Unilateral Cleavage Furrows in Multinucleate Cells.

Multinucleate cells can be produced in Dictyostelium by electric pulse-induced fusion. In these cells, unilateral cleavage furrows are formed at spaces between areas that are controlled by aster microtubules. A peculiarity of unilateral cleavage furrows is their propensity to join laterally with other furrows into rings to form constrictions. This means cytokinesis is biphasic in multinucleate cells, the final abscission of daughter cells being independent of the initial direction of furrow progression. Myosin-II and the actin filament cross-linking protein cortexillin accumulate in unilateral furrows, as they do in the normal cleavage furrows of mononucleate cells. In a myosin-II-null background, multinucleate or mononucleate cells were produced by cultivation either in suspension or on an adhesive substrate. Myosin-II is not essential for cytokinesis either in mononucleate or in multinucleate cells but stabilizes and confines the position of the cleavage furrows. In fused wild-type cells, unilateral furrows ingress with an average velocity of 1.7 µm × min-1, with no appreciable decrease of velocity in the course of ingression. In multinucleate myosin-II-null cells, some of the furrows stop growing, thus leaving space for the extensive broadening of the few remaining furrows.

N-terminal polar amino acids of the C2 insert of nonmuscle myosin II-C2 regulate its functional properties.

In this study, we investigated the regions in the alternatively spliced C2 insert of nonmuscle myosin (NM) II-C conferring unique functional properties to the protein. We used constructs carrying deletions within different regions of C2 in neuronal cells; namely, the polar N terminus, the proline/serine-rich middle, and the nonpolar C terminus. We compared the wild-type NM II-C2 and deletion mutants with respect to ATPase activity, coassembly with NM II-B, regulation by myosin light-chain kinase (MLCK), and solubility, to determine the C2 region(s) involved in these processes. In addition, we examined the ability of the mutants to rescue the neurite-shortening phenotype upon NM II-C2 knockdown in Neuro-2a cells. Our data highlight the importance of the polar N terminus in NM II-C2 function.

Loss of Myh14 Increases Susceptibility to Noise-Induced Hearing Loss in CBA/CaJ Mice.

MYH14 is a member of the myosin family, which has been implicated in many motile processes such as ion-channel gating, organelle translocation, and the cytoskeleton rearrangement. Mutations in MYH14 lead to a DFNA4-type hearing impairment. Further evidence also shows that MYH14 is a candidate noise-induced hearing loss (NIHL) susceptible gene. However, the specific roles of MYH14 in auditory function and NIHL are not fully understood. In the present study, we used CRISPR/Cas9 technology to establish a Myh14 knockout mice line in CBA/CaJ background (now referred to as Myh14-/- mice) and clarify the role of MYH14 in the cochlea and NIHL. We found that Myh14-/- mice did not exhibit significant hearing loss until five months of age. In addition, Myh14-/- mice were more vulnerable to high intensity noise compared to control mice. More significant outer hair cell loss was observed in Myh14-/- mice than in wild type controls after acoustic trauma. Our findings suggest that Myh14 may play a beneficial role in the protection of the cochlea after acoustic overstimulation in CBA/CaJ mice.

The actomyosin machinery is required for Drosophila retinal lumen formation.

Multicellular tubes consist of polarized cells wrapped around a central lumen and are essential structures underlying many developmental and physiological functions. In Drosophila compound eyes, each ommatidium forms a luminal matrix, the inter-rhabdomeral space, to shape and separate the key phototransduction organelles, the rhabdomeres, for proper visual perception. In an enhancer screen to define mechanisms of retina lumen formation, we identified Actin5C as a key molecule. Our results demonstrate that the disruption of lumen formation upon the reduction of Actin5C is not linked to any discernible defect in microvillus formation, the rhabdomere terminal web (RTW), or the overall morphogenesis and basal extension of the rhabdomere. Second, the failure of proper lumen formation is not the result of previously identified processes of retinal lumen formation: Prominin localization, expansion of the apical membrane, or secretion of the luminal matrix. Rather, the phenotype observed with Actin5C is phenocopied upon the decrease of the individual components of non-muscle myosin II (MyoII) and its upstream activators. In photoreceptor cells MyoII localizes to the base of the rhabdomeres, overlapping with the actin filaments of the RTW. Consistent with the well-established roll of actomyosin-mediated cellular contraction, reduction of MyoII results in reduced distance between apical membranes as measured by a decrease in lumen diameter. Together, our results indicate the actomyosin machinery coordinates with the localization of apical membrane components and the secretion of an extracellular matrix to overcome apical membrane adhesion to initiate and expand the retinal lumen.

