Overview. My lab studies the process of cytokinesis, the final step of cell division, where one cell is separated into two. This process is fundamentally important throughout life: it drives development and helps maintain adult tissues, while cytokinesis failure can promote birth defects and tumor formation. Much of the previous work on cytokinesis has been carried out in isolated cells in the artificial space of a culture dish. These cells do not make cell-cell contacts with neighboring cells and are flat, growing in two dimensions on an unnatural, hard substrate (Fig. 1). Furthermore, many cultured cell lines are transformed to allow for continuous growth in culture, and therefore the mechanisms that regulate the cell cycle and cell division are abnormal. In epithelial tissues, cells are polarized, grow in three dimensions, and form cell-cell junctions with their neighbors (Fig. 1). In my lab, we want to understand how cytokinesis works in intact epithelial tissues where the dividing cell gives and receives inputs from its neighboring cells. It is critical to understand how cytokinesis works in an epithelial context because over 85% of cancers arise from epithelial tissues, and both cytokinesis failure and cell-cell junction defects can contribute to tumor formation and metastasis.
Regulation of Localized Rho GTPase Activity in Dividing Epithelial Cells. We know that precisely localized zones of active RhoA, a small GTPase that promotes actomyosin contractility, are required both for cytokinesis and for proper cell-cell junctions (Fig. 2). However, we don’t understand the signaling and mechanical mechanisms that regulate localized Rho GTPase activity during cytokinesis in epithelial cells. Our long-term goal is to determine the mechanisms by which localized Rho GTPase activation is regulated in space and time during cytokinesis in the intact vertebrate epithelium. In this context, the dividing cell must properly regulate formation and constriction of the contractile ring while also maintaining and remodeling cell-cell junctions with neighboring cells so that epithelial barrier function is not breached.
RhoA cycles between active (GTP-bound) and inactive (GDP-bound) forms, and its activity state is regulated by guanine nucleotide exchange factors (GEFs) that activate RhoA and GTPase activating proteins (GAPs) that inactivate RhoA (Fig. 2). The biological output of RhoA depends on where RhoA-GTP is localized in the cell. When RhoA is locally activated, it undergoes a conformational change enabling it to recruit effector proteins that promote F-actin polymerization and Myosin-2 activation (Fig. 2). Force generated by actomyosin contraction then pinches one cell into two during cytokinesis.
RhoA signaling also affects cell-cell junction integrity by regulating the contraction of an apical actomyosin belt (Fig. 3). Both tight junctions (TJs), which are responsible for regulating epithelial barrier function, and adherens junctions (AJs), which provide adhesion between epithelial cells, are linked to the apical actomyosin belt (Fig. 3). Some tension in the apical actomyosin belt is required for proper junction formation and maintenance, but too much tension can cause junctions to break apart. We aim to develop a better understanding of the GEFs, GAPs, kinases, and other regulatory proteins that are responsible for initiating and maintaining localized RhoA activation and actomyosin tension at the proper time and place for successful cytokinesis and optimal junction integrity.
Approach and Model Organism. The approaches we use to address these questions include biochemistry, molecular biology, and cell biology, with an emphasis on live confocal imaging at the cellular level. One key strength of our work is that it employs fluorescent probes to detect the subcellular localization and dynamics of active Rho GTPases and other key molecular players in live cells. Additionally, we have developed a toolkit of multiple GEFs, GAPs, and other Rho GTPase regulators, as well as cell-cell junction components. By perturbing the proper regulation of Rho GTPase activation and observing and quantifying the effects on cells, we can dissect the molecular mechanisms that regulate RhoA activation during cytokinesis and at cell-cell junctions.
Another major strength of our work is that it is carried out in a natural environment, the developing Xenopus laevis (African clawed frog) embryo (Fig. 4). In this context, a dividing cell makes cell-cell junctions with its neighbors as part of a larger polarized epithelial tissue where cells are interconnected and communicate via mechanical and signaling interactions. Xenopus embryos offer multiple advantages for studying cytokinesis including external development, rapid and synchronous cell division, large cell size for detailed microscopy, and the ability to examine different developmental stages. Investigating how cytokinesis works in epithelial tissues is clinically relevant because over 85% of cancers are carcinomas, which arise from epithelial cells. Indeed, multiple proteins that are involved in regulating RhoA activity during cytokinesis and at cell-cell junctions are misregulated in human cancers. Importantly, the proteins that regulate cytokinesis and cell-cell junctions in Xenopus are highly conserved with those in human cells. Therefore, the insights we gain from these studies will further our understanding of how human cells divide and how misregulation of cytokinesis and cell-cell junctions may contribute to cancer in humans.
Sound interesting? Contact Dr. Miller to discuss joining the lab.