Involvement of the septation initiation network in events during cytokinesis in fission yeast.
ABSTRACT: The septation initiation network (SIN), comprising a GTPase and a cascade of three protein kinases, regulates cell division in fission yeast Schizosaccharomyces pombe, but questions remain about its influence on cytokinesis. Here, we made quantitative measurements of the numbers of Cdc7p kinase molecules (a marker for SIN activity) on spindle pole bodies (SPBs), and on the timing of assembly, maturation and constriction of contractile rings via six different proteins tagged with fluorescent proteins. When SIN activity is low in spg1-106 mutant cells at 32°C, cytokinetic nodes formed contractile rings ?3?min slower than wild-type cells. During the maturation period, these rings maintained normal levels of the myosin-II mEGFP-Myo2p but accumulated less of the F-BAR protein Cdc15p-GFP than in wild-type cells. The Cdc15p-GFP fluorescence then disintegrated into spots as mEGFP-Myo2p dissociated slowly. Some rings started to constrict at the normal time, but most failed to complete constriction. When high SIN activity persists far longer than normal on both SPBs in cdc16-116 mutant cells at 32°C, contractile rings assembled and constricted normally, but disassembled slowly, delaying cell separation.
Project description:Cytokinesis in many eukaryotes involves the function of an actomyosin-based contractile ring. In fission yeast, actomyosin ring maturation and stability require a conserved signaling pathway termed the SIN (septation initiation network). The SIN consists of a GTPase (Spg1p) and three protein kinases, all of which localize to the mitotic spindle pole bodies (SPBs). Two of the SIN kinases, Cdc7p and Sid1p, localize asymmetrically to the newly duplicated SPB in late anaphase. How this asymmetry is achieved is not understood, although it is known that their symmetric localization impairs cytokinesis.Here we characterize a new Forkhead-domain-associated protein, Csc1p, and identify SIN-inhibitory PP2A complex (SIP), which is crucial for the establishment of SIN asymmetry. Csc1p localizes to both SPBs early in mitosis, is lost from the SPB that accumulates Cdc7p, and instead accumulates at the SPB lacking Cdc7p. Csc1p is required for the dephosphorylation of the SIN scaffolding protein Cdc11p and is thereby required for the recruitment of Byr4p, a component of the GTPase-activating subunit for Spg1p, to the SPB.Because Cdc7p does not bind to GDP-Spg1p, we propose that the SIP-mediated Cdc11p dephosphorylation and the resulting recruitment of Byr4p are among the earliest steps in the establishment of SIN asymmetry.
Project description:In most cell types, mitosis and cytokinesis are tightly coupled such that cytokinesis occurs only once per cell cycle. The fission yeast Schizosaccharomyces pombe divides using an actomyosin-based contractile ring and is an attractive model for the study of the links between mitosis and cytokinesis. In fission yeast, the anaphase-promoting complex/cyclosome (APC/C) and the septation initiation network (SIN), a spindle pole body (SPB)-associated GTPase-driven signaling cascade, function sequentially to ensure proper coordination of mitosis and cytokinesis. Here, we find a novel interplay between the tetratricopeptide repeat (TPR) domain-containing subunit of the APC/C, Nuc2p, and the SIN, that appears to not involve other subunits of the APC/C. Overproduction of Nuc2p led to an increase in the presence of multinucleated cells, which correlated with a defect in actomyosin ring maintenance and localization of the SIN component protein kinases Cdc7p and Sid1p to the SPBs, indicative of defective SIN signaling. Conversely, loss of Nuc2p function led to increased SIN signaling, characterized by the persistent localization of Cdc7p and Sid1p on SPBs and assembly of multiple actomyosin rings and division septa. Nuc2p appears to function independently of the checkpoint with FHA and ring finger (CHFR)-related protein Dma1p, a known inhibitor of the SIN in fission yeast. Genetic and biochemical analyses established that Nuc2p might influence the nucleotide state of Spg1p GTPase, a key regulator of the SIN. We propose that Nuc2p, by inhibiting the SIN after cell division, prevents further deleterious cytokinetic events, thereby contributing to genome stability.