Functional and genetic interactions of TOR in the budding yeast Saccharomyces cerevisiae with myosin type II-deficiency (myo1Δ).

BACKGROUND: Yeast has numerous mechanisms to survive stress. Deletion of myosin type II (myo1Δ) in Saccharomyces cerevisiae results in a cell that has defective cytokinesis. To survive this genetically induced stress, this budding yeast up regulates the PKC1 cell wall integrity pathway (CWIP). More recently, our work indicated that TOR, another stress signaling pathway, was down regulated in myo1Δ strains. Since negative signaling by TOR is known to regulate PKC1, our objectives in this study were to understand the cross-talk between the TOR and PKC1 signaling pathways and to determine if they share upstream regulators for mounting the stress response in myo1Δ strains. RESULTS: Here we proved that TORC1 signaling was down regulated in the myo1Δ strain. While a tor1Δ mutant strain had increased viability relative to myo1Δ, a combined myo1Δtor1Δ mutant strain showed significantly reduced cell viability. Synthetic rescue of the tor2-21(ts) lethal phenotype was observed in the myo1Δ strain in contrast to the chs2Δ strain, a chitin synthase II null mutant that also activates the PKC1 CWIP and exhibits cytokinesis defects very similar to myo1Δ, where the rescue effect was not observed. We observed two pools of Slt2p, the final Mitogen Activated Protein Kinase (MAPK) of the PKC1 CWIP; one pool that is up regulated by heat shock and one that is up regulated by the myo1Δ stress. The cell wall stress sensor WSC1 that activates PKC1 CWIP under other stress conditions was shown to act as a negative regulator of TORC1 in the myo1Δ mutant. Finally, the repression of TORC1 was inversely correlated with the activation of PKC1 in the myo1Δ strain. CONCLUSIONS: Regulated expression of TOR1 was important in the activation of the PKC1 CWIP in a myo1Δ strain and hence its survival. We found evidence that the PKC1 and TORC1 pathways share a common upstream regulator associated with the cell wall stress sensor WSC1. Surprisingly, essential TORC2 functions were not required in the myo1Δ strain. By understanding how yeast mounts a concerted stress response, one can further design pharmacological cocktails to undermine their ability to adapt and to survive.

Myosin II is a negative regulator of oligodendrocyte morphological differentiation.

During their development as myelinating cells, oligodendrocyte progenitors (OPC) undergo dramatic changes in the organization of their cytoskeleton. These changes involve an increase in cell branching and in lamella extension, which is important for the ability of oligodendrocytes to myelinate multiple axons in the CNS. We have previously shown that the levels of the actin-associated motor protein nonmuscle myosin II (NMII) decrease as oligodendrocyte differentiate and that inhibition of NMII activity increases branching and myelination, suggesting that NMII is a negative regulator of oligodendrocyte differentiation. In agreement with this interpretation, we have found that overexpression of NMII prevents oligodendrocyte branching and differentiation and that OPC maturation is accelerated in NMII knockout mice as shown by a significant increase in the percentage of mature MBP(+) cells. Although several pathways have been implicated in oligodendrocyte morphogenesis, their specific contribution to the regulation of NMII activity has not been directly examined. We tested the hypothesis that the activity of NMII in OPC is controlled by Fyn kinase via downregulation of RhoA-ROCK-NMII phosphorylation. We found that treatment with PP2 or knockdown of Fyn using siRNA prevents the decrease in myosin phosphorylation normally observed during OPC differentiation and that the inhibition of branching induced by overexpression of constitutively active RhoA can be reversed by treatment with Y27632 or blebbistatin. Taken together, our results demonstrate that Fyn kinase downregulates NMII activity, thus promoting oligodendrocyte morphological differentiation.

Global mRNA expression analysis in myosin II deficient strains of Saccharomyces cerevisiae reveals an impairment of cell integrity functions.

