Of Mice and Men

Non-muscle myosin II has diverse functions in cell contractility, morphology, cytokinesis and migration. Mammalian cells have three isoforms of non-muscle myosin II, termed IIA, IIB and IIC, encoded by three different genes. These isoforms share considerable homology and some overlapping functions, yet they exhibit differences in enzymatic properties, subcellular localization, molecular interaction and tissue distribution 1-6. Our studies have focused on the IIA isoform, and they reveal unique regulatory roles in cell-cell adhesion and cell migration that are associated with cross-talk of the actomyosin system with microtubules. In humans, various mutations in the MYH9 gene that encodes the myosin IIA heavy chain cause autosomal dominant disease, whereas in mice, the complete deficiency is embryonic lethal but heterozygous mice are nearly normal. We discuss here the differences between mouse and human phenotypes and how the wealth of mechanistic knowledge about myosin II based on in vitro studies and mouse models can help us understand the molecular and cellular pathophysiology of myosin IIA deficiency in humans.     

Of Mice and Men: Relevance of Cellular and Molecular Characterizations of Myosin IIA to MYH9-Related Human Disease

Non-muscle myosin II has diverse functions in cell contractility, morphology, cytokinesis and migration. Mammalian cells have three isoforms of non-muscle myosin II, termed IIA, IIB and IIC, encoded by three different genes. These isoforms share considerable homology and some overlapping functions, yet they exhibit differences in enzymatic properties, subcellular localization, molecular interaction and tissue distribution.^1–6 Our studies have focused on the IIA isoform, and they reveal unique regulatory roles in cell-cell adhesion and cell migration that are associated with cross-talk of the actomyosin system with microtubules. In humans, various mutations in the MYH9 gene that encodes the myosin IIA heavy chain cause autosomal dominant disease, whereas in mice, the complete deficiency is embryonic lethal but heterozygous mice are nearly normal. We discuss here the differences between mouse and human phenotypes and how the wealth of mechanistic knowledge about myosin II based on in vitro studies and mouse models can help us understand the molecular and cellular pathophysiology of myosin IIA deficiency in humans.Key Words: myosin IIA, MYH9, microtubules, actin, cytoskeleton, platelets, cell-cell adhesionTo study the distinct roles and contribution of each isoform and to avoid the problem of compensation by overlapping functions, it is simplest to work with cells that exclusively express one isoform. However, very few cell types naturally lack myosin IIA, making it necessary to develop a knockout mouse model for myosin IIA or to use knockdown techniques. Unfortunately, the myosin IIA knockout mutation is embryonic-lethal at an early stage (E6.5–E7.5), and the embryo fails to organize normal germ layers, so tissue-derived cells are not available.⁷ Nevertheless, the IIA-null embryonic stem cells that were used to generate the knockout model have proven very useful for elucidating some basic features and general phenomena of myosin IIA-deficient cells when analyzed as single cells or clusters (embryoid bodies).We and others^1–6,8 have recently reported that ablation of the myosin IIA isoform by gene targeting, siRNA knockdown or chemical inhibition results in pronounced defects in cellular contractility, focal adhesions, actin stress fiber organization and tail retraction, in line with the classic role of myosin II.⁸ More surprising was our finding that myosin IIA-deficient cells displayed substantially increased cell migration and exaggerated membrane ruffling, which was dependent on the small G-protein Rac1, its activator Tiam1 and the microtubule motor kinesin Eg5 (Fig. 1). We demonstrated that myosin IIA deficiency stabilized microtubules and shifted the balance between actomyosin and microtubules, increasing the persistence of microtubules in active membrane ruffles. Myosin IIB could partially compensate for the absence of the IIA isoform in cellular contractility under conditions of suppressed microtubule polymerization, but it could not replace myosin IIA in modulating cell migration. We concluded that myosin IIA negatively regulates cell migration and maintains a balance between the actomyosin and microtubule systems by regulating microtubule dynamics. The phenomenon of increased cell migration was not limited only to single migrating cells, but also to ES cells clustered as embryoid bodies. In a previous paper⁷ we have shown that myosin IIA is also specifically required for normal maintenance of cell-cell adhesions and the normal localization of major junction proteins such as E-cadherin, β-catenin and ZO-2. In its absence, cells actively dispersed from embryoid bodies and migrated outward. The failure to form normal cell-cell adhesions in the mouse knockout model resulted in malformation of the embryonic germ layers and early lethality of the deficient embryos.Open in a separate windowFigure 1General mechanism of myosin IIA as a negative regulator of microtubule expansion. Under normal conditions (right), microtubule (red) expansion is restricted by the actomyosin (blue) system. However, when myosin IIA is ablated (left), few actin stress fibers and focal adhesions are formed and