University of Groningen Rap 1-dependent pathways coordinate cytokinesis in Dictyostelium

Cytokinesis is the final step of mitosis when a mother cell is separated into two daughter cells. Major cytoskeletal changes are essential for cytokinesis; it is, however, not well understood how the microtubules and actomyosin cytoskeleton are exactly regulated in time and space. In this paper, we show that during the early stages of cytokinesis, in roundedup Dictyostelium discoideum cells, the small G-protein Rap1 is activated uniformly at the cell cortex. When cells begin to elongate, active Rap1 becomes restricted from the furrow region, where the myosin contractile ring is subsequently formed. In the final stages of cytokinesis, active Rap1 is only present at the cell poles. Mutant cells with decreased Rap1 activation at the poles showed strongly decreased growth rates. Hyperactivation of Rap1 results in severe growth delays and defective spindle formation in adherent cells and cell death in suspension. Furthermore, Rap mutants show aberrant regulation of the actomyosin cytoskeleton, resulting in extended furrow ingression times and asymmetrical cell division. We propose that Rap1 drives cytokinesis progression by coordinating the three major cytoskeletal components: microtubules, actin, and myosin II. Importantly, mutated forms of Rap also affect cytokinesis in other organisms, suggesting a conserved role for Rap in cell division. INTRODUCTION Cell division is a fundamental process that is required for cell pro­ liferation and differentiation of cell types. In anaphase, formation of the spindle apparatus pulls the chromosomes toward the poles of the dividing cell and triggers the beginning of cytokinesis, the final step in the separation of a mother cell into two daughter cells. Fol­ lowing the assembly of microtubule filaments in the expanding mi­ totic spindle, bundles of actin and non–muscle myosin filaments create a contractile ring that constricts the plasma membrane at the furrow region, while actin filaments are formed at the poles of the cells. This temporal and spatial regulation of the cytoskeleton is essential for the separation of the daughter cells (Glotzer, 2005; Kanada et al., 2005; Wang, 2005). Most of the current models there­ fore incorporate these global, polar, and equatorial processes in orchestrating the division process (Wang, 2005; Surcel et al., 2010); it is, however, not well understood how they are coordinated in time and space. To investigate this in more detail, we used the model organism Dictyostelium discoideum. Cell division is one example of cell polarization that depends on the organization of the cytoskeletal elements. Establishment and maintenance of eukaryotic cell polarization can be induced by small fluctuations of an asymmetric stimulus. A stimulus can be either intra­ cellular, like signals from the dividing nucleus during cell division, or extracellular, such as chemical gradients during chemotaxis. Similar processes are involved in establishing polarity during chemotaxis and in regulating asymmetry during cytokinesis (Wang, 2005). Myosin II filaments and cortexillin are localized at the back of chemotactic cells and at the furrow of dividing cells, whereas PIP3 (phosphatidylinositol (3,4,5)­triphosphate), dynacortin, coronin, Scar, and active Ras and Rac are localized at the leading edge and poles during chemotaxis and cell division, respectively (Janetopoulos et al., 2005; Sasaki et al., 2007; King et al., 2010; Surcel et al., 2010). Dictyostelium Rap1 is a well­known component in establishing cell polarity and regulating cytoskeletal rearrangements during chemotaxis (Lee and Jeon, 2012). During chemotaxis, Dictyostelium Rap1 is involved in the regulation of adhesion, myosin II disassembly, and PI3K (phosphatidylinositol­3­ kinase) activation (Kortholt and van Haastert, 2008; Lee and Jeon, 2012), all processes that are also critical for cytokinesis. Consistently, Monitoring Editor Carole Parent National Institutes of Health Received: Aug 15, 2014 Revised: Sep 30, 2014 Accepted: Oct 2, 2014 This article was published online ahead of print in MBoC in Press (http://www .molbiolcell.org/cgi/doi/10.1091/mbc.E14-08-1285) on October 8, 2014. Address correspondence to: Arjan Kortholt (A.Kortholt@rug.nl). © 2014 Plak et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0). “ASCB®,” “The American Society for Cell Biology®,” and “Molecular Biology of the Cell®” are registered trademarks of The American Society for Cell Biology. Abbreviations used: BSA, bovine serum albumin; DAPI, 4′,6-diamidino-2-phenylindole; DOX, doxycycline; GFP, green fluorescent protein; MTOCs, microtubule-organizing centers; RFP, red fluorescent protein.