Actomyosin Activity Research Articles

Self-Organized Nuclear Positioning Synchronizes the Cell Cycle in Drosophila Embryos

The synchronous cleavage divisions of early embryogenesis require coordination of the cell-cycle oscillator, the dynamics of the cytoskeleton, and the cytoplasm.Yet, it remains unclear how spatially restricted biochemical signals are integrated with physical properties of the embryo to generate collective dynamics. Here, we show that synchronization of the cell cycle in Drosophila embryos requires accurate nuclear positioning, which is regulated by the cell-cycle oscillator through cortical contractility and cytoplasmic flows. We demonstrate that biochemical oscillations are initiated by local Cdk1 inactivation and spread through the activity of phosphatase PP1 to generate cortical myosin II gradients. These gradients cause cortical and cytoplasmic flows that control proper nuclear positioning. Perturbations of PP1 activity and optogenetic manipulations of cortical actomyosin disrupt nuclear spreading, resulting in loss of cell-cycle synchrony. We conclude that mitotic synchrony is established by a self-organized mechanism that integrates the cell-cycle oscillator and embryo mechanics.

The Skeletal Muscle Super Relaxed State (SRX) is Localized to the C-Zone

Under relaxing (Low/No Calcium) conditions, ATP hydrolysis by myosin in striated muscle shows two distinct subpopulations: a faster “Disordered Relaxed” (DRX) population, and a strongly inhibited “Super Relaxed” (SRX) population. This SRX state contributes to the low metabolic rate of resting muscle and SRX dysregulation may underlie some myopathies. Presently, the spatial arrangement of these two hydrolytic states at the sarcomere level is unclear, although it has been reported that Myosin Binding Protein C (MyBP-C) may stabilize the SRX state, which implies possible localization to the C-zone where MyBP-C exists. Here, we imaged single molecules of fluorescently labeled ATP being hydrolyzed by myosin in relaxed myofibrils from rat soleus skeletal muscle. First, we showed that BODIPY-ATP supports actomyosin activity comparable to non-fluorescent ATP in an in vitro motility assay. Then, we estimated the myosin BODIPY-ATP hydrolysis rate by measuring the lifetimes of individual BODIPY-ATP molecules binding to myofibrils followed presumably by the release of BODIPY-ADP. Lifetimes were well fit as two populations with lifetimes of 33.5±4.1s and 181±42s; consistent with reported kinetic rates for DRX and SRX myosin in skeletal muscle. Each individual ATP binding event was then mapped to its distance from the center of the sarcomere (M-line), as imaged by fluorescent antibody labeling of myomesin. Interestingly, ATP hydrolysis lifetimes within the C-zone (200 - 500nm from the M-line) were significantly longer than those of the flanking non-MyBP-C containing regions. Within the C-zone, hydrolysis lifetimes were well fit as two equal sized populations, with lifetimes of 34.4±14.2s and 178±54s, while in the non-MyBP-C containing regions, 95% of the events were well fit as a single population with a lifetime of 34.6±5.2s. Our data suggest that the strongly inhibited SRX myosins exist predominantly within the C-zone.

Resolving the Actin Lattice and Identifying the Relative Position of MYBP-C's N-Terminus in Cardiac Muscle using Storm Microscopy

