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.
Cell division is a fundamental process for life; cells that fail to divide become multinucleated and can be found in both normal tissue, such as the lactating mammary gland, and in diseased tissue, such as tumors. Recent evidence shows that the microenvironment around mammary epithelial cells regulates cell division, specifically that stiff microenvironments disrupt cytokinetic abscission and generate multinucleated cells. The proposed study will investigate how the chemical and mechanical microenvironment regulates cell division, to uncover new therapeutic targets for diseases associated with multinucleation.
