Saravanan Palani

Actomyosin ring driven cytokinesis in budding yeast

Cytokinesis is the final process in the cell cycle that physically divides one cell into two. In budding yeast, cytokinesis is driven by a contractile actomyosin ring (AMR) and the simultaneous formation of a primary septum, which serves as template for cell wall deposition. AMR assembly, constriction, primary septum formation and cell wall deposition are successive processes and tightly coupled to cell cycle progression to ensure the correct distribution of genetic material and cell organelles among the two rising cells prior to cell division. The role of the AMR in cytokinesis and the molecular mechanisms that drive AMR constriction and septation are the focus of current research. This review summarizes the recent progresses in our understanding of how budding yeast cells orchestrate the multitude of molecular mechanisms that control AMR driven cytokinesis in a spatio-temporal manner to achieve an error free cell division.

Isolation of Cytokinetic Actomyosin Rings from Saccharomyces cerevisiae and Schizosaccharomyces pombe.

Cytokinesis is the final stage of cell division, through which cellular constituents of mother cells are partitioned into two daughter cells resulting in the increase in cell number. In animal and fungal cells cytokinesis is mediated by an actomyosin contractile ring, which is attached to the overlying cell membrane. Contraction of this ring after chromosome segregation physically severs the mother cell into two daughters. Here we describe methods for the isolation and partial purification of the actomyosin ring from the fission yeast Schizosaccharomyces pombe and the budding yeast Saccharomyces cerevisiae, which can serve as in vitro systems to facilitate biochemical and ultrastructural analysis of cytokinesis in these genetically tractable model systems.

Curvature-induced expulsion of actomyosin bundles during cytokinetic ring contraction

Many eukaryotes assemble a ring-shaped actomyosin network that contracts to drive cytokinesis. Unlike actomyosin in sarcomeres, which cycles through contraction and relaxation, the cytokinetic ring disassembles during contraction through an unknown mechanism. Here we find in Schizosaccharomyces japonicus and Schizosaccharomyces pombe that, during actomyosin ring contraction, actin filaments associated with actomyosin rings are expelled as micron-scale bundles containing multiple actomyosin ring proteins. Using functional isolated actomyosin rings we show that expulsion of actin bundles does not require continuous presence of cytoplasm. Strikingly, mechanical compression of actomyosin rings results in expulsion of bundles predominantly at regions of high curvature. Our work unprecedentedly reveals that the increased curvature of the ring itself promotes its disassembly. It is likely that such a curvature-induced mechanism may operate in disassembly of other contractile networks. DOI: http://dx.doi.org/10.7554/eLife.21383.001

Myo2p is the major motor involved in actomyosin ring contraction in fission yeast

Summary Cytokinesis in many eukaryotes requires an actomyosin-based contractile ring [1]. In fission yeast, cytokinesis involves the type II myosins Myo2p and Myp2p and the type V myosin Myo51p [2]. A recent study by Laplante et al.[3], using deletion mutants of myp2 and myo51 and the mis-sense mutant myo2-E1 [4], concluded that each myosin has distinct functions and proposed that Myp2p plays the dominant role in actomyosin ring contraction. Here we present evidence that Myo2p, not Myp2p, is likely to be the major motor driving actomyosin ring contractility. Since the previous work [3] was performed at 25°C, the permissive temperature for myo2-E1, we compared cytokinesis timings in myo2-E1 and myo2Δ at 25°C and found that myo2-E1 is only partially compromised at 25°C. Furthermore, we find that myp2Δ and myp2Δ myo51Δ double mutants contract actomyosin rings at ∼90% of the rate of wild-type cells at 30°C and 36°C, suggesting that Myp2p plays a minimal role in ring contraction at these temperatures. Finally, ring contraction in our myo2-E1 strain took longer at 25°C than previously reported [3]. Although faster-acting alleles of myo2 will be required to evaluate its contribution at 25°C, our work establishes that Myo2p is the major motor involved in ring contraction, under most, if not all, conditions.