Cardiac myosin-binding protein C (MyBP-C) is a critical modulator of cardiac muscle contractility. MyBP-C is localized to the C-zone's in the sarcomere through the binding of its C-terminal domains to the myosin rods that form the filament backbone. In vitro studies suggest MyBP-C's N-terminal domains modulate actomyosin activity through binding to actin and/or the myosin heads. Direct evidence for these potential binding partner interactions in vivo is lacking. In cross-sectional view of muscle, actin and myosin filaments form a hexagonal lattice. The relative positions of actin and MyBP-C's N-terminus were imaged within this lattice in cardiac muscle preparations using Stochastic Optical Reconstruction Microscopy (STORM) and single particle averaging. Hearts from transgenic mice expressing MyBP-C with an N-terminal Myc tag were chemically fixed, frozen in liquid nitrogen with sucrose as a cryoprotectant and thinly sliced to produce 400 nm-thick cross-sections. MyBP-C and actin were labeled with anti-Myc-Alexa647 and phalloidin-Alexa488 respectively. Two-color STORM movies were collected using total internal reflection microscopy, and images of actin and MyBP-C were reconstructed. The actin lattice was identified from the actin reconstructions by cross-correlation with a hexagonal template. Individual actin hexagons were cropped from the images and averaged to improve the resolution to 4 nm. The average distance between two adjacent actin filaments within the hexagon was 28 ± 1 nm, in agreement with the 25 ± 4 nm distance measured by negative stain EM. To identify the position of MyBP-C's Myc-tagged N-terminus, the signal from the second color channel was averaged and overlaid on the actin hexagon. The signal from MyBP-C's N-terminus was diffuse but had strong colocalization with actin. These data support a model where MyBP-C's N-terminal domains are flexible but interact with actin in situ.

Role of intrinsic disorder in muscle sarcomeres.

The role and utility of intrinsically disordered regions (IDRs) is reviewed for two groups of sarcomeric proteins, such as members of tropomodulin/leiomodin (Tmod/Lmod) protein homology group and myosin binding protein C (MyBP-C). These two types of sarcomeric proteins represent very different but strongly interdependent functions, being responsible for maintaining structure and operation of the muscle sarcomere. The role of IDRs in the formation of complexes between thin filaments and Tmods/Lmods is discussed within the framework of current understanding of the thin filament length regulation. For MyBP-C, the function of IDRs is discussed in the context of MYBP-C-dependent sarcomere contraction and actomyosin activation.

Multiple feedback mechanisms fine-tune Rho signaling to regulate morphogenetic outcomes.

Rho signaling is a conserved mechanism for generating forces through activation of contractile actomyosin. How this pathway can produce different cell morphologies is poorly understood. In the Drosophila embryonic epithelium, we investigate how Rho signaling controls force asymmetry to drive morphogenesis. We study a distinct morphogenetic process termed 'alignment'. This process results in striking columns of rectilinear cells connected by aligned cell-cell contacts. We found that this is driven by contractile actomyosin cables that elevate tension along aligning interfaces. Our data show that polarization of Rho effectors, Rok and Dia, directs formation of these cables. Constitutive activation of these effectors causes aligning cells to instead invaginate. This suggests that moderating Rho signaling is essential to producing the aligned geometry. Therefore, we tested for feedback that could fine-tune Rho signaling. We discovered that F-actin exerts negative feedback on multiple nodes in the pathway. Further, we present evidence that suggests that Rok in part mediates feedback from F-actin to Rho in a manner independent of Myo-II. Collectively, our work suggests that multiple feedback mechanisms regulate Rho signaling, which may account for diverse morphological outcomes.

The RhoGEF protein Plekhg5 regulates apical constriction of bottle cells during gastrulation.

Apical constriction regulates epithelial morphogenesis during embryonic development, but how this process is controlled is not understood completely. Here, we identify a Rho guanine nucleotide exchange factor (GEF) gene plekhg5 as an essential regulator of apical constriction of bottle cells during Xenopus gastrulation. plekhg5 is expressed in the blastopore lip and its expression is sufficient to induce ectopic bottle cells in epithelia of different germ layers in a Rho-dependent manner. This activity is not shared by arhgef3, which encodes another organizer-specific RhoGEF. Plekhg5 protein is localized in the apical cell cortex via its pleckstrin homology domain, and the GEF activity enhances its apical recruitment. Plekhg5 induces apical actomyosin accumulation and cell elongation. Knockdown of plekhg5 inhibits activin-induced bottle cell formation and endogenous blastopore lip formation in gastrulating frog embryos. Apical accumulation of actomyosin, apical constriction and bottle cell formation fail to occur in these embryos. Taken together, our data indicate that transcriptional regulation of plekhg5 expression at the blastopore lip determines bottle cell morphology via local polarized activation of Rho by Plekhg5, which stimulates apical actomyosin activity to induce apical constriction.

