Actomyosin Ring Assembly Research Articles

Coordinated cortical ER remodeling facilitates actomyosin ring assembly

The cortical endoplasmic reticulum (cER) is a reticulated network closely attached to the plasma membrane (PM). In the fission yeast Schizosaccharomyces pombe (S.pombe), ER-PM contacts have been suggested to restrict both the allocation and compaction of large-sized actomyosin assemblies along the lateral cell cortex. However, how cells orchestrate ER-PM contact remodeling in accordance with actomyosin coalescence for contractile ring assembly is unclear. Here, we reveal that actomyosin compaction directs the remodeling of the free tubular cER edges, whereas active exocytosis subsequently promotes the reorganization of the eisosome-bound cER rims by weakening their association or repatterning the eisosome-coated PM furrows. cER-eisosome contacts also act to reserve tubular cER edges and, hence, the ER shaping machinery at the lateral cell cortex. By manipulating or rerouting exocytosis in mutants with compromised actomyosin compaction, due to either the loss of myosin II activity or sheet-like cER morphology, we show that exocytosis facilitates ring formation likely by creating free tubular cER rims allowing robust cER remodeling. We thus propose that coordinated cER remodeling driven by both actomyosin forces and active exocytosis ensures proper contractile ring assembly. Our work also provides mechanistic insights into cER-related modulation in actomyosin ring assembly.

Tension modulation of actomyosin ring assembly and RhoGTPases activity: Perspectives from the Xenopus oocyte wound healing model

Cells are remarkably resilient structures; they are able to recover from injuries to their plasma membrane (PM) and cytoskeleton that would normally constitute existential threats. This capacity is exemplified by Xenopus laevis oocytes which can recover from very large PM defects through exocytotic and endocytic events and can repair damaged cortical cytoskeleton structures through the formation of a contractile actomyosin ring (AMR). Formation of the AMR involves the localized Ca2+ -dependent activation of RhoA and Cdc42, and the pre-patterning of guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). However, this model fails to account for observations that suggest a link between cytoskeletal dynamics, intracellular tension, and AMR formation. It also does not explain why the formation of an AMR is not involved in the cytoskeletal repair program of adherent cells. We show here evidence for the support of tension as an essential regulatory signal for the formation of AMR. Indeed, oocytes in which global tension has been experimentally reduced were unable to form a functional AMR following injury, showing severely diminished RhoA activity at the wound site. These new insights place the cytoskeleton at the center of events involving changes in cell shape such as cytokinesis which also involves the formation and closure of an AMR.

The kinetic landscape and interplay of protein networks in cytokinesis

SummaryCytokinesis is executed by protein networks organized into functional modules. Individual proteins within each module have been characterized to various degrees. However, the collective behavior and interplay of the modules remain poorly understood. In this study, we conducted quantitative time-lapse imaging to analyze the accumulation kinetics of more than 20 proteins from different modules of cytokinesis in budding yeast. This analysis has led to a comprehensive picture of the kinetic landscape of cytokinesis, from actomyosin ring (AMR) assembly to cell separation. It revealed that the AMR undergoes biphasic constriction and that the switch between the constriction phases is likely triggered by AMR maturation and primary septum formation. This analysis also provided further insights into the functions of actin filaments and the transglutaminase-like protein Cyk3 in cytokinesis and, in addition, defined Kre6 as the likely enzyme that catalyzes β-1,6-glucan synthesis to drive cell wall maturation during cell growth and division.

Autocrine insulin pathway signaling regulates actin dynamics in cell wound repair.

Cells are exposed to frequent mechanical and/or chemical stressors that can compromise the integrity of the plasma membrane and underlying cortical cytoskeleton. The molecular mechanisms driving the immediate repair response launched to restore the cell cortex and circumvent cell death are largely unknown. Using microarrays and drug-inhibition studies to assess gene expression, we find that initiation of cell wound repair in the Drosophila model is dependent on translation, whereas transcription is required for subsequent steps. We identified 253 genes whose expression is up-regulated (80) or down-regulated (173) in response to laser wounding. A subset of these genes were validated using RNAi knockdowns and exhibit aberrant actomyosin ring assembly and/or actin remodeling defects. Strikingly, we find that the canonical insulin signaling pathway controls actin dynamics through the actin regulators Girdin and Chickadee (profilin), and its disruption leads to abnormal wound repair. Our results provide new insight for understanding how cell wound repair proceeds in healthy individuals and those with diseases involving wound healing deficiencies.

Equatorial Assembly of the Cell-Division Actomyosin Ring in the Absence of Cytokinetic Spatial Cues

SummaryThe 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.

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

ABSTRACTCytokinesis 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.