Project description:The molecular organization of cytokinesis proteins governs contractile ring function. We used single molecule localization microscopy in live cells to elucidate the molecular organization of cytokinesis proteins and relate it to the constriction rate of the contractile ring. Wild-type fission yeast cells assemble contractile rings by the coalescence of cortical proteins complexes called nodes whereas cells without Anillin/Mid1p (Δmid1) lack visible nodes yet assemble contractile rings competent for constriction from the looping of strands. We leveraged the Δmid1 contractile ring assembly mechanism to determine how two distinct molecular organizations, nodes versus strands, can yield functional contractile rings. Contrary to previous interpretations, nodes assemble in Δmid1 cells. Our results suggest that Myo2p heads condense upon interaction with actin filaments and an excess number of Myo2p heads bound to actin filaments hinders constriction thus reducing the constriction rate. Our work establishes a predictive correlation between the molecular organization of nodes and the behavior of the contractile ring.
Project description:Spatial and temporal regulation of cytokinesis is essential for cell division, yet the mechanisms that control the formation and constriction of the contractile ring are incompletely understood. In the fission yeast Schizosaccharomyces pombe proteins that contribute to the cytokinetic contractile ring accumulate during interphase in nodes-precursor structures around the equatorial cortex. During mitosis, additional proteins join these nodes, which condense to form the contractile ring. The cytokinesis protein Blt1p is unique in being present continuously in nodes from early interphase through to the contractile ring until cell separation. Blt1p was shown to stabilize interphase nodes, but its functions later in mitosis were unclear. We use analytical ultracentrifugation to show that purified Blt1p is a tetramer. We find that Blt1p interacts physically with Sid2p and Mob1p, a protein kinase complex of the septation initiation network, and confirm known interactions with F-BAR protein Cdc15p. Contractile rings assemble normally in blt1? cells, but the initiation of ring constriction and completion of cell division are delayed. We find three defects that likely contribute to this delay. Without Blt1p, contractile rings recruited and retained less Sid2p/Mob1p and Clp1p phosphatase, and ?-glucan synthase Bgs1p accumulated slowly at the cleavage site.
Project description:We observed live fission yeast expressing pairs of functional fluorescent fusion proteins to test the popular model that the cytokinetic contractile ring assembles from a single myosin II progenitor or a Cdc12p-Cdc15p spot. Under our conditions, the anillin-like protein Mid1p establishes a broad band of small dots or nodes in the cortex near the nucleus. These nodes mature by the addition of conventional myosin II (Myo2p, Cdc4p, and Rlc1p), IQGAP (Rng2p), pombe Cdc15 homology protein (Cdc15p), and formin (Cdc12p). The nodes coalesce laterally into a compact ring when Cdc12p and profilin Cdc3p stimulate actin polymerization. We did not observe assembly of contractile rings by extension of a leading cable from a single spot or progenitor. Arp2/3 complex and its activators accumulate in patches near the contractile ring early in anaphase B, but are not concentrated in the contractile ring and are not required for assembly of the contractile ring. Their absence delays late steps in cytokinesis, including septum formation and cell separation.
Project description:The mechanics that govern the constriction of the contractile ring remain poorly understood yet are critical to understanding the forces that drive cytokinesis. We used laser ablation in fission yeast cells to unravel these mechanics focusing on the role of Cdc15p as a putative anchoring protein. Our work shows that the severed constricting contractile ring recoils to a finite point leaving a gap that can heal if less than ∼1 µm. Severed contractile rings in Cdc15p-depleted cells exhibit an exaggerated recoil, which suggests that the recoil is limited by the anchoring of the ring to the plasma membrane. Based on a physical model of the severed contractile ring, we propose that Cdc15p impacts the stiffness of the contractile ring more than the viscous drag.