microtubules are free to extend closer to the cell membrane and stimulate membrane ruffling in a Rac/Tiam1/ Eg5 kinesin dependent manner. Myosin IIA ablation also increases nondirectional cell migration.Myosin IIA deficiency is not merely an in vitro phenomenon, but it can also cause human disease. Four main autosomal dominant disorders are related to mutations in MYH9: May-Hegglin, Fechtner, Sebastian and Epstein. The common feature in all four syndromes is macrothrombocytopenia, and granulocyte inclusion bodies characterize the first three syndromes. Some patients show later onset of deafness, cataracts and glomerulonephritis. One might expect that the wealth of data published on the cellular roles of myosin II isoforms would have deciphered the molecular basis of these syndromes; however, applying the knowledge from mice to men remains, as often happens, quite limited. While heterozygous mice exhibit a nearly normal phenotype,⁹ in humans, mutations in the gene MYH9 are autosomal dominant, i.e., heterozygous individuals are affected.One hypothesis is that mutations result in the production of an aberrant non-functional or malfunctioning protein product with dominant-negative effects after dimerizing with the remaining normal protein. Another hypothesis might be that as evolution advanced and different isoforms developed, each isoform acquired a distinct role during development and adulthood, the functional overlaps between them became smaller, and as a result compensation is limited. The degree of compensation that allows near-normal murine development might not be sufficient in humans, and haploinsufficiency causes disease. Both hypotheses may prove valid, as suggested by a study of several different mutations: haploinsufficiency of myosin-IIA in the megakaryocytic lineage is the mechanism of macrothrombocytopenia, whereas inclusion bodies in granulocytes are the consequence of a dominant-negative effect.¹⁰Despite the differences in myosin IIA deficiency phenotypes between humans and mice we would like to suggest that some of the cellular and molecular mechanisms that we and others have characterized may help to explain the human disease. Cells that exclusively express the IIA isoform, such as platelets and granulocytes, are primarily affected, while sensory hearing loss, cataract and glomerulonephritis are found only in some patients and with later onset. This dependence on a single isoform explains why the platelet disorder of macrothrombocytopenia is common to all four syndromes and appears at birth.To understand how our findings on the crosstalk between the actomyosin and microtubule cytoskeletal systems are relevant to the platelet disorder, we should look carefully into the process of platelet formation and changes in the involved cytoskeletal balance (Fig. 2A). Platelets are released by megakaryocytes via cytoplasmic extensions called proplatelets in a process that requires profound changes in cytoskeletal organization. Microtubules provide the main structural component of proplatelets and extend into the elongating cellular processes.^11,12 At this stage, we suggest that myosin IIA is downregulated to allow microtubule expansion, and indeed, it was found that either elevation of myosin-IIA activity by exogenous expression, or mimicking constitutive phosphorylation of its regulatory myosin light chain (MLC), significantly attenuates proplatelet formation.¹³ However, myosin IIA activity is needed at the following branching stage where its actomyosin- dependent action is used to bifurcate the proplatelet shaft; actin filament aggregates occur periodically along the proplatelet shaft and appear to be used as “muscles” to bend the microtubules; myosin is the most likely source for the collapsing forces applied to these joints.¹² In the absence of myosin IIA, a defect in megakaryocyte fragmentation occurs.¹⁴ Abnormally large and misshapen platelets termed giant platelets are characteristics of MYH9-related macrothrombocytopenia and represent fragmentation defects: immunohistochemical studies of these spheroid platelets revealed disorganization of both microtubule and actin systems.¹⁵ An intriguing finding in platelets from several MYH9-syndrome patients is increased association of myosin with actin filaments in resting platelets; however, the concomitant failure of myosin to associate further with the reorganized cytoskeleton upon stimulation, together with the inability of multiple signaling molecules to be integrated with the cytoskeleton,¹⁶ may indicate faulty cross-talk between the defective myosin and other cytoskeletal components (e.g., microtubules).Open in a separate windowFigure 2Cytoskeletal cross-talk in MYH9-related disorders. (A) Normal proplatelet and platelet formation involves cytoskeletal rearrangement. Megakaryocytes (MK) undergo endomitosis and cytoplasmic maturation. An array of microtubules originally emanates radially from the centrosome (red). Just before platelet formation, the centrosome disassembles, and microtubules form a circumferential bundle near the cell membrane. The reticular organization of actin changes to form aggregates at one pole of the cell opposite the pseudopod. Sliding of microtubules propels elongation of cytoplasmic extensions that bend and branch with the aid of actomyosin, which concentrates at the base of the shafts of the proplatelet (PP). In the absence of functional myosin