Wu Lab

Contractile-ring formation depends on actin polymerization by formins and the motor activities of myosin-II and myosin V (Coffman et al., J. Cell Biol. 2013; Wang et al., J. Cell Biol. 2014), but we do not know how they function during ring constriction. We will make the quantitative measurements of constricting rings required for mathematical modeling: the molecule numbers and turnover kinetics of formins, myosins, α-actinin, and cofilin, their relative positions by super-resolution microscopy, their behaviors and contractile-ring tension in mutant cells, and the biochemical activities of myosins. Then we will use these parameters to test models to account for ring constriction with 3D computer simulations. Completion of this project will elucidate the basic principles and contributions of key actin-binding proteins in contractile-ring constriction.

The prevailing model for membrane expansion during cytokinesis is that vesicles tethered by the exocyst complex insert into the plasma membrane adjacent to the contractile ring at the leading edge of the cleavage furrow. However, we have observed vesicles deposited along the whole cleavage furrow behind the constricting ring by high spatiotemporal resolution microscopy and have evidence that both the exocyst and Transport Particle Protein II (TRAPP-II) complexes participate (Wang et al., PLOS Biology, 2016). By contrast, endocytosis is more active at the rim of the cleavage furrow. In this project, we will use genetics, confocal microscopy, super-resolution microscopy, and electron tomography to determine how the balance between exocytosis and endocytosis expands the plasma membrane at the division site, determine the contributions of exocyst and TRAPP-II complexes in tethering vesicles for plasma-membrane deposition, and investigate how targeted plasma-membrane deposition coordinates with ring constriction during cytokinesis.

The extracellular matrix contributes to cytokinesis in both animal cells and fungi, where the septum serves as extracellular matrix. Depletion of chondroitin proteoglycan core proteins or the secreted matrix protein hemicentin leads to cytokinesis failure in worms and mice. However, the mechanism coordinating ring constriction and extracellular matrix remodeling is poorly understood since >100 proteins are involved in these two cytokinetic stages. We hypothesize that glucan synthases delivered by vesicle trafficking coordinate ring constriction with septum formation to ensure cell integrity during cell division. We discovered three novel and conserved proteins, coiled-coil protein Rng10, glucan synthase-associated protein Sbg1, and Rng13, that may help explain the still obscure relationship between ring constriction and septum formation by regulating the localizations or stability of glucan synthases. We will characterize how the three proteins and their domains contribute to ring constriction, ring disassembly, and septum formation. Then we will elucidate how glucan synthases help anchor the contractile ring and coordinate ring constriction with septum formation. Completion of this project will reveal how glucan synthases and their regulators coordinate ring constriction and septum formation for successful cytokinesis. Conserved β- and α-glucan synthases make the glucans of the septum and cell wall in fission yeast as well as in fungal pathogens. The novel proteins and their binding partners that we discovered are conserved in fungal pathogens and thus may be ideal anti-fungal drug targets.

Rho GTPases are important regulators in all stages of cytokinesis. They are “molecular switches” that switch between the active GTP-bound and the inactive GDP-bound forms. The activity of Rho GTPases is regulated by Rho guanine nucleotide exchange factors (GEFs) and Rho GTPase activating proteins (GAPs). In human cells, the exact roles of Rho GTPases and their GEFs and GAPs in cytokinesis are still poorly understood due to the redundancies (>70 Rho GEFs in humans) and limited genetic and molecular tools in mammalian cells. We have found that Rho GEFs Rgf3, Gef2, Gef3, and RhoGAP Rga7 are involved in various stages of cytokinesis in fission yeast. Interestingly, the localizations and activities of these GEFs and GAPs are regulated by their adaptors/binding partners. We will continue investigating roles of these and other GEFs, GAPs, and their adaptors in cytokinesis.

Single-cell (or cellular) wound healing/repair is an essential process related to cytokinesis in components and the overall pathways involved. We will use mutants or laser ablation to precisely wound specific sites at different stages of the cell cycle. Understanding the mechanism of wound healing is fundamental to biology and human health because wounds can result from diverse sources at cellular and tissue levels: environmental stress, trauma, surgery, infection, and muscle contraction. In addition, wounding can promote tumor formation and metastasis, and tumor cells could survive chemotherapy and radiation therapy using wound-healing pathways. What we learn about single-cell wound healing in fission yeast will help us understand wound responses in human cells. Therefore, new therapies could eventually be designed to facilitate wound healing, lessen the incidence of tumorigenesis, and manage tumor cells based on our conceptual breakthroughs on the molecular links between cytokinesis and single-cell wound-healing responses.