能经历EMT，并且能够经历EMT。 正常释放。因此，我假设ECM的硬度沿着EMT信号传导影响了细胞的能力， 蜗牛结合到Septin-6的启动子上，并影响正常分泌所需因子的平衡。我会 使用合成微环境结合测定来测量蛋白质-DNA相互作用以及 成像技术，以了解蜗牛和微环境如何破坏细胞分裂。在具体目标1中， 将使用染色质免疫沉淀（ChIP）测定、启动子-报告基因测定和测序（ChIP-seq 和RNA-seq）以确定Snail和刚度如何协同调节乳腺癌中septin-6的表达。 上皮细胞在《特定目标2》中，我将把联合收割机合成基质与定量成像和时间推移相结合 分析以确定僵硬的微环境和Snail改变的机制。成功 完成这些目标将加深我们对微环境如何调节免疫反应的理解 失败，这将提供更多的了解胞质分裂的过程，也可能表明治疗 多核化相关疾病的靶点。同时，这项研究计划与我的 在一个多机构的MD/PhD环境中的培训计划，这将加强我对 定量细胞和分子生物学，以及该领域如何通知和通知临床实践， 内科。

Project Summary: Cytokinetic abscission, the final stage of cell division, is a complex process that requires the assembly of proteins in a sequential fashion to successfully sever the intercellular bridge that connects the two daughter cells. Abscission failure generates multinucleated cells, which are observed in normal tissues, such as human epidermis, myoblasts, and the terminal epithelium of a lactating mammary gland, as well as in several human tumors, including those of the breast. Multinucleated cells that continue to divide will lead to aneuploidy, a sign of chromosomal instability, underlining the importance of understanding how microenvironmental conditions lead to abscission failure and multinucleation. We recently found that mammary epithelial cells induced to undergo epithelial-mesenchymal transition (EMT) on stiff microenvironments, similar to what is found at the terminal ends of a normal mammary gland or in breast tumors, fail to complete cytokinesis, resulting in multinucleated cells. EMT signaling through Snail leads to an increase in the expression of septin-6, a filament- forming GTPase that is recruited to the intercellular bridge, specifically the midbody, during cytokinesis. Cells cultured on soft substrata do not upregulate septin-6 expression, fail to undergo EMT, and are able to undergo normal abscission. I therefore hypothesize that ECM stiffness along with EMT signaling affects the ability of Snail to bind to the promoter of septin-6 and affects the balance of factors required for normal abscission. I will use synthetic microenvironments combined with assays to measure protein-DNA interactions as well as imaging techniques to understand how Snail and the microenvironment disrupt abscission. In Specific Aim 1, I will use chromatin immunoprecipitation (ChIP) assays, promoter-reporter assays, and sequencing (ChIP-seq and RNA-seq) to determine how Snail and stiffness synergistically regulate expression of septin-6 in mammary epithelial cells. In Specific Aim 2, I will combine synthetic substrata with quantitative imaging and timelapse analysis to determine the mechanism by which stiff microenvironments and Snail alter abscission. Successfully completing these aims will deepen our understanding of how the microenvironment regulates abscission failure, which will provide additional insight into the process of cytokinesis and may also suggest therapeutic targets for diseases associated with multinucleation. At the same time, this research plan synergizes with my training plan within a multi-institutional MD/PhD environment, which will strengthen my understanding of quantitative cell and molecular biology, as well as how this field informs and is informed by clinical practice in internal medicine.