IIA (dashed line), proplatelets and normal platelets (PL) fail to form. The defect in fragmentation leads to the formation of giant platelets characteristically found in MYH9-related diseases. (B) The development and maintenance of cell-cell adhesions are crucial for preserving normal epithelial tissue structures. Myosin IIA generates tension by pulling against actin fibers and plays a key role in cell-cell adhesion via cadherins with possible cross-talk with microtubules. Its absence may result in morphological and functional changes in the epithelial structures that affect the organs shown, although the mechanisms of pathogenesis remain to be clarified.Other clinical manifestations of MYH9 mutations involve epithlial structures in the inner ear, lens and kidney, causing hearing loss, cataracts, and nephritis. Normal function of epithelial tissues depends heavily on cell-cell adhesion, which is responsible for tissue continuity and for the formation of sealing zones around cavities; proper localization of junctional components such as E-cadherin, β-catenin and ZO molecules is essential. As we and others have previously shown, myosin IIA is crucial for the formation and maintenance of cell-cell adhesions.^7,17 Numerous studies have demonstrated the involvement of both myosin II and microtubules in regulation of assembly and disassembly of cell-cell adhesions.¹⁸ Dynamic microtubules regulate the concentration of E-cadherin at cell-cell contacts¹⁹ by regulating the activation of myosin II, which is critical for the ability of cells to concentrate E-cadherin at these sites.²⁰ Microtubules were shown to regulate disassembly of epithelial apical junctions by contractile actin ring formation.²¹ We infer that a cytoskeletal balance sustained by cross-talk between microtubules and actomyosin or sub-populations of actomyosin may play a key role in maintaining cell-cell adhesions (Fig. 2B), but the specific role of myosin IIA in this particular cytoskeletal cross-talk needs further examination.The physiological (and frequently pathological) process of epithelial-to-mesenchymal transition involves disruption of cell-cell adhesion complexes. It induces morphological changes, cell scattering and cell migration. During the formation of anterior polar cataracts and posterior capsular cataracts, lens epithelial cells transdifferentiate and proliferate into plaques of large spindle-shaped cells or myofibroblasts through epithelial-mesenchymal transition (EMT). Loss of E-cadherin is frequently involved in this process and can be repressed by TGFβ1 in lens epithelial cells during the formation of cataracts.²² The specific role of myosin IIA in maintaining transparency of the lens is unknown; however, a recent paper²³ showed that inhibition of myosin light chain kinase (MLCK) resulted in the development of nuclear lens opacity and abnormal fiber cell organization.Another member of the cadherin family, cadherin23, is an important element in hair cell stereocilia of the inner ear, where it functions as an inter-stereocilia link.²⁴ MYH9 expression was identified in the developing and mature inner ear of mice, suggesting a role for this protein in the development and maintenance of auditory function.²⁵ Although no reports have yet linked cadherin to myosin II function in the inner ear, it is plausible that such an interaction could exist analogous to other cell-cell adhesions, and may hint at a possible molecular explanation for the hearing loss in MYH9 deficiency.MYH9 is widely expressed in the kidney, mainly in the glomerulus and peritubular vessels. Within the glomerulus, MYH9 mRNA and protein are mostly expressed in the epithelial visceral cells.²⁶ Glomerular parietal epithelial EMT is a key event in the development of glomerulonephritis, contributing to the formation of myofibroblasts from epithelial cells and to glomerular fibrosis.²⁷ Expression of the cadherin complex was found to be diminished in human crescentic glomerulonephritis.²⁸ Taken together, defects in cell-cell adhesion that lead to disruption of an epithelial structure enable EMT and may provide a possible link of mutated MYH9 to glomerulonephritis.The complete ablation of myosin IIA in vitro and in mouse models does not produce phenotypes equivalent to those listed above due to the partial deficiencies resulting from various mutations in humans, and as mentioned previously, a single missing allele in mice does not cause parallel severe effects, perhaps due to the absence of dominant-negative effects. Nevertheless, the fact that the human cells that exclusively express the IIA isoform are immediately affected suggests similarity to the null phenotype. Late onset of other symptoms in tissues with mixed isoform expression could be the result of haploinsufficiency, partial impairment of myosin IIA enzymatic activity,²⁹ and/or the existence of isoform compensation. Nonetheless, the striking phenotypes identified in myosin IIA-ablated models can provide a framework for thinking about the pathophysiology of known human mutations with regard to novel cellular functions of the IIA isoform in maintaining cytoskeletal balance in single cells and cell-cell adhesions in multi-cellular structures. Consequently, it should be fruitful to examine human myosin IIA mutations expressed in knock-in mouse models by analyses at the cellular and tissue level, since they may explain the human disease.     