Adherens Junction Length during Tissue Contraction Is Controlled by the Mechanosensitive Activity of Actomyosin and Junctional Recycling

SummaryDuring epithelial contraction, cells generate forces to constrict their surface and, concurrently, fine-tune the length of their adherens junctions to ensure force transmission. While many studies have focused on understanding force generation, little is known on how junctional length is controlled. Here, we show that, during amnioserosa contraction in Drosophila dorsal closure, adherens junctions reduce their length in coordination with the shrinkage of apical cell area, maintaining a nearly constant junctional straightness. We reveal that junctional straightness and integrity depend on the endocytic machinery and on the mechanosensitive activity of the actomyosin cytoskeleton. On one hand, upon junctional stretch and decrease in E-cadherin density, actomyosin relocalizes from the medial area to the junctions, thus maintaining junctional integrity. On the other hand, when junctions have excess material and ruffles, junction removal is enhanced, and high junctional straightness and tension are restored. These two mechanisms control junctional length and integrity during morphogenesis.

Theoretical Analysis of Stress Distribution and Cell Polarization Surrounding a Model Wound

A growing amount of experimental evidence shows that the local elastic field acting on cells governs their spatial organization and polarity in a tissue. Interestingly, experiments on wound healing reveal a universal formation of thick actomyosin bundles around the margins of epithelial gaps. Although the forces involved in this process have been measured, the mechanisms governing cellular alignment and contractile ring formation are still not fully understood. To theoretically investigate this process, we have carried out a self-consistent calculation of the elastic field that is actively generated around a circular gap in a contractile cell monolayer that is adhered to an elastic substrate, taking into account the responsiveness of actomyosin activity to the locally generated stress. We model actomyosin contractility by a radial distribution of point force dipoles that may alter in magnitude and orientation in response to the local elastic stress. In addition, the model takes into account the forces exerted by leader cells on the margins of the cell monolayer. Our model suggests that the presence of a hole in the center of a contractile cell monolayer creates a mechanical tendency for actomyosin forces to polarize tangentially around the hole margin. In addition, it predicts that this tendency optimizes with substrate rigidity, thickness, and strength of cell adhesion to the substrate. Our calculations support the view that the universal formation of a peripheral contractile ring is a consequence of actomyosin contractility in the bulk and its inherent responsiveness to the local stress.

Planar cell polarity signalling coordinates heart tube remodelling through tissue-scale polarisation of actomyosin activity

Development of a multiple-chambered heart from the linear heart tube is inherently linked to cardiac looping. Although many molecular factors regulating the process of cardiac chamber ballooning have been identified, the cellular mechanisms underlying the chamber formation remain unclear. Here, we demonstrate that cardiac chambers remodel by cell neighbour exchange of cardiomyocytes guided by the planar cell polarity (PCP) pathway triggered by two non-canonical Wnt ligands, Wnt5b and Wnt11. We find that PCP signalling coordinates the localisation of actomyosin activity, and thus the efficiency of cell neighbour exchange. On a tissue-scale, PCP signalling planar-polarises tissue tension by restricting the actomyosin contractility to the apical membranes of outflow tract cells. The tissue-scale polarisation of actomyosin contractility is required for cardiac looping that occurs concurrently with chamber ballooning. Taken together, our data reveal that instructive PCP signals couple cardiac chamber expansion with cardiac looping through the organ-scale polarisation of actomyosin-based tissue tension.

Smooth muscle-like Ca2+-regulation of actin\u2013myosin interaction in adult jellyfish striated muscle

Cnidaria is an animal phylum, whose members probably have the most ancestral musculature. We prepared and characterized, for the first time to our knowledge, native actomyosin from the striated myoepithelium of the adult moon jelly Aurelia sp. The actomyosin contained myosin, paramyosin-like protein, Ser/Thr-kinase, actin, and two isoforms of tropomyosin, but not troponin, which is known to activate contraction dependent on intracellular Ca2+ signaling in almost all striated muscles of bilaterians. Notably, the myosin comprised striated muscle-type heavy chain and smooth muscle-type regulatory light chains. In the presence of Ca2+, the Mg-ATPase activity of actomyosin was stimulated and Ser21 of the regulatory light chain was concomitantly phosphorylated by the addition of calmodulin and myosin light chain kinase prepared from chicken smooth muscle. Collectively, these results suggest that, similar to smooth muscle, the contraction of jellyfish striated muscle is regulated by Ca2+-dependent phosphorylation of the myosin light chain.