BACKGROUND: The Saccharomyces cerevisiae MYO1 gene encodes the myosin II heavy chain (Myo1p), a protein required for normal cytokinesis in budding yeast. Myo1p deficiency in yeast (myo1Delta) causes a cell separation defect characterized by the formation of attached cells, yet it also causes abnormal budding patterns, formation of enlarged and elongated cells, increased osmotic sensitivity, delocalized chitin deposition, increased chitin synthesis, and hypersensitivity to the chitin synthase III inhibitor Nikkomycin Z. To determine how differential expression of genes is related to these diverse cell wall phenotypes, we analyzed the global mRNA expression profile of myo1Delta strains. RESULTS: Global mRNA expression profiles of myo1Delta strains and their corresponding wild type controls were obtained by hybridization to yeast oligonucleotide microarrays. Results for selected genes were confirmed by real time RT-PCR. A total of 547 differentially expressed genes (p < or = 0.01) were identified with 263 up regulated and 284 down regulated genes in the myo1Delta strains. Gene set enrichment analysis revealed the significant over-representation of genes in the protein biosynthesis and stress response categories. The SLT2/MPK1 gene was up regulated in the microarray, and a myo1Deltaslt2Delta double mutant was non-viable. Overexpression of ribosomal protein genes RPL30 and RPS31 suppressed the hypersensitivity to Nikkomycin Z and increased the levels of phosphorylated Slt2p in myo1Delta strains. Increased levels of phosphorylated Slt2p were also observed in wild type strains under these conditions. CONCLUSION: Following this analysis of global mRNA expression in yeast myo1Delta strains, we conclude that 547 genes were differentially regulated in myo1Delta strains and that the stress response and protein biosynthesis gene categories were coordinately regulated in this mutant. The SLT2/MPK1 gene was confirmed to be essential for myo1Delta strain viability, supporting that the up regulated stress response genes are regulated by the PKC1 cell integrity pathway. Suppression of Nikkomycin Z hypersensitivity together with Slt2p phosphorylation was caused by the overexpression of ribosomal protein genes RPL30 and RPS31. These ribosomal protein mRNAs were down regulated in the myo1Delta arrays, suggesting that down regulation of ribosomal biogenesis may affect cell integrity in myo1Delta strains.

Shrinkage insensitivity of NKCC1 in myosin II-depleted cytoplasts from Ehrlich ascites tumor cells.

Protein phosphorylation/dephosphorylation and cytoskeletal reorganization regulate the Na(+)-K(+)-2Cl(-) cotransporter (NKCC1) during osmotic shrinkage; however, the mechanisms involved are unclear. We show that in cytoplasts, plasma membrane vesicles detached from Ehrlich ascites tumor cells (EATC) by cytochalasin treatment, NKCC1 activity evaluated as bumetanide-sensitive (86)Rb influx was increased compared with the basal level in intact cells yet could not be further increased by osmotic shrinkage. Accordingly, cytoplasts exhibited no regulatory volume increase after shrinkage. In cytoplasts, cortical F-actin organization was disrupted, and myosin II, which in shrunken EATC translocates to the cortical region, was absent. Moreover, NKCC1 activity was essentially insensitive to the myosin light chain kinase (MLCK) inhibitor ML-7, a potent blocker of shrinkage-induced NKCC1 activity in intact EATC. Cytoplast NKCC1 activity was potentiated by the Ser/Thr protein phosphatase inhibitor calyculin A, partially inhibited by the protein kinase A inhibitor H89, and blocked by the broad protein kinase inhibitor staurosporine. Cytoplasts exhibited increased protein levels of NKCC1, Ste20-related proline- and alanine-rich kinase (SPAK), and oxidative stress response kinase 1, yet they lacked the shrinkage-induced plasma membrane translocation of SPAK observed in intact cells. The basal phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK) was increased in cytoplasts compared with intact cells, yet in contrast to the substantial activation in shrunken intact cells, p38 MAPK could not be further activated by shrinkage of the cytoplasts. Together these findings indicate that shrinkage activation of NKCC1 in EATC is dependent on the cortical F-actin network, myosin II, and MLCK.

Dissection of amoeboid movement into two mechanically distinct modes.