EPH/EPHRIN regulates cellular organization by actomyosin contractility effects on cell contacts

EPH/EPHRIN signaling is essential to many aspects of tissue self-organization and morphogenesis, but little is known about how EPH/EPHRIN signaling regulates cell mechanics during these processes. Here, we use a series of approaches to examine how EPH/EPHRIN signaling drives cellular self-organization. Contact angle measurements reveal that EPH/EPHRIN signaling decreases the stability of heterotypic cell:cell contacts through increased cortical actomyosin contractility. We find that EPH/EPHRIN-driven cell segregation depends on actomyosin contractility but occurs independently of directed cell migration and without changes in cell adhesion. Atomic force microscopy and live cell imaging of myosin localization support that EPH/EPHRIN signaling results in increased cortical tension. Interestingly, actomyosin contractility also nonautonomously drives increased EPHB2:EPHB2 homotypic contacts. Finally, we demonstrate that changes in tissue organization are driven by minimization of heterotypic contacts through actomyosin contractility in cell aggregates and by mouse genetics experiments. These data elucidate the biomechanical mechanisms driving EPH/EPHRIN-based cell segregation wherein differences in interfacial tension, regulated by actomyosin contractility, govern cellular self-organization.     

Caldesmon inhibits nonmuscle cell contractility and interferes with the formation of focal adhesions.

Caldesmon is known to inhibit the ATPase activity of actomyosin in a Ca(2+)-calmodulin-regulated manner. Although a nonmuscle isoform of caldesmon is widely expressed, its functional role has not yet been elucidated. We studied the effects of nonmuscle caldesmon on cellular contractility, actin cytoskeletal organization, and the formation of focal adhesions in fibroblasts. Transient transfection of nonmuscle caldesmon prevents myosin II-dependent cell contractility and induces a decrease in the number and size of tyrosine-phosphorylated focal adhesions. Expression of caldesmon interferes with Rho A-V14-mediated formation of focal adhesions and stress fibers as well as with formation of focal adhesions induced by microtubule disruption. This inhibitory effect depends on the actin- and myosin-binding regions of caldesmon, because a truncated variant lacking both of these regions is inactive. The effects of caldesmon are blocked by the ionophore A23187, thapsigargin, and membrane depolarization, presumably because of the ability of Ca(2+)-calmodulin or Ca(2+)-S100 proteins to antagonize the inhibitory function of caldesmon on actomyosin contraction. These results indicate a role for nonmuscle caldesmon in the physiological regulation of actomyosin contractility and adhesion-dependent signaling and further demonstrate the involvement of contractility in focal adhesion formation.     

Cortical actomyosin breakage triggers shape oscillations in cells and cell fragments

Cell shape and movements rely on complex biochemical pathways that regulate actin, microtubules, and substrate adhesions. Some of these pathways act through altering the cortex contractility. Here we examined cellular systems where contractility is enhanced by disassembly of the microtubules. We found that adherent cells, when detached from their substrate, developed a membrane bulge devoid of detectable actin and myosin. A constriction ring at the base of the bulge oscillated from one side of the cell to the other. The movement was accompanied by sequential redistribution of actin and myosin to the membrane. We observed this oscillatory behavior also in cell fragments of various sizes, providing a simplified, nucleus-free system for biophysical studies. Our observations suggest a mechanism based on active gel dynamics and inspired by symmetry breaking of actin gels growing around beads. The proposed mechanism for breakage of the actomyosin cortex may be used for cell polarization.     

Functional role for stable microtubules in lens fiber cell elongation

The process of tissue morphogenesis, especially for tissues reliant on the establishment of a specific cytoarchitecture for their functionality, depends a balanced interplay between cytoskeletal elements and their interactions with cell adhesion molecules. The microtubule cytoskeleton, which has many roles in the cell, is a determinant of directional cell migration, a process that underlies many aspects of development. We investigated the role of microtubules in development of the lens, a tissue where cell elongation underlies morphogenesis. Our studies with the microtubule depolymerizing agent nocodazole revealed an essential function for the acetylated population of stable microtubules in the elongation of lens fiber cells, which was linked to their regulation of the activation state of myosin. Suppressing myosin activation with the inhibitor blebbistatin could attenuate the loss of acetylated microtubules by nocodazole and rescue the effect of this microtubule depolymerization agent on both fiber cell elongation and lens integrity. Our results also suggest that acetylated microtubules impact lens morphogenesis through their interaction with N-cadherin junctions, with which they specifically associate in the region where lens fiber cell elongate. Disruption of the stable microtubule network increased N-cadherin junctional organization along lateral borders of differentiating lens fiber cells, which was prevented by suppression of myosin activity. These results reveal a role for the stable microtubule population in lens fiber cell elongation, acting in tandem with N-cadherin cell-cell junctions and the actomyosin network, giving insight into the cooperative role these systems play in tissue morphogenesis.     

