Surprisingly little is known about how cytokinesis works in an intact epithelial environment where the dividing cell is connected to its neighboring cells via cell-cell junctions. Notably, failed cytokinesis can promote tumor formation; over 85% of cancers arise from epithelial tissues, and cell-cell junction defects can contribute to cancer metastasis. Therefore, the objective of the proposed research is to characterize the molecular mechanisms by which epithelial cells maintain and remodel their cell-cell junctions during cell division. Our central hypothesis is that the scaffolding protein Anillin regulates proper spatiotemporal patterning of active RhoA (RhoA-GTP) and is essential for regulating junction structure, remodeling, and tension in dividing and non-dividing epithelial cells. Our focus on RhoA, a small GTPase that promotes actomyosin contractility, stems from evidence that precisely localized zones of active RhoA are required both for cytokinesis and for formation and maintenance of cell-cell junctions. Our group is uniquely positioned to tackle the stated objective because we use high-resolution live imaging of active RhoA dynamics in the intact vertebrate epithelium of the Xenopus laevis embryo. We will test our central hypothesis by pursuing three specific aims.
In Aim 1, we will determine Anillin's function in maintaining cell-cell junctions. Or exciting preliminary data indicate that the scaffolding protein Anillin, which is known to play an important role in regulating cytokinesis, also plays a novel role in regulating cell-cell junction integrity. We will test the hypothesis that Anillin regulates cell-cell junctions by controlling th distribution of junctional RhoA-GTP and stabilizing the apical actomyosin belt.
In Aim 2, we will characterize how dynamic cell-cell junction remodeling is regulated in dividing cells. We will test the hypothesis that junction protein dynamics are altered in dividing cells due to changes in tension, and proper regulation of localized RhoA-GTP by Anillin is required for junction remodeling.
In Aim 3, we will identify mechanisms by which dividing cells regulate and respond to tension changes. Here, we will test the hypothesis that RhoA is activated in response to mechanical force, and Anillin stabilizes RhoA-GTP and promotes increased junctional tension. Using innovative approaches that include live imaging with a fluorescent probe that specifically highlights active RhoA in the cell, characterizing for the first time how cell-cell junction protei dynamics are regulated during vertebrate cell division, and examining RhoA-mediated cellular mechanics - all in intact vertebrate epithelial tissue - will allow us to gain new insights about hw cell division works in epithelial cells. Completion of the proposed research is expected to identif novel mechanisms that regulate localized RhoA activity and the actomyosin-mediated tension required for cell-cell junction maintenance and remodeling during cytokinesis in the intact vertebrate epithelium.
The proposed research is relevant to public health because understanding how cytokinesis is normally regulated in epithelial tissues may lead to insights about how cytokinesis failure and cell-cell junction defects contribute to tumor formation and metastasis. The proteins we are studying, RhoA and Anillin, are misregulated in human cancers. Therefore, our work identifying mechanisms by which RhoA activity is regulated in dividing epithelial cells has the potential to help identify novel molecular targets for cancer therapeutics.
| Higashi, Tomohito; Miller, Ann L (2017) Tricellular junctions: how to build junctions at the TRICkiest points of epithelial cells. Mol Biol Cell 28:2023-2034 |
| Arnold, Torey R; Stephenson, Rachel E; Miller, Ann L (2017) Rho GTPases and actomyosin: Partners in regulating epithelial cell-cell junction structure and function. Exp Cell Res 358:20-30 |
| Stephenson, Rachel E; Miller, Ann L (2017) Tools for live imaging of active Rho GTPases in Xenopus. Genesis 55: |
| Breznau, Elaina B; Murt, Megan; Blasius, T Lynne et al. (2017) The MgcRacGAP SxIP motif tethers Centralspindlin to microtubule plus ends in Xenopus laevis. J Cell Sci 130:1809-1821 |
| Higashi, Tomohito; Arnold, Torey R; Stephenson, Rachel E et al. (2016) Maintenance of the Epithelial Barrier and Remodeling of Cell-Cell Junctions during Cytokinesis. Curr Biol 26:1829-42 |
| Machta, Benjamin B; Gray, Ellyn; Nouri, Mariam et al. (2016) Conditions that Stabilize Membrane Domains Also Antagonize n-Alcohol Anesthesia. Biophys J 111:537-545 |