Project description:We investigated the role of regulatory light-chain (Rlc1p) and heavy-chain phosphorylation in controlling fission yeast myosin-II (Myo2p) motor activity and function during cytokinesis. Phosphorylation of Rlc1p leads to a fourfold increase in Myo2p's in vitro motility rate, which ensures effective contractile ring constriction and function. Surprisingly, unlike with smooth muscle and nonmuscle myosin-II, RLC phosphorylation does not influence the actin-activated ATPase activity of Myo2p. A truncated form of Rlc1p lacking its extended N-terminal regulatory region (including phosphorylation sites) supported maximal Myo2p in vitro motility rates and normal contractile ring function. Thus, the unphosphorylated N-terminal extension of Rlc1p can uncouple the ATPase and motility activities of Myo2p. We confirmed the identity of one out of two putative heavy-chain phosphorylation sites previously reported to control Myo2p function and cytokinesis. Although in vitro studies indicated that phosphorylation at Ser-1444 is not needed for Myo2p motor activity, phosphorylation at this site promotes the initiation of contractile ring constriction.
Project description:In animal cells, cytokinesis occurs by constriction of an actomyosin ring. In fission yeast cells, ring constriction is triggered by the septum initiation network (SIN), an SPB-associated GTPase-regulated kinase cascade that coordinates exit from mitosis with cytokinesis. We have identified a novel protein, Etd1p, required to trigger actomyosin ring constriction in fission yeasts. This protein is localised at the cell tips during interphase. In mitosis, it relocates to the medial cortex region and, coincident with cytokinesis, it assembles into the actomyosin ring by association to Cdc15p. Relocation of Etd1p from the plasma membrane to the medial ring is triggered by SIN signalling and, reciprocally, relocation of the Sid2p-Mob1p kinase complex from the SPB to the division site, a late step in the execution of the SIN, requires Etd1p. These results suggest that Etd1p coordinates the mitotic activation of SIN with the initiation of actomyosin ring constriction. Etd1p peaks during cytokinesis and is degraded by the ubiquitin-dependent 26S-proteasome pathway at the end of septation, providing a mechanism to couple inactivation of SIN to completion of cytokinesis.
Project description:In fission yeast cells cortical nodes containing the protein Blt1p and several kinases appear early in G2, mature into cytokinetic nodes by adding anillin Mid1p, myosin-II, formin Cdc12p, and other proteins, and condense into a contractile ring by movements that depend on actin and myosin-II. Previous studies concluded that cells without Mid1p lack cytokinetic nodes and assemble rings unreliably from myosin-II strands but left open questions. Why do strands form outside the equatorial region? Why is ring assembly unreliable without Mid1p? We found in ?mid1 cells that Cdc12p accumulates in cytokinetic nodes scattered in the cortex and produces actin filaments that associate with myosin-II, Rng2p, and Cdc15p to form strands located between the nodes. Strands incorporate nodes, and in ~67% of cells, strands slowly close into rings that constrict without the normal ~25-min maturation period. Ring assembly is unreliable and slow without Mid1p because the scattered Cdc12p nodes generate strands spread widely beyond the equator, and growing strands depend on random encounters to merge with other strands into a ring. We conclude that orderly assembly of the contractile ring in wild-type cells depends on Mid1p to recruit myosin-II, Rng2p, and Cdc15p to nodes and to place cytokinetic nodes around the cell equator.
Project description:Myosin-II (Myo2p) and tropomyosin are essential for contractile ring formation and cytokinesis in fission yeast. Here we used a combination of in vivo and in vitro approaches to understand how these proteins function at contractile rings. We find that ring assembly is delayed in Myo2p motor and tropomyosin mutants, but occurs prematurely in cells engineered to express two copies of myo2. Thus, the timing of ring assembly responds to changes in Myo2p cellular levels and motor activity, and the emergence of tropomyosin-bound actin filaments. Doubling Myo2p levels suppresses defects in ring assembly associated with a tropomyosin mutant, suggesting a role for tropomyosin in maximizing Myo2p function. Correspondingly, tropomyosin increases Myo2p actin affinity and ATPase activity and promotes Myo2p-driven actin filament gliding in motility assays. Tropomyosin achieves this by favoring the strong actin-bound state of Myo2p. This mode of regulation reflects a role for tropomyosin in specifying and stabilizing actomyosin interactions, which facilitates contractile ring assembly in the fission yeast system.