Motor Activity Dependent and Independent Functions of Myosin II Contribute to Actomyosin Ring Assembly and Contraction in Schizosaccharomyces pombe

Summary Cytokinesis depends on a contractile actomyosin ring in many eukaryotes [1, 2, 3]. Myosin II is a key component of the actomyosin ring, although whether it functions as a motor or as an actin cross-linker to exert its essential role is disputed [1, 4, 5]. In Schizosaccharomyces pombe, the myo2-E1 mutation affects the upper 50 kDa sub-domain of the myosin II heavy chain, and cells carrying this lethal mutation are defective in actomyosin ring assembly at the non-permissive temperature [6, 7]. myo2-E1 also affects actomyosin ring contraction when rings isolated from permissive temperature-grown cells are incubated with ATP [8]. Here we report isolation of a compensatory suppressor mutation in the lower 50 kDa sub-domain (myo2-E1-Sup1) that reverses the inability of myo2-E1 to form colonies at the restrictive temperature. myo2-E1-Sup1 is capable of assembling normal actomyosin rings, although rings isolated from myo2-E1-Sup1 are defective in ATP-dependent contraction in vitro. Furthermore, the product of myo2-E1-Sup1 does not translocate actin filaments in motility assays in vitro. Superimposition of myo2-E1 and myo2-E1-Sup1 on available rigor and blebbistatin-bound myosin II structures suggests that myo2-E1-Sup1 may represent a novel actin translocation-defective allele. Actomyosin ring contraction and viability of myo2-E1-Sup1 cells depend on the late cytokinetic S. pombe myosin II isoform, Myp2p, a non-essential protein that is normally dispensable for actomyosin ring assembly and contraction. Our work reveals that Myo2p may function in two different and essential modes during cytokinesis: a motor activity-independent form that can promote actomyosin ring assembly and a motor activity-dependent form that supports ring contraction.

A New Membrane Protein Sbg1 Links the Contractile Ring Apparatus and Septum Synthesis Machinery in Fission Yeast

Cytokinesis in many organisms requires a plasma membrane anchored actomyosin ring, whose contraction facilitates cell division. In yeast and fungi, actomyosin ring constriction is also coordinated with division septum assembly. How the actomyosin ring interacts with the plasma membrane and the plasma membrane-localized septum synthesizing machinery remains poorly understood. In Schizosaccharomyces pombe, an attractive model organism to study cytokinesis, the β-1,3-glucan synthase Cps1p / Bgs1p, an integral membrane protein, localizes to the plasma membrane overlying the actomyosin ring and is required for primary septum synthesis. Through a high-dosage suppressor screen we identified an essential gene, sbg1+ (suppressor of beta glucan synthase 1), which suppressed the colony formation defect of Bgs1-defective cps1-191 mutant at higher temperatures. Sbg1p, an integral membrane protein, localizes to the cell ends and to the division site. Sbg1p and Bgs1p physically interact and are dependent on each other to localize to the division site. Loss of Sbg1p results in an unstable actomyosin ring that unravels and slides, leading to an inability to deposit a single contiguous division septum and an important reduction of the β-1,3-glucan proportion in the cell wall, coincident with that observed in the cps1-191 mutant. Sbg1p shows genetic and / or physical interaction with Rga7p, Imp2p, Cdc15p, and Pxl1p, proteins known to be required for actomyosin ring integrity and efficient septum synthesis. This study establishes Sbg1p as a key member of a group of proteins that link the plasma membrane, the actomyosin ring, and the division septum assembly machinery in fission yeast.