Acto-Myosin Driven Functional Nanoclusters of GPI-Anchored Proteins Are Generated by Integrin Receptor Signaling

GPI-anchored protein (GPI-AP) nanoclusters are generated by cortical acto-myosin activity. While our understanding of the physical principles behind this process is emerging, the molecular machinery required for the generation of these nanoclusters is unknown. Here, we show that ligand mediated membrane receptor signaling triggers nanocluster formation. Both soluble and surface-tethered RGD ligands bind the β1-integrin receptor and activate focal adhesion and src- kinases, resulting in RhoA signaling. This cascade ultimately triggers actin-nucleation via specific formins, driving nanoclustering of both GPI-APs and a model transmembrane protein with an actin-binding domain. Integrin signaling concurrently results in talin mediated activation of vinculin. This is necessary for the coupling of the dynamic actin machinery to the inner leaflet driving GPI-AP nanoclustering. Disruption of GPI-AP nanoclustering in either GPI-anchor remodeling mutants or in cells that express vinculin mutants, provide evidence that these nanoclusters are necessary for activating cell spreading, a hallmark of integrin function.

Tetraspanin-enriched microdomains regulate digitation junctions.

Tetraspanins co-emerged with multi-cellular organisms during evolution and are typically localized at the cell–cell interface, [corrected] and form tetraspanin-enriched microdomains (TEMs) by associating with each other and other membrane molecules. Tetraspanins affect various biological functions, but how tetraspanins engage in multi-faceted functions at the cellular level is largely unknown. When cells interact, the membrane microextrusions at the cell-cell interfaces form dynamic, digit-like structures between cells, which we term digitation junctions (DJs). We found that (1) tetraspanins CD9, CD81, and CD82 and (2) TEM-associated molecules integrin α3β1, CD44, EWI2/PGRL, and PI-4P are present in DJs of epithelial, endothelial, and cancer cells. Tetraspanins and their associated molecules also regulate the formation and development of DJs. Moreover, (1) actin cytoskeleton, RhoA, and actomyosin activities and (2) growth factorreceptor-Src-MAP kinase signaling, but not PI-3 kinase, regulate DJs. Finally, we showed that DJs consist of various forms in different cells. Thus, DJs are common, interactive structures between cells, and likely affect cell adhesion, migration, and communication. TEMs probably modulate various cell functions through DJs. Our findings highlight that DJ morphogenesis reflects the transition between cell-matrix adhesion and cell-cell adhesion and involves both cell-cell and cell-matrix adhesion molecules.

α-Catenin-dependent cytoskeletal tension controls Yap activity in the heart.

Shortly after birth, muscle cells of the mammalian heart lose their ability to divide. At the same time, the N-cadherin/catenin cell adhesion complex accumulates at the cell termini, creating a specialized type of cell-cell contact called the intercalated disc (ICD). To investigate the relationship between ICD maturation and proliferation, αE-catenin (Ctnna1) and αT-catenin (Ctnna3) genes were deleted to generate cardiac-specific α-catenin double knockout (DKO) mice. DKO mice exhibited aberrant N-cadherin expression, mislocalized actomyosin activity and increased cardiomyocyte proliferation that was dependent on Yap activity. To assess effects on tension, cardiomyocytes were cultured on deformable polyacrylamide hydrogels of varying stiffness. When grown on a stiff substrate, DKO cardiomyocytes exhibited increased cell spreading, nuclear Yap and proliferation. A low dose of either a myosin or RhoA inhibitor was sufficient to block Yap accumulation in the nucleus. Finally, activation of RhoA was sufficient to increase nuclear Yap in wild-type cardiomyocytes. These data demonstrate that α-catenins regulate ICD maturation and actomyosin contractility, which, in turn, control Yap subcellular localization, thus providing an explanation for the loss of proliferative capacity in the newborn mammalian heart.