Myosin II is present in gastric parietal cells and required for lamellipodial dynamics associated with cell activation

Nonmuscle myosin II has been shown to participate in organizing the actin cytoskeleton in polarized epithelial cells. Vectorial acid secretion in cultured parietal cells involves translocation of proton pumps from cytoplasmic vesicular membranes to the apical plasma membrane vacuole with coordinated lamellipodial dynamics at the basolateral membrane. Here we identify nonmuscle myosin II in rabbit gastric parietal cells. Western blots with isoform-specific antibodies indicate that myosin IIA is present in both cytosolic and particulate membrane fractions whereas the IIB isoform is associated only with particulate fractions. Immunofluorescent staining demonstrates that myosin IIA is diffusely located throughout the cytoplasm of resting parietal cells. However, after stimulation, myosin IIA is rapidly redistributed to lamellipodial extensions at the cell periphery; virtually all the cytoplasmic myosin IIA joins the newly formed basolateral membrane extensions. 2,3-Butanedione monoximine (BDM), a myosin-ATPase inhibitor, greatly diminishes the lamellipodial dynamics elicited by stimulation and retains the pattern of myosin IIA cytoplasmic staining. However, BDM had no apparent effect on the stimulation associated redistribution of H,K-ATPase from a cytoplasmic membrane compartment to apical membrane vacuoles. The myosin light chain kinase inhibitor 1-(5-iodonaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine (ML-7) also did not alter the stimulation-associated recruitment of H,K-ATPase to apical membrane vacuoles, but unlike BDM it had relatively minor inhibitory effects on lamellipodial dynamics. We conclude that specific disruption of the basolateral actomyosin cytoskeleton has no demonstrable effect on recruitment of H,K-ATPase-rich vesicles into the apical secretory membrane. However, myosin II plays an important role in regulating lamellipodial dynamics and cortical actomyosin associated with parietal cell activation. acid secretion; cytoskeleton; ion channels and pumps     

Rho kinase differentially regulates phosphorylation of nonmuscle myosin II isoforms A and B during cell rounding and migration

The actin-myosin cytoskeleton is generally accepted to produce the contractile forces necessary for cellular processes such as cell rounding and migration. All vertebrates examined to date are known to express at least two isoforms of non-muscle myosin II, referred to as myosin IIA and myosin IIB. Studies of myosin IIA and IIB in cultured cells and null mice suggest that these isoforms perform distinct functions. However, how each myosin II isoform contributes individually to all the cellular functions attributed to "myosin II" has yet to be fully characterized. Using isoform-specific small-interfering RNAs, we found that depletion of either isoform resulted in opposing migration phenotypes, with myosin IIA- and IIB-depleted cells exhibiting higher and lower wound healing migration rates, respectively. In addition, myosin IIA-depleted cells demonstrated impaired thrombin-induced cell rounding and undertook a more motile morphology, exhibiting decreased amounts of stress fibers and focal adhesions, with concomitant increases in cellular protrusions. Cells depleted of myosin IIB, however, were efficient in thrombin-induced cell rounding, displayed a more retractile phenotype, and maintained focal adhesions but only in the periphery. Last, we present evidence that Rho kinase preferentially regulates phosphorylation of the regulatory light chain associated with myosin IIA. Our data suggest that the myosin IIA and IIB isoforms are regulated by different signaling pathways to perform distinct cellular activities and that myosin IIA is preferentially required for Rho-mediated contractile functions.     

TRPM7, a novel regulator of actomyosin contractility and cell adhesion

Actomyosin contractility regulates various cell biological processes including cytokinesis, adhesion and migration. While in lower eukaryotes, alpha-kinases control actomyosin relaxation, a similar role for mammalian alpha-kinases has yet to be established. Here, we examined whether TRPM7, a cation channel fused to an alpha-kinase, can affect actomyosin function. We demonstrate that activation of TRPM7 by bradykinin leads to a Ca(2+)- and kinase-dependent interaction with the actomyosin cytoskeleton. Moreover, TRPM7 phosphorylates the myosin IIA heavy chain. Accordingly, low overexpression of TRPM7 increases intracellular Ca2+ levels accompanied by cell spreading, adhesion and the formation of focal adhesions. Activation of TRPM7 induces the transformation of these focal adhesions into podosomes by a kinase-dependent mechanism, an effect that can be mimicked by pharmacological inhibition of myosin II. Collectively, our results demonstrate that regulation of cell adhesion by TRPM7 is the combined effect of kinase-dependent and -independent pathways on actomyosin contractility.     