The current dominant model of cell locomotion proposes that actin polymerization pushes against the membrane at the leading edge producing filopodia and lamellipodia that move the cell forward. Despite its success, this model does not fully explain the complex process of amoeboid motility, such as that occurring during embryogenesis and metastasis. Here, we show that Dictyostelium cells moving in a physiological milieu continuously produce ;blebs' at their leading edges, and demonstrate that focal blebbing contributes greatly to their locomotion. Blebs are well-characterized spherical hyaline protrusions that occur when a patch of cell membrane detaches from its supporting cortex. Their formation requires the activity of myosin II, and their physiological contribution to cell motility has not been fully appreciated. We find that pseudopodia extension, cell body retraction and overall cell displacement are reduced under conditions that prevent blebbing, including high osmolarity and blebbistatin, and in myosin-II-null cells. We conclude that amoeboid motility comprises two mechanically different processes characterized by the production of two distinct cell-surface protrusions, blebs and filopodia-lamellipodia.

Myosin and the PAR proteins polarize microfilament-dependent forces that shape and position mitotic spindles in Caenorhabditis elegans.

In Caenorhabditis elegans, the partitioning proteins (PARs), microfilaments (MFs), dynein, dynactin, and a nonmuscle myosin II all localize to the cortex of early embryonic cells. Both the PARs and the actomyosin cytoskeleton are required to polarize the anterior-posterior (a-p) body axis in one-cell zygotes, but it remains unknown how MFs influence embryonic polarity. Here we show that MFs are required for the cortical localization of PAR-2 and PAR-3. Furthermore, we show that PAR polarity regulates MF-dependent cortical forces applied to astral microtubules (MTs). These forces, which appear to be mediated by dynein and dynactin, produce changes in the shape and orientation of mitotic spindles. Unlike MFs, dynein, and dynactin, myosin II is not required for the production of these forces. Instead, myosin influences embryonic polarity by limiting PAR-3 to the anterior cortex. This in turn produces asymmetry in the forces applied to MTs at each pole and allows PAR-2 to accumulate in the posterior cortex of a one-cell zygote and maintain asymmetry.

Dictyostelium myosin II mutations that uncouple the converter swing and ATP hydrolysis cycle.

During the ATP hydrolysis cycle of the Dictyostelium myosin II motor domain, two conserved alpha-helices, the SH1/SH2 helix and the relay helix, rotate in a coordinated way to induce the swing motion of the converter domain. A network of hydrophobic and ionic interactions in these two helices and the converter may ensure that the motions of these helices are effectively transmitted to the converter. To examine the roles of these interactions in the ATPase-dependent converter swing, we disrupted two conserved hydrophobic linkages among them by means of a point mutation (I499A or F692A). The resulting mutations induced only limited changes in the kinetic parameters of ATP hydrolysis, except for a marked increase of basal MgATPase activity. However, the mutant myosins completely lost their in vitro and in vivo motor functions. Measurements of the intrinsic tryptophan fluorescence and the GFP-based FRET revealed that the converter domain of these mutants did not swing during steady-state ATP hydrolysis or in the presence of tightly trapped Mg.ADP.V(i), which shows that the point mutations induced the uncoupling of the converter swing and ATP hydrolysis cycle. These results highlight the importance of these hydrophobic linkages for transmitting the coordinated twist motions of the helices to the converter as well as the requirement of this converter swing for force generation.

Tail chimeras of Dictyostelium myosin II support cytokinesis and other myosin II activities but not full development.

Dictyostelium lacking myosin II cannot grow in suspension culture, develop beyond the mound stage or cap concanavalin A receptors and chemotaxis is impaired. Recently, we showed that the actin-activated MgATPase activity of myosin chimeras in which the tail domain of Dictyostelium myosin II heavy chain is replaced by the tail domain of either Acanthamoeba or chicken smooth muscle myosin II is unregulated and about 20 times higher than wild-type myosin. The Acanthamoeba chimera forms short bipolar filaments similar to, but shorter than, filaments of Dictyostelium myosin and the smooth muscle chimera forms much larger side-polar filaments. We now find that the Acanthamoeba chimera expressed in myosin null cells localizes to the periphery of vegetative amoeba similarly to wild-type myosin but the smooth muscle chimera is heavily concentrated in a single cortical patch. Despite their different tail sequences and filament structures and different localization of the smooth muscle chimera in interphase cells, both chimeras support growth in suspension culture and concanavalin A capping and colocalize with the ConA cap but the Acanthamoeba chimera subsequently disperses more slowly than wild-type myosin and the smooth muscle chimera apparently not at all. Both chimeras also partially rescue chemotaxis. However, neither supports full development. Thus, neither regulation of myosin activity, nor regulation of myosin polymerization nor bipolