The force required to separate one cell into two cells during cytokinesis comes from a contractile ring composed primarily of F-actin and myosin-2 . Formation of this contractile ring is dependent on the small GTPase Rho, which is activated in a precise zone at the cell equator prior to cytokinesis. Active Rho then templates the actomyosin contractile ring by activating effector proteins including formins, which are actin nucleators, and the kinases ROCK and citron, which activate myosin-2.
Rho GTPases. Rho cycles between active (GTP-bound) and inactive (GDP-bound) forms, with the aid of guanine nucleotide exchange factors (GEFs) that activate Rho and GTPase activating proteins (GAPs) that inactivate Rho.
The signal that activates Rho specifically at the equatorial cortex is thought to be delivered by the centralspindlin complex, which consists of the microtubule plus-end-directed kinesin, MKLP1, and the Rho GAP, MgcRacGAP. Centralspindlin accumulates at the plus ends of the microtubules of the mitotic spindle, especially at regions of microtubule overlap. MgcRacGAP binds the Rho GEF Ect2 and positions it properly to activate the equatorial Rho activity zone, which in turn activates the actomyosin contractile ring. We recently showed that in addition to positioning Ect2, MgcRacGAP plays a key role in limiting the spread of the Rho activity zone. MgcRacGAP's GAP activity is required to locally inactivate Rho, promoting cycling of Rho between the active and inactive states throughout cytokinesis – a process we termed GTPase Flux. This ensures that the Rho zone remains precisely focused, which is necessary for a functional contractile ring.
Regulation of cytokinesis by Rho GTPase Flux. Focused activation of Rho, which requires MgcRacGAP's GAP activity to drive Rho through the GTPase cycle, is required for proper formation of the contractile ring and for successful cytokinesis. Kymographs show accumulation of active Rho over time in control embryos and embryos expressing wild type or GAP-DEAD MgcRacGAP.
While regulation of Rho activity by centralspindlin/Ect2 is clearly an important cytokinesis regulatory mechanism, we think there are key additional layers of Rho regulation that aid in making the process of cytokinesis robust. In addition to the initial signal(s) that activate Rho, there are likely multiple 'amplifiers' and 'focusers' that work to activate Rho at precisely the right place and time and keep it from being activated in ectopic locations. Moreover, the contribution of these 'amplifiers' and 'focusers' may differ in different systems based on organism, cell size, spindle geometry, and whether the dividing cell makes cell-cell contacts with neighboring cells. Our lab aims to study the proteins and mechanisms that are involved in Rho regulation during cytokinesis. In addition, we are interested in studying how misregulation of proteins involved in cytokinesis and/or cytokinesis failure may promote tumor formation.
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. We use embryos from the African clawed frog, Xenopus laevis, as a vertebrate model system. Xenopus embryos offer multiple advantages for these studies including external development, rapid and synchronous cell division, large cell size for detailed imaging, the ability to examine cells in embryos of different developmental stages, and the capacity to knock down proteins of interest using antisense morpholinos.
The Xenopus laevis model system. Xenopus embryos are an excellent system for studying cytokinesis. This system allows us to exploit different developmental stages including the blastomeres of the early embryo, the epithelial cells of the gastrula-stage embryo, and the epithelial cells of the tadpole stage, in which we can observe tumor formation. (Frog picture is courtesy of http://www.xenopus.com/links.htm and picture of Xenopus embryos is courtesy of http://www.bio.davidson.edu/people/balom)
Gene replacement in the intact vertebrate epithelium. We use morpholino knockdown to examine loss of function embryos and gene replacement to examine embryos where the endogenous protein is replaced with a mutant or fluorescently-tagged version of the protein expressed at near endogenous levels.
We think this is an excellent model system to address the types of questions described above, and we are poised to make unique contributions in this field. In contrast to much of the work on cytokinesis in the literature, which has been carried out in cultured cells growing on coverslips, our work using Xenopus embryos is carried out in a natural environment where the dividing cell makes cell-cell contacts with its neighbors as part of an intact polarized epithelium in a vertebrate organism. This is important because the dividing cell may integrate signals from surrounding cells and, in turn, send signals that influence other cells in the epithelium. Moreover, studying how cytokinesis works in normal cells and what happens when cytokinesis fails in an epithelial context is clinically relevant because cancers arising from epithelial cells, carcinomas, make up about 85% of all cancers. Importantly, the key proteins that regulate cytokinesis 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 may contribute to cancer in humans.
Sound interesting? Contact Dr. Miller to discuss joining the lab.