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

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

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

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

Equatorial assembly of the cell-division actomyosin ring in the absence of cytokinetic spatial cues

The position of the division site dictates the size and fate of daughter cells in many organisms. In animal cells, division-site placement involves overlapping mechanisms, including signaling from the central spindle microtubules, astral microtubules, and spindle poles and through polar contractions [1, 2, 3]. In fission yeast, division-site positioning requires overlapping mechanisms involving the anillin-related protein Mid1 and the tip complex (comprising the Kelch-repeat protein Tea1, the Dyrk-kinase Pom1, and the SH3-domain protein Tea4) [4, 5, 6, 7, 8, 9, 10, 11]. In addition to these factors, cell shape has also been shown to participate in the maintenance of the position of the actomyosin ring [12, 13, 14]. The first principles guiding actomyosin ring placement, however, have not been elucidated in any organism. Because actomyosin ring positioning, ring assembly, and cell morphogenesis are genetically separable in fission yeast, we have used it to derive actomyosin ring placement mechanisms from first principles. We report that, during ring assembly in the absence of cytokinetic cues (anillin-related Mid1 and tip-complex proteins), actin bundles follow the path of least curvature and assemble actomyosin rings in an equatorial position in spherical protoplasts and along the long axis in cylindrical cells and compressed protoplasts. The equatorial position of rings is abolished upon treatment of protoplasts with an actin-severing compound or by slowing down actin polymerization. We propose that the physical properties of actin filaments/bundles play key roles in actomyosin ring assembly and positioning, and that key cytokinetic molecules may modulate the length of actin filaments to promote ring assembly along the short axis

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

Motor activity dependent and independent functions for Myosin II in cytokinesis contribute to actomyosin ring assembly and contraction in Schizosaccharomyces pombe

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

Rapid production of pure recombinant actin isoforms in Pichia pastoris

Actins are major eukaryotic cytoskeletal proteins, which perform 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 presently for biochemical studies of actin cytoskeleton from other organisms / cell types. 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 using 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 S. cerevisiae, S. 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 actin dendritic 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

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

Phosphoregulation of tropomyosin is crucial for actin cable turnover and division site placement

Tropomyosin is a coiled-coil actin binding protein key to the stability of actin filaments. Whereas, in muscle cells, tropomyosin is subject to calcium regulation, its regulation in non-muscle cells is not understood. Here, we provide evidence that the fission yeast tropomyosin, Cdc8, is regulated by phosphorylation of a serine residue. Failure of phosphorylation leads to an increased number and stability of actin cables and causes misplacement of the division site in certain genetic backgrounds. Phosphorylation of Cdc8 weakens its interaction with actin filaments. Furthermore, we show through in vitro reconstitution that phosphorylation-mediated release of Cdc8 from actin filaments facilitates access of the actin severing protein Adf1 and subsequent filament disassembly. These studies establish that phosphorylation may be a key mode of regulation of non-muscle tropomyosins, which in fission yeast controls actin filament stability and division site placement

Molecular Tuning of Actin Dynamics in Leukocyte Migration as Revealed by Immune-Related Actinopathies

International audienceMotility is a crucial activity of immune cells allowing them to patrol tissues as they differentiate, sample or exchange information, and execute their effector functions. Although all immune cells are highly migratory, each subset is endowed with very distinct motility patterns in accordance with functional specification. Furthermore individual immune cell subsets adapt their motility behaviour to the surrounding tissue environment. This review focuses on how the generation and adaptation of diversified motility patterns in immune cells is sustained by actin cytoskeleton dynamics. In particular, we review the knowledge gained through the study of inborn errors of immunity (IEI) related to actin defects. Such pathologies are unique models that help us to uncover the contribution of individual actin regulators to the migration of immune cells in the context of their development and function

Difficulties in Generating Specific Antibodies for Immunohistochemical Detection of Nitrosylated Tubulins

<div><p>Protein S-nitrosylation, the covalent attachment of a nitroso moiety to thiol groups of specific cysteine residues, is one of the major pathways of nitric oxide signaling. Hundreds of proteins are subject to this transient post-translational modification and for some the functional consequences have been identified. Biochemical assays for the analysis of protein S-nitrosylation have been established and can be used to study if and under what conditions a given protein is S-nitrosylated. In contrast, the equally desirable subcellular localization of specific S-nitrosylated protein isoforms has not been achieved to date. In the current study we attempted to specifically localize S-nitrosylated α- and β-tubulin isoforms in primary neurons after fixation. The approach was based on <i>in situ</i> replacement of the labile cysteine nitroso modification with a stable tag and the subsequent use of antibodies which recognize the tag in the context of the tubulin polypeptide sequence flanking the cysteine residue of interest. We established a procedure for tagging S-nitrosylated proteins in cultured primary neurons and obtained polyclonal anti-tag antibodies capable of specifically detecting tagged proteins on immunoblots and in fixed cells. However, the antibodies were not specific for tubulin isoforms. We suggest that different tagging strategies or alternative methods such as fluorescence resonance energy transfer techniques might be more successful.</p></div