Mechanoregulated inhibition of formin facilitates contractile actomyosin ring assembly

Cytokinesis physically separates dividing cells by forming a contractile actomyosin ring. The fission yeast contractile ring has been proposed to assemble by Search-Capture-Pull-Release from cytokinesis precursor nodes that include the molecular motor type-II myosin Myo2 and the actin assembly factor formin Cdc12. By successfully reconstituting Search-Capture-Pull in vitro, we discovered that formin Cdc12 is a mechanosensor, whereby myosin pulling on formin-bound actin filaments inhibits Cdc12-mediated actin assembly. We mapped Cdc12 mechanoregulation to its formin homology 1 domain, which facilitates delivery of new actin subunits to the elongating actin filament. Quantitative modeling suggests that the pulling force of the myosin propagates through the actin filament, which behaves as an entropic spring, and thereby may stretch the disordered formin homology 1 domain and impede formin-mediated actin filament elongation. Finally, live cell imaging of mechano-insensitive formin mutant cells established that mechanoregulation of formin Cdc12 is required for efficient contractile ring assembly in vivo.

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

SummaryCytokinesis 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.

ER-PM Contacts Define Actomyosin Kinetics for Proper Contractile Ring Assembly

The cortical endoplasmic reticulum (ER), an elaborate network of tubules and cisternae [1], establishes contact sites with the plasma membrane (PM) through tethering machinery involving a set of conserved integral ER proteins [2]. The physiological consequences of forming ER-PM contacts are not fully understood. Here, we reveal a kinetic restriction role of ER-PM contacts over ring compaction process for proper actomyosin ring assembly in Schizosaccharomyces pombe. We show that fission yeast cells deficient in ER-PM contacts exhibit aberrant equatorial clustering of actin cables during ring assembly and are particularly susceptible to compromised actin filament crosslinking activity. Using quantitative image analyses and computer simulation, we demonstrate that ER-PM contacts function to modulate the distribution of ring components and to constrain their compaction kinetics. We propose that ER-PM contacts have evolved as important physical modulators to ensure robust ring assembly.

Measurements of Myosin-II Motor Activity During Cytokinesis in Fission Yeast.

Fission yeast myosin-II (Myo2p) represents the critical actin-based motor protein that drives actomyosin ring assembly and constriction during cytokinesis. We detail three different methods to measure Myo2p motor function. Actin-activated ATPases provide a readout of actomyosin ATPase motor activity in a bulk assay; actin filament motility assays reveal the speed and efficiency of myosin-driven actin filament gliding (when motors are anchored); myosin-bead motility assays reveal the speed and efficiency of myosin ensembles traveling along actin filaments (when actin is anchored). Collectively, these methods allow us to combine the standard in vivo approaches common to fission yeast with in vitro biochemical methods to learn more about the mechanistic action of myosin-II during cytokinesis.

The Putative Exchange Factor Gef3p Interacts with Rho3p GTPase and the Septin Ring during Cytokinesis in Fission Yeast

The small GTP-binding proteins of the Rho family and its regulatory proteins play a central role in cytokinetic actomyosin ring assembly and cytokinesis. Here we show that the fission yeast guanine nucleotide exchange factor Gef3p interacts with Rho3p at the division site. Gef3p contains a putative DH homology domain and a BAR/IMD-like domain. The protein localized to the division site late in mitosis, where it formed a ring that did not constrict with actomyosin ring (cytokinetic actomyosin ring) invagination; instead, it split into a double ring that resembled the septin ring. Gef3p co-localized with septins and Mid2p and required septins and Mid2p for its localization. Gef3p interacts physically with the GTP-bound form of Rho3p. Although Gef3p is not essential for cell separation, the simultaneous disruption of gef3(+) and Rho3p-interacting proteins, such as Sec8p, an exocyst component, Apm1p, a subunit of the clathrin adaptor complex or For3p, an actin-polymerizing protein, yielded cells with strong defects in septation and polarity respectively. Our results suggest that interactions between septins and Rho-GEFs provide a new targeting mechanism for GTPases in cytokinesis, in this case probably contributing to Rho3p function in vesicle tethering and vesicle trafficking in the later steps of cell separation.

Cdk1-dependent phosphorylation of Iqg1 governs actomyosin ring assembly prior to cytokinesis

Contraction of the actomyosin ring (AMR) provides the centripetal force that drives cytokinesis. In budding yeast (Saccharomyces cerevisiae), assembly and contraction of the AMR is coordinated with membrane deposition and septum formation at the bud neck. A central player in this process is Iqg1, which promotes recruitment of actin to the myosin ring and links AMR assembly with that of septum-forming components. We observed early actin recruitment in response to inhibition of cyclin-dependent kinase 1 (Cdk1) activity, and we find that the Cdk1-dependent phosphorylation state of Iqg1 is a determining factor in the timing of bud neck localization of both Iqg1 and actin, with both proteins accumulating prematurely in cells expressing nonphosphorylatable Iqg1 mutants. We also identified the primary septum regulator Hof1 as a binding partner of Iqg1, providing a regulatory link between the septation and contractile pathways that cooperate to complete cytokinesis.