Actin turnover maintains actin filament homeostasis during cytokinetic ring contraction

Cytokinesis in many eukaryotes involves a tension-generating actomyosin-based contractile ring. Many components of actomyosin rings turn over during contraction, although the significance of this turnover has remained enigmatic. Here, using Schizosaccharomyces japonicus, we investigate the role of turnover of actin and myosin II in its contraction. Actomyosin ring components self-organize into ∼1-µm-spaced clusters instead of undergoing full-ring contraction in the absence of continuous actin polymerization. This effect is reversed when actin filaments are stabilized. We tested the idea that the function of turnover is to ensure actin filament homeostasis in a synthetic system, in which we abolished turnover by fixing rings in cell ghosts with formaldehyde. We found that these rings contracted fully upon exogenous addition of a vertebrate myosin. We conclude that actin turnover is required to maintain actin filament homeostasis during ring contraction and that the requirement for turnover can be bypassed if homeostasis is achieved artificially.

Detection of phosphorylation status of cytokinetic components

Yeast cells can be easily cultured, synchronized, and genetically modified making them a convenient model system to study molecular mechanisms underlying cytokinesis. Here, we describe simple methods that allow the analysis of the phosphorylation profile of cytokinetic proteins, both in vivo and in vitro, using standard laboratory equipment. In addition, we compare the ability of three different, standard, and optimized acrylamide gel conditions to separate phosphorylated forms, using the protein Inn1 as an example.

Steric hindrance in the upper 50 kDa domain of the motor Myo2p leads to cytokinesis defects in fission yeast

ABSTRACT Cytokinesis in many eukaryotes requires a contractile actomyosin ring that is placed at the division site. In fission yeast, which is an attractive organism for the study of cytokinesis, actomyosin ring assembly and contraction requires the myosin II heavy chain Myo2p. Although myo2-E1, a temperature-sensitive mutant defective in the upper 50 kDa domain of Myo2p, has been studied extensively, the molecular basis of the cytokinesis defect is not understood. Here, we isolate myo2-E1-Sup2, an intragenic suppressor that contains the original mutation in myo2-E1 (G345R) and a second mutation in the upper 50 kDa domain (Y297C). Unlike myo2-E1-Sup1, a previously characterized myo2-E1 suppressor, myo2-E1-Sup2 reverses actomyosin ring contraction defects in vitro and in vivo. Structural analysis of available myosin motor domain conformations suggests that a steric clash in myo2-E1, which is caused by the replacement of a glycine with a bulky arginine, is relieved in myo2-E1-Sup2 by mutation of a tyrosine to a smaller cysteine. Our work provides insight into the function of the upper 50 kDa domain of Myo2p, informs a molecular basis for the cytokinesis defect in myo2-E1, and may be relevant to the understanding of certain cardiomyopathies. Summary: An unbiased genetic screen uncovers a mutation that reverses a steric clash in the myosin II motor head, which was previously reported to cause defective actomyosin ring-dependent cytokinesis in fission yeast.

Rapid production of pure recombinant actin isoforms in Pichia pastoris

ABSTRACT Actins are major eukaryotic cytoskeletal proteins, and they are involved in many important cell functions, including cell division, cell polarity, wound healing and muscle contraction. Despite obvious drawbacks, muscle actin, which is easily purified, is used extensively for biochemical studies of the non-muscle actin cytoskeleton. Here, we report a rapid and cost-effective method to purify heterologous actins expressed in the yeast Pichia pastoris. Actin is expressed as a fusion with the actin-binding protein thymosin β4 and purified by means of an affinity tag introduced in the fusion. Following cleavage of thymosin β4 and the affinity tag, highly purified functional full-length actin is liberated. We purify actins from Saccharomyces cerevisiae and Schizosaccharomyces pombe, and the β- and γ-isoforms of human actin. We also report a modification of the method that facilitates expression and purification of arginylated actin, a form of actin thought to regulate dendritic actin networks in mammalian cells. The methods we describe can be performed in all laboratories equipped for molecular biology, and should greatly facilitate biochemical and cell biological studies of the actin cytoskeleton. Summary:Here, we describe a method to purify recombinant actin to homogeneity by expression in Pichia pastoris. The purified actin is polymerisation competent and should facilitate biochemical and cell biological studies of the actin cytoskeleton.