Jian-Qiu Wu

Professor
Faculty

Research Description

Outstanding Questions in Cytokinesis
Our long-term goal is to reveal the molecular mechanisms of cytokinesis. Cytokinesis partitions cellular constituents into two daughter cells at the end of the cell cycle. From yeast to humans, cytokinesis requires the coordination of six key events: division-plane selection, actomyosin contractile-ring assembly, ring constriction and associated disassembly, targeted plasma-membrane deposition, extracellular matrix formation, and daughter-cell separation (Fig. 1). The molecular mechanisms of the last four events are least understood because the pathways overlap and the events are currently poorly resolved in time and space.

Fig. 1. Key events in fission yeast cytokinesis.

Fig. 1. Key events in fission yeast cytokinesis. Cytokinesis nodes and the actomyosin contractile ring are in red, nuclei in gray, actin filaments in green, plasma membrane in black, cell wall in light blue, primary septum in dark blue, and secondary septa in yellow.

 

The Fission Yeast S. pombe as a Model System to Study Cytokinesis
The fission yeast S. pombe has emerged as a favorite model system for the analysis of cytokinesis. Not only is it genetically tractable and favorable for microscopic analysis, but it also has highly efficient homologous recombination for gene targeting, a small (13.8 Mb) and fully sequenced eukaryotic genome, and perhaps most importantly, it carries out cytokinesis much like animal cells. Cytokinesis begins with the formation of nodes at the cell equator (Figs. 1 and 2). We discovered how cytokinesis nodes condense into a contractile ring by a “search, capture, pull, and release” (SCPR) mechanism (Vavylonis et al., Science 2008). Constriction of the ring is coupled with invagination of the plasma membrane and formation of a trilaminar septum, but the mechanisms coordinating these processes are unknown. Digestion of the primary septum leads to cell separation (Fig. 1). We are studying cytokinesis in S. pombe using complementary genetic, microscopic (confocal, EM, & super-resolution), biochemical, and mathematical approaches. The concepts learned from fission yeast are applicable to the coordination of actomyosin contractile structures, plasma-membrane dynamics, and extracellular matrix remodeling in animal-cell cytokinesis, cell motility, cell morphogenesis, and wound healing since most proteins are conserved.

Fig. 2. Model for cytokinesis node assembly and architecture.

Fig. 2. Model for cytokinesis node assembly and architecture. Protein distances, stoichiometries (except Cdc12), and timings of appearance are depicted (Laporte et al., J. Cell Biol. 2011).

Significance of Studying Cytokinesis
Cytokinesis is essential for cell proliferation and differentiation. Cells carefully regulate cytokinesis because failure of cytokinesis can lead to tetraploidy, a prelude to aneuploidy and transformation of normal cells into cancer cells. Cancer cells often misregulate structural and regulatory components of cytokinesis. In contrast, regulated inhibition of cytokinesis contributes to normal mammalian development and tissue repair, such as in the proliferation and differentiation of polyploid hepatocytes, megakaryocytes, certain brain cells, and other cell types. Because most proteins involved in cytokinesis are evolutionarily conserved, principles established in fission yeast can be applied to higher eukaryotes, including humans. In addition, aspects of cytokinesis specific to fungi are excellent targets for anti-fungal drugs that have minimal impact on mammalian hosts. This latter point is important because ~1.2 billion people worldwide suffer from fungal diseases and 1.5 to 2 million die of a fungal infection every year. Inhibitors (caspofungin, micafungin, Aculeacin A, and others) of conserved β-glucan synthases that make the septum for cytokinesis are used to treat fungal infections, but anti-fungal drug development has largely stalled since the 1990s and drug resistance is emerging. Novel and basic knowledge of septum/cell wall synthesis is urgently needed. Thus, discoveries on both conserved and organism-specific attributes of cytokinesis may be harnessed to improve human health.

 

Current Research Projects

Project 1. Characterize the molecular mechanism of contractile-ring constriction.
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, a-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.

Project 2. Characterize the molecular mechanism that coordinates contractile-ring constriction and plasma-membrane deposition.
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.

Project 3. Elucidate the molecular mechanism that coordinates contractile-ring constriction and septum formation.
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.

Project 4. Investigate how Rho GTPases regulate cytokinesis.
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.

Project 5. Elucidate the molecular mechanism of single-cell wound healing.
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.

 

Current Research Support

  • 2016-2020     R01 grant GM118746 from the National Institutes of Health
  • 2013-2018     American Cancer Society, Research Scholar Grant RSG-13-005-01-CCG

 

Publications

Areas of Expertise
  • Cytokinesis and single-cell wound healing
Education
  • Ph.D. - The University of North Carolina at Chapel Hill
  • Postdoctoral - Yale University

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614 292 4466
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Columbus, OH 43210