Feedback Regulation of SIN by Etd1 and Rho1 in Fission Yeast

Abstract

In fission yeast, the septation initiation network (SIN) is thought to promote cytokinesis by downstream activation of Rho1, a conserved GTPase that controls cell growth and division. Here we show that Etd1 and PP2A-Pab1, antagonistic regulators of SIN, are Rho1 regulators. Our genetic and biochemical studies indicate that a C-terminal region of Etd1 may activate Rho1 by directly binding it, whereas an N-terminal domain confers its ability to localize at the growing tips and the division site where Rho1 functions. In opposition to Etd1, our results indicate that PP2A-Pab1 inhibits Rho1. The SIN cascade is upstream-regulated by the Spg1 GTPase. In the absence of Etd1, activity of Spg1 drops down prematurely, thereby inactivating SIN. Interestingly, we find that ectopic activation of Rho1 restores Spg1 activity in Etd1-depleted cells. By using a cytokinesis block strategy, we show that Rho1 is essential to feedbackactivate Spg1 during actomyosin ring constriction. Therefore, activation of Spg1 by Rho1, which in turn is regulated by Etd1, uncovers a novel feedback loop mechanism that ensures SIN activity while cytokinesis is progressing.F ISSION yeast cells grow by elongating at their cell ends before dividing by forming a medially placed division septum during cytokinesis. New-born cells initiate growth in a monopolar manner from the old cell end that existed before division. During G2, cells initiate growth from the new end in a process known as NETO (new end take-off). These cells then grow in a bipolar mode until mitosis, when they stop growth and initiate cell division by septation (Mitchison and Nurse 1985;Hayles and Nurse 2001). Both polar growth and septation require proper cell membrane and cell-wall synthesis to ensure cell integrity. In Schizosaccharomyces pombe, the cell wall is composed of b-glucan, a-glucan, and mannoproteins. Synthesis of the major cell wall and primary septum polymer, 1,3-b-glucan, requires the activity of Drc1/Cps1/Bgs1 1,3-b-glucan synthase, which in turn is regulated by the guanosine triphosphate (GTP)-binding protein Rho1 (Garcia et al. 2006;Cortes et al. 2007). As with all GTPases, Rho1 acts as a binary switch, cycling between inactive GDP-bound and active GTPbound conformational states (Yang et al. 2003). This transition is controlled by three types of proteins: GEFs (guanine exchange factors), which catalyze the exchange of GDP for GTP, rendering the protein active; GAPs (GTPase activating proteins), which enhance nucleotide hydrolysis, inactivating the GTPase; and guanine nucleotide dissociation inhibitors, which appear to block spontaneous activation (Garcia et al. 2006).Upon the onset of mitosis, growth ceases at the cell ends and is re-established in the middle of the cell, where the actomyosin ring forms. At the end of mitosis, once the two sets of chromosomes have segregated, actomyosin ring constriction and septum formation is triggered by the septation initiation network (SIN) from the spindle pole body (SPB) (Krapp et al. 2004;Wolfe and Gould 2005). The nucl...

Spatial control of translation repression and polarized growth by conserved NDR kinase Orb6 and RNA-binding protein Sts5

“…SIN signaling remains active until completion of cytokinesis, which is marked by closure of the contractile ring and a fully formed cell septum (García-Cortés and McCollum, 2009; Alcaide-Gavilán et al, 2014). After cytokinesis, the primary septum must be degraded for cell separation to occur.…”

Analysis ofS. pombeSIN protein SPB-association reveals two genetically separable states of the SIN

“…Previous studies have indicated that Etd1p is required both for maintenance of Cdc7p-GFP at the SPBs in anaphase and for the intensity increase of Cdc7p-GFP in anaphase (Alcaide-Gavilan et al, 2013;Daga et al, 2005;Lahoz et al, 2010). Although these studies differ qualitatively in their conclusions, they both implicate Etd1p in establishing the late state of the SIN.…”

Cytokinesis: Robust cell shape regulation

“…In fission yeast, the contractile network assembly is triggered by the septation initiation network (SIN) through the activation of Rho1 GTPase. Recent evidence has shown that Rho1 can also affect upstream regulators of SIN, thereby providing feedback to control actomyosin assembly and furrow ingression [64]. Thus, different systems have evolved intricate methods to ensure that cytokinesis proceeds with high fidelity.…”