Spatial Fluctuations at Vertices of Epithelial Layers: Quantification of Regulation by Rho Pathway

In living matter, shape fluctuations induced by acto-myosin are usually studied in vitro via reconstituted gels, whose properties are controlled by changing the concentrations of actin, myosin, and cross-linkers. Such an approach deliberately avoids consideration of the complexity of biochemical signaling inherent to living systems. Acto-myosin activity inside living cells is mainly regulated by the Rho signaling pathway, which is composed of multiple layers of coupled activators and inhibitors. Here, we investigate how such a pathway controls the dynamics of confluent epithelial tissues by tracking the displacements of the junction points between cells. Using a phenomenological model to analyze the vertex fluctuations, we rationalize the effects of different Rho signaling targets on the emergent tissue activity by quantifying the effective diffusion coefficient, and the persistence time and length of the fluctuations. Our results reveal an unanticipated correlation between layers of activation/inhibition and spatial fluctuations within tissues. Overall, this work connects regulation via biochemical signaling with mesoscopic spatial fluctuations, with potential application to the study of structural rearrangements in epithelial tissues.

APKC-mediated displacement and actomyosin-mediated retention polarize Miranda in Drosophila neuroblasts.

Cell fate assignment in the nervous system of vertebrates and invertebrates often hinges on the unequal distribution of molecules during progenitor cell division. We address asymmetric fate determinant localization in the developing Drosophila nervous system, specifically the control of the polarized distribution of the cell fate adapter protein Miranda. We reveal a step-wise polarization of Miranda in larval neuroblasts and find that Miranda's dynamics and cortical association are differently regulated between interphase and mitosis. In interphase, Miranda binds to the plasma membrane. Then, before nuclear envelope breakdown, Miranda is phosphorylated by aPKC and displaced into the cytoplasm. This clearance is necessary for the subsequent establishment of asymmetric Miranda localization. After nuclear envelope breakdown, actomyosin activity is required to maintain Miranda asymmetry. Therefore, phosphorylation by aPKC and differential binding to the actomyosin network are required at distinct phases of the cell cycle to polarize fate determinant localization in neuroblasts.

A dual role of the extracellular domain of Drosophila Crumbs for morphogenesis of the embryonic neuroectoderm.

ABSTRACTEpithelia are highly polarised tissues and several highly conserved polarity protein complexes serve to establish and maintain polarity. The transmembrane protein Crumbs (Crb), the central component of the Crb protein complex, is required, among others, for the maintenance of polarity in most epithelia in the Drosophila embryo. However, different epithelia exhibit different phenotypic severity upon loss of crb. Using a transgenomic approach allowed us to more accurately define the role of crb in different epithelia. In particular, we provide evidence that the loss of epithelial tissue integrity in the ventral epidermis of crb mutant embryos is due to impaired actomyosin activity and an excess number of neuroblasts. We demonstrate that the intracellular domain of Crb could only partially rescue this phenotype, while it is able to completely restore tissue integrity in other epithelia. Based on these results we suggest a dual role of the extracellular domain of Crb in the ventral neuroectoderm. First, it is required for apical enrichment of the Crb protein, which in turn regulates actomyosin activity and thereby ensures tissue integrity; and second, the extracellular domain of Crb stabilises the Notch receptor and thereby ensures proper Notch signalling and specification of the correct number of neuroblasts.

Rapid tenderizing of goose breast muscle based on actomyosin dissociation by low-frequency ultrasonication

The effect of low-frequency ultrasonication on tenderness, myofibrillar fragmentation and dissociation of actomyosin from goose breast muscle was investigated. Tenderness is the total mechanical strength of the skeletal muscle tissue. The compression and depression induced by ultrasound produces microbubbles in the structure that propagate shock waves of high energy throughout the meat tissue, thereby causing myofibrillar damage. Ultrasound (20 kHz) at 800 W for 20 min had a greater impact on the improvement of meat tenderness than untreated meat (control) during the 0–24 h storage time (4 °C). Significantly higher pH values and myofibrillar fragmentation indices were observed in ultrasonicated meat (6.44 and 22.7%) compared with the control (6.03 and 18.2%) after 24 h of storage. The lowest cooking loss and shear force were obtained in ultrasonicated meat (15.7% and 1970 g) vs. the control (23.9% and 2470 g) at 12 h storage. Furthermore, the ultrasound effect also significantly increased the count of free actin and decreased the ATPase activity of actomyosin during storage. Atomic force microscopy demonstrated that small subunits of actin significantly increased with ultrasonication. This study provides a new perspective of tenderization with the dissociation of actomyosin by ultrasonication combined with proper storage time.