The RhoGEF TEM4 Regulates Endothelial Cell Migration by Suppressing Actomyosin Contractility

Persistent cellular migration requires efficient protrusion of the front of the cell, the leading edge where the actin cytoskeleton and cell-substrate adhesions undergo constant rearrangement. Rho family GTPases are essential regulators of the actin cytoskeleton and cell adhesion dynamics. Here, we examined the role of the RhoGEF TEM4, an activator of Rho family GTPases, in regulating cellular migration of endothelial cells. We found that TEM4 promotes the persistence of cellular migration by regulating the architecture of actin stress fibers and cell-substrate adhesions in protruding membranes. Furthermore, we determined that TEM4 regulates cellular migration by signaling to RhoC as suppression of RhoC expression recapitulated the loss-of-TEM4 phenotypes, and RhoC activation was impaired in TEM4-depleted cells. Finally, we showed that TEM4 and RhoC antagonize myosin II-dependent cellular contractility and the suppression of myosin II activity rescued the persistence of cellular migration of TEM4-depleted cells. Our data implicate TEM4 as an essential regulator of the actin cytoskeleton that ensures proper membrane protrusion at the leading edge of migrating cells and efficient cellular migration via suppression of actomyosin contractility.     

Cellular Contractility Requires Ubiquitin Mediated Proteolysis

Background

Cellular contractility, essential for cell movement and proliferation, is regulated by microtubules, RhoA and actomyosin. The RhoA dependent kinase ROCK ensures the phosphorylation of the regulatory Myosin II Light Chain (MLC) Ser19, thereby activating actomyosin contractions. Microtubules are upstream inhibitors of contractility and their depolymerization or depletion cause cells to contract by activating RhoA. How microtubule dynamics regulates RhoA remains, a major missing link in understanding contractility.

Principal Findings

We observed that contractility is inhibited by microtubules not only, as previously reported, in adherent cells, but also in non-adhering interphase and mitotic cells. Strikingly we observed that contractility requires ubiquitin mediated proteolysis by a Cullin-RING ubiquitin ligase. Inhibition of proteolysis, ubiquitination and neddylation all led to complete cessation of contractility and considerably reduced MLC Ser19 phosphorylation.

Conclusions

Our results imply that cells express a contractility inhibitor that is degraded by ubiquitin mediated proteolysis, either constitutively or in response to microtubule depolymerization. This degradation seems to depend on a Cullin-RING ubiquitin ligase and is required for cellular contractions.     

Functional divergence of human cytoplasmic myosin II: kinetic characterization of the non-muscle IIA isoform

Cytoplasmic (or non-muscle) myosin II isoforms are widely expressed molecular motors playing essential cellular roles in cytokinesis and cortical tension maintenance. Two of the three human non-muscle myosin II isoforms (IIA and IIB) have been investigated at the protein level. Transient kinetics of non-muscle myosin IIB showed that this motor has a very high actomyosin ADP affinity and slow ADP release. Here we report the kinetic characterization of the non-muscle myosin IIA isoform. Similar to non-muscle myosin IIB, non-muscle myosin IIA shows high ADP affinity and little enhancement of the ADP release rate by actin. The ADP release rate constant, however, is more than an order of magnitude higher than the steady-state ATPase rate. This implies that non-muscle myosin IIA spends only a small fraction of its ATPase cycle time in strongly actin-bound states, which is in contrast to non-muscle myosin IIB. Non-muscle myosin II isoforms thus appear to have distinct enzymatic properties that may be of importance in carrying out their cellular functions.     

Convergence and Extrusion Are Required for Normal Fusion of the Mammalian Secondary Palate

The fusion of two distinct prominences into one continuous structure is common during development and typically requires integration of two epithelia and subsequent removal of that intervening epithelium. Using confocal live imaging, we directly observed the cellular processes underlying tissue fusion, using the secondary palatal shelves as a model. We find that convergence of a multi-layered epithelium into a single-layer epithelium is an essential early step, driven by cell intercalation, and is concurrent to orthogonal cell displacement and epithelial cell extrusion. Functional studies in mice indicate that this process requires an actomyosin contractility pathway involving Rho kinase (ROCK) and myosin light chain kinase (MLCK), culminating in the activation of non-muscle myosin IIA (NMIIA). Together, these data indicate that actomyosin contractility drives cell intercalation and cell extrusion during palate fusion and suggest a general mechanism for tissue fusion in development.     