UCS Protein Rng3p Is Essential for Myosin-II Motor Activity during Cytokinesis in Fission Yeast

UCS proteins have been proposed to operate as co-chaperones that work with Hsp90 in the de novo folding of myosin motors. The fission yeast UCS protein Rng3p is essential for actomyosin ring assembly and cytokinesis. Here we investigated the role of Rng3p in fission yeast myosin-II (Myo2p) motor activity. Myo2p isolated from an arrested rng3-65 mutant was capable of binding actin, yet lacked stability and activity based on its expression levels and inactivity in ATPase and actin filament gliding assays. Myo2p isolated from a myo2-E1 mutant (a mutant hyper-sensitive to perturbation of Rng3p function) showed similar behavior in the same assays and exhibited an altered motor conformation based on limited proteolysis experiments. We propose that Rng3p is not required for the folding of motors per se, but instead works to ensure the activity of intrinsically unstable myosin-II motors. Rng3p is specific to conventional myosin-II and the actomyosin ring, and is not required for unconventional myosin motor function at other actin structures. However, artificial destabilization of myosin-I motors at endocytic actin patches (using a myo1-E1 mutant) led to recruitment of Rng3p to patches. Thus, while Rng3p is specific to myosin-II, UCS proteins are adaptable and can respond to changes in the stability of other myosin motors.

Insight into Actin Organization and Function in Cytokinesis from Analysis of Fission Yeast Mutants

Actin is a key cytoskeletal protein with multiple roles in cellular processes such as polarized growth, cytokinesis, endocytosis, and cell migration. Actin is present in all eukaryotes as highly dynamic filamentous structures, such as linear cables and branched filaments. Detailed investigation of the molecular role of actin in various processes has been hampered due to the multifunctionality of the protein and the lack of alleles defective in specific processes. The actin cytoskeleton of the fission yeast, Schizosaccharomyces pombe, has been extensively characterized and contains structures analogous to those in other cell types. In this study, primarily with the view to uncover actin function in cytokinesis, we generated a large bank of fission yeast actin mutants that affect the organization of distinct actin structures and/or discrete physiological functions of actin. Our screen identified 17 mutants with specific defects in cytokinesis. Some of these cytokinesis mutants helped in dissecting the function of specific actin structures during ring assembly. Further genetic analysis of some of these actin mutants revealed multiple genetic interactions with mutants previously known to affect the actomyosin ring assembly. We also characterize a mutant allele of actin that is suppressed upon overexpression of Cdc8p-tropomyosin, underscoring the utility of this mutant bank. Another 22 mutant alleles, defective in polarized growth and/or other functions of actin obtained from this screen, are also described in this article. This mutant bank should be a valuable resource to study the physiological and biochemical functions of actin.

The Kin1 kinase and the calcineurin phosphatase cooperate to link actin ring assembly and septum synthesis in fission yeast

The Kin1 protein kinase of fission yeast, which regulates cell surface cohesiveness during interphase cell growth, is also present at the cell division site during mitosis; however, its function in cell division has remained elusive. In FK506-mediated calcineurin deficient cells, mitosis is extended and ring formation is transiently compromised but septation remains normal. Here, we show that Kin1 inhibition in these cells leads to polyseptation and defects in membrane closure. Actomyosin ring disassembly is prevented and ultimately the daughter cells fail to separate. We show that the Pmk1 MAP kinase pathway and the type V myosin Myo4 act downstream of the cytokinetic function of Kin1. Kin1 inhibition also promotes polyseptation in myo3Δ, a type II myosin heavy-chain mutant defective in ring assembly. In contrast, Kin1 inactivation rescues septation in a myosin light-chain cdc4-8 thermosensitive mutant. A structure/function analysis of the Kin1 protein sequence identified a novel motif outside the kinase domain that is important for its polarised localisation and its catalytic activity. This motif is remarkably conserved in all fungal Kin1 homologues but is absent in related kinases of metazoans. We conclude that calcineurin and Kin1 activities must be tightly coordinated to link actomyosin ring assembly with septum synthesis and membrane closure and to ensure separation of the daughter cells.

Molecular control of animal cell cytokinesis

Cytokinesis is the process by which mitotic cells physically split in two following chromosome segregation. Dividing animal cells first ingress a cytokinetic furrow and then separate the plasma membrane by abscission. The general cytological events and several conserved molecular factors involved in cytokinesis have been known for many years. However, recent progress in microscopy, chemical genetics, biochemical reconstitution and biophysical methodology has tremendously increased our understanding of the underlying molecular mechanisms. We discuss how recent insights have led to refined models of the distinct steps of animal cell cytokinesis, including anaphase spindle reorganization, division plane specification, actomyosin ring assembly and contraction, and abscission. We highlight how molecular signalling pathways coordinate the individual events to ensure faithful partitioning of the genome to emerging daughter cells.