Control of cytoskeletal dynamics during cellular responses to pore forming toxins

ABSTRACTFollowing damage by pore forming toxins (PFTs) host cells engage repair processes and display profound cytoskeletal remodeling and concomitant plasma membrane (PM) blebbing. We have recently demonstrated that host cells utilize similar mechanisms to control cytoskeletal dynamics in response to PFTs and during cell migration. This involves assembly of cortical actomyosin bundles, reorganisation of the endoplasmic reticulum (ER) network, and the interaction between the ER chaperone Gp96 and the molecular motor Non-muscle Myosin Heavy Chain IIA (NMHCIIA). Consequently, Gp96 regulates actomyosin activity, PM blebbing and cell migration, and protects PM integrity against PFTs. In addition, we observed that PFTs increase association of Gp96 and ER vacuoles with the cell surface or within PM blebs loosely attached to the cell body. Similarly, gut epithelial cells damaged by PFTs in vivo were shown to release microvilli structures or directly purge cytoplasmic content. Cytoplasmic purging involves profound cytoskeletal remodeling and ER vacuolation, suggesting that our observations recapitulate recovery processes in vivo. Here, we discuss our findings in light of the current understanding of PM repair mechanisms and in vivo recovery responses to PFTs.

Actomyosin drives cancer cell nuclear dysmorphia and threatens genome stability

Altered nuclear shape is a defining feature of cancer cells. The mechanisms underlying nuclear dysmorphia in cancer remain poorly understood. Here we identify PPP1R12A and PPP1CB, two subunits of the myosin phosphatase complex that antagonizes actomyosin contractility, as proteins safeguarding nuclear integrity. Loss of PPP1R12A or PPP1CB causes nuclear fragmentation, nuclear envelope rupture, nuclear compartment breakdown and genome instability. Pharmacological or genetic inhibition of actomyosin contractility restores nuclear architecture and genome integrity in cells lacking PPP1R12A or PPP1CB. We detect actin filaments at nuclear envelope rupture sites and define the Rho-ROCK pathway as the driver of nuclear damage. Lamin A protects nuclei from the impact of actomyosin activity. Blocking contractility increases nuclear circularity in cultured cancer cells and suppresses deformations of xenograft nuclei in vivo. We conclude that actomyosin contractility is a major determinant of nuclear shape and that unrestrained contractility causes nuclear dysmorphia, nuclear envelope rupture and genome instability.

An Actomyosin-Arf-GEF Negative Feedback Loop for Tissue Elongation under Stress

In response to a pulling force, a material can elongate, hold fast, or fracture. During animal development, multi-cellular contraction of one region often stretches neighboring tissue. Such local contraction occurs by induced actomyosin activity, but molecular mechanisms are unknown for regulating the physical properties of connected tissue for elongation under stress. We show that cytohesins, and their Arf small G protein guanine nucleotide exchange activity, are required for tissues to elongate under stress during both Drosophila dorsal closure (DC) and zebrafish epiboly. In Drosophila, protein localization, laser ablation, and genetic interaction studies indicate that the cytohesin Steppke reduces tissue tension by inhibiting actomyosin activity at adherens junctions. Without Steppke, embryogenesis fails, with epidermal distortions and tears resulting frommyosin misregulation. Remarkably, actomyosin network assembly is necessary and sufficient for local Steppke accumulation, where live imaging shows Steppke recruitment within minutes. This rapid negative feedback loop provides a molecular mechanism for attenuating the main tension generator of animal tissues. Such attenuation relaxes tissues and allows orderly elongation under stress.