Pak1 regulates focal adhesion strength, myosin IIA distribution, and actin dynamics to optimize cell migration

Cell motility requires the spatial and temporal coordination of forces in the actomyosin cytoskeleton with extracellular adhesion. The biochemical mechanism that coordinates filamentous actin (F-actin) assembly, myosin contractility, adhesion dynamics, and motility to maintain the balance between adhesion and contraction remains unknown. In this paper, we show that p21-activated kinases (Paks), downstream effectors of the small guanosine triphosphatases Rac and Cdc42, biochemically couple leading-edge actin dynamics to focal adhesion (FA) dynamics. Quantitative live cell microscopy assays revealed that the inhibition of Paks abolished F-actin flow in the lamella, displaced myosin IIA from the cell edge, and decreased FA turnover. We show that, by controlling the dynamics of these three systems, Paks regulate the protrusive activity and migration of epithelial cells. Furthermore, we found that expressing Pak1 was sufficient to overcome the inhibitory effects of excess adhesion strength on cell motility. These findings establish Paks as critical molecules coordinating cytoskeletal systems for efficient cell migration.     

Non‐muscle myosin IIB helps mediate TNF cell death signaling independent of actomyosin contractility (AMC)

Non‐muscle myosin II (NM II) helps mediate survival and apoptosis in response to TNF‐alpha (TNF), however, NM II's mechanism of action in these processes is not fully understood. NM II isoforms are involved in a variety of cellular processes and differences in their enzyme kinetics, localization, and activation allow NM II isoforms to have distinct functions within the same cell. The present study focused on isoform specific functions of NM IIA and IIB in mediating TNF induced apoptosis. Results show that siRNA knockdown of NM IIB, but not NM IIA, impaired caspase cleavage and nuclear condensation in response to TNF. NM II's function in promoting cell death signaling appears to be independent of actomyosin contractility (AMC) since treatment of cells with blebbistatin or cytochalasin D failed to inhibit TNF induced caspase cleavage. Immunoprecipitation studies revealed associations of NM IIB with clathrin, FADD, and caspase 8 in response to TNF suggesting a role for NM IIB in TNFR1 endocytosis and the formation of the death inducing signaling complex (DISC). These findings suggest that NM IIB promotes TNF cell death signaling in a manner independent of its force generating property. J. Cell. Biochem. 9999: 1365–1375, 2010. © 2010 Wiley‐Liss, Inc.     

Actomyosin contractility spatiotemporally regulates actin network dynamics in migrating cells

Coupling interactions among mechanical and biochemical factors are important for the realization of various cellular processes that determine cell migration. Although F-actin network dynamics has been the focus of many studies, it is not yet clear how mechanical forces generated by actomyosin contractility spatiotemporally regulate this fundamental aspect of cell migration. In this study, using a combination of fluorescent speckle microscopy and particle imaging velocimetry techniques, we perturbed the actomyosin system and examined quantitatively the consequence of actomyosin contractility on F-actin network flow and deformation in the lamellipodia of actively migrating fish keratocytes. F-actin flow fields were characterized by retrograde flow at the front and anterograde flow at the back of the lamellipodia, and the two flows merged to form a convergence zone of reduced flow intensity. Interestingly, activating or inhibiting actomyosin contractility altered network flow intensity and convergence, suggesting that network dynamics is directly regulated by actomyosin contractility. Moreover, quantitative analysis of F-actin network deformation revealed that the deformation was significantly negative and predominant in the direction of cell migration. Furthermore, perturbation experiments revealed that the deformation was a function of actomyosin contractility. Based on these results, we suggest that the actin cytoskeletal structure is a mechanically self-regulating system, and we propose an elaborate pathway for the spatiotemporal self-regulation of the actin cytoskeletal structure during cell migration. In the proposed pathway, mechanical forces generated by actomyosin interactions are considered central to the realization of the various mechanochemical processes that determine cell motility.     

Guanine Nucleotide Exchange Factor-H1 Regulates Cell Migration via Localized Activation of RhoA at the Leading Edge