Evidence that a septin diffusion barrier is dispensable for cytokinesis in budding yeast.

Septins are essential for cytokinesis in Saccharomyces cerevisiae, but their precise roles remain elusive. Currently, it is thought that before cytokinesis, the hourglass-shaped septin structure at the mother-bud neck acts as a scaffold for assembly of the actomyosin ring (AMR) and other cytokinesis factors. At the onset of cytokinesis, the septin hourglass splits to form a double ring that sandwiches the AMR and may function as diffusion barriers to restrict diffusible cytokinesis factors to the division site. Here, we show that in cells lacking the septin Cdc10 or the septin-associated protein Bud4, the septins form a ring-like structure at the mother-bud neck that fails to re-arrange into a double ring early in cytokinesis. Strikingly, AMR assembly and constriction, the localization of membrane-trafficking and extracellular-matrix-remodeling factors, cytokinesis, and cell-wall-septum formation all occur efficiently in cdc10Δ and bud4Δ mutants. Thus, diffusion barriers formed by the septin double ring do not appear to be critical for S. cerevisiae cytokinesis. However, an AMR mutation and a septin mutation have synergistic effects on cytokinesis and the localization of cytokinesis proteins, suggesting that tethering to the AMR and a septin diffusion barrier may function redundantly to localize proteins to the division site.

IQGAP-Related Rng2p Organizes Cortical Nodes and Ensures Position of Cell Division in Fission Yeast

Correct positioning of the cell division machinery is crucial for genomic stability and cell fate determination. The fission yeast Schizosaccharomyces pombe, like animal cells, divides using an actomyosin ring and is an attractive model to study eukaryotic cytokinesis. In S. pombe, positioning of the actomyosin ring depends on the anillin-related protein Mid1p. Mid1p arrives first at the medial cortex and recruits actomyosin ring components to node-like structures, although how this is achieved is unknown. Here we show that the IQGAP-related protein Rng2p, an essential component of the actomyosin ring, is a key element downstream of Mid1p. Rng2p physically interacts with Mid1p and is required for the organization of other actomyosin ring components into cortical nodes. Failure of localization of Rng2p to the nodes prevents medial retention of Mid1p and leads to actomyosin ring assembly in a node-independent manner at nonmedial locations. We conclude that Mid1p recruits Rng2p to cortical nodes at the division site and that Rng2p, in turn, recruits other components of the actomyosin ring to cortical nodes, thereby ensuring correct placement of the division site.

The mitosis-to-interphase transition is coordinated by cross talk between the SIN and MOR pathways in Schizosaccharomyces pombe

The mechanisms that regulate cytoskeletal remodeling during the transition between mitosis and interphase are poorly understood. In fission yeast the MOR pathway promotes actin polarization to cell tips in interphase, whereas the SIN signaling pathway drives actomyosin ring assembly and cytokinesis. We show that the SIN inhibits MOR signaling in mitosis by interfering with Nak1 kinase-mediated activation of the most downstream MOR component, the NDR family kinase Orb6. Inactivation of the MOR may be a key function of the SIN because attenuation of MOR signaling rescued the cytokinetic defects of SIN mutants and allowed weak SIN signaling to trigger ectopic cytokinesis. Furthermore, failure to inhibit the MOR is toxic when the cell division apparatus is compromised. Together, our results reveal a mutually antagonistic relationship between the SIN and MOR pathways, which is important for completion of cytokinesis and coordination of cytoskeletal remodeling at the mitosis-to-interphase transition.

The Cortical ER Network Limits the Permissive Zone for Actomyosin Ring Assembly

Precise positioning of the cellular division plane is important for accurate segregation of genetic material and determination of daughter cell fates. Here we report a surprising connection between division site positioning and the organization of the cortical endoplasmic reticulum (ER). The cortical ER is an interconnected network of flat cisternae and highly curved tubules sharing a continuous lumen. Stabilization of high curvature by reticulon and DP1 family proteins contributes to formation of tubules. We show that in the fission yeast Schizosaccharomyces pombe, the ER network is maintained by a set of three membrane proteins: reticulon/Rtn1p, DP1/Yop1p, and a newly identified evolutionarily conserved protein, Tts1p. Cells lacking the ER domain sustained by these proteins exhibit severe defects in division plane positioning as a result of abnormal dispersion of a key regulator of division site selection, Mid1p, along the cell cortex. This triggers delocalized assembly of actomyosin cables and compromises their compaction into a single medially positioned ring. We propose that the cortical ER network restricts the lateral motion of Mid1p and hence generates a permissive zone for actomyosin ring assembly precisely at the cell equator.