The Role of EMT signaling and ECM Stiffness in Cytokinetic Abscisssion

项目总结： 细胞分裂是细胞分裂的最后阶段，是一个复杂的过程，需要组装 成功地切断了连接两个女儿的细胞间桥梁 细胞。脱落失败会产生多核细胞，这在正常组织中可以观察到，如人类 哺乳期乳腺的表皮、成肌细胞和末梢上皮，以及几个人的 肿瘤，包括乳房肿瘤。持续分裂的多核细胞将导致非整倍体，这是一个迹象 染色体不稳定，强调了了解微环境条件如何 导致脱落失败和多核。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信号导致Septin-6表达增加，Septin-6是一种细丝-6。 形成GTP酶，在胞质分裂时被招募到细胞间桥，特别是中体。单元格 在软基质上培养不上调Septin-6的表达，不能进行EMT，能够进行EMT 正常的脱落。因此，我假设ECM僵硬与EMT信号一起影响 蜗牛与Septin-6启动子结合，影响正常脱落所需因子的平衡。这就做 使用合成微环境和分析相结合的方法来测量蛋白质-DNA相互作用以及 成像技术，以了解蜗牛和微环境是如何破坏脱落的。在具体目标1中，我 将使用染色质免疫沉淀(CHIP)分析、启动子-报告分析和测序(CHIP-SEQ 和RNA-seq)，以确定Snail和Stiffness如何协同调节乳腺中Septin-6的表达 上皮细胞。在具体目标2中，我将把合成底物与定量成像和时间流逝相结合 分析确定僵硬的微环境和蜗牛改变脱落的机制。成功 完成这些目标将加深我们对微环境如何调节脱落的理解 失败，这将为细胞质分裂过程提供更多的洞察，也可能提示治疗 与多核手术相关的疾病的靶点。同时，这项研究计划与我的 多机构MD/PHD环境下的培训计划，这将加深我对 定量细胞和分子生物学，以及这一领域如何在临床实践中被告知和被告知 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.