Cell migration involves the cooperative reorganization of the actin and microtubule cytoskeletons, as well as the turnover of cell–substrate adhesions, under the control of Rho family GTPases. RhoA is activated at the leading edge of motile cells by unknown mechanisms to control actin stress fiber assembly, contractility, and focal adhesion dynamics. The microtubule-associated guanine nucleotide exchange factor (GEF)-H1 activates RhoA when released from microtubules to initiate a RhoA/Rho kinase/myosin light chain signaling pathway that regulates cellular contractility. However, the contributions of activated GEF-H1 to coordination of cytoskeletal dynamics during cell migration are unknown. We show that small interfering RNA-induced GEF-H1 depletion leads to decreased HeLa cell directional migration due to the loss of the Rho exchange activity of GEF-H1. Analysis of RhoA activity by using a live cell biosensor revealed that GEF-H1 controls localized activation of RhoA at the leading edge. The loss of GEF-H1 is associated with altered leading edge actin dynamics, as well as increased focal adhesion lifetimes. Tyrosine phosphorylation of focal adhesion kinase and paxillin at residues critical for the regulation of focal adhesion dynamics was diminished in the absence of GEF-H1/RhoA signaling. This study establishes GEF-H1 as a critical organizer of key structural and signaling components of cell migration through the localized regulation of RhoA activity at the cell leading edge.     

Nerve growth factor promotes reorganization of the axonal microtubule array at sites of axon collateral branching

The localized debundling of the axonal microtubule array and the entry of microtubules into axonal filopodia are two defining features of collateral branching. We report that nerve growth factor (NGF), a branch‐inducing signal, increases the frequency of microtubule debundling along the axon shaft of chicken embryonic sensory neurons. Sites of debundling correlate strongly with the localized targeting of microtubules into filopodia. Platinum replica electron microscopy suggests physical interactions between debundled microtubules and axonal actin filaments. However, as evidenced by depolymerization of actin filaments and inhibition of myosin II, actomyosin force generation does not promote debundling. In contrast, loss of actin filaments or inhibition of myosin II activity promotes debundling, indicating that axonal actomyosin forces suppress debundling. MAP1B is a microtubule associated protein that represses axon branching. Following treatment with NGF, microtubules penetrating filopodia during the early stages of branching exhibited lower levels of associated MAP1B. NGF increased and decreased the levels of MAP1B phosphorylated at a GSK‐3β site (pMAP1B) along the axon shaft and within axonal filopodia, respectively. The levels of MAP1B and pMAP1B were not altered at sites of debundling, relative to the rest of the axon. Unlike the previously determined effects of NGF on the axonal actin cytoskeleton, the effects of NGF on microtubule debundling were not affected by inhibition of protein synthesis. Collectively, these data indicate that NGF promotes localized axonal microtubule debundling, that actomyosin forces antagonize microtubule debundling, and that NGF regulates pMAP1B in axonal filopodia during the early stages of collateral branch formation. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 75: 1441–1461, 2015     

Stable and dynamic microtubules coordinately shape the myosin activation zone during cytokinetic furrow formation

The cytokinetic furrow arises from spatial and temporal regulation of cortical contractility. To test the role microtubules play in furrow specification, we studied myosin II activation in echinoderm zygotes by assessing serine19-phosphorylated regulatory light chain (pRLC) localization after precisely timed drug treatments. Cortical pRLC was globally depressed before cytokinesis, then elevated only at the equator. We implicated cell cycle biochemistry (not microtubules) in pRLC depression, and differential microtubule stability in localizing the subsequent myosin activation. With no microtubules, pRLC accumulation occurred globally instead of equatorially, and loss of just dynamic microtubules increased equatorial pRLC recruitment. Nocodazole treatment revealed a population of stable astral microtubules that formed during anaphase; among these, those aimed toward the equator grew longer, and their tips coincided with cortical pRLC accumulation. Shrinking the mitotic apparatus with colchicine revealed pRLC suppression near dynamic microtubule arrays. We conclude that opposite effects of stable versus dynamic microtubules focuses myosin activation to the cell equator during cytokinesis.     

F-actin polymerization and retrograde flow drive sustained PLCγ1 signaling during T cell activation

Activation of T cells by antigen-presenting cells involves assembly of signaling molecules into dynamic microclusters (MCs) within a specialized membrane domain termed the immunological synapse (IS). Actin and myosin IIA localize to the IS, and depletion of F-actin abrogates MC movement and T cell activation. However, the mechanisms that coordinate actomyosin dynamics and T cell receptor signaling are poorly understood. Using pharmacological inhibitors that perturb individual aspects of actomyosin dynamics without disassembling the network, we demonstrate that F-actin polymerization is the primary driver of actin retrograde flow, whereas myosin IIA promotes long-term integrity of the IS. Disruption of F-actin retrograde flow, but not myosin IIA contraction, arrested MC centralization and inhibited sustained Ca(2+) signaling at the level of endoplasmic reticulum store release. Furthermore, perturbation of retrograde flow inhibited PLCγ1 phosphorylation within MCs but left Zap70 activity intact. These studies highlight the importance of ongoing actin polymerization as a central driver of actomyosin retrograde flow, MC centralization, and sustained Ca(2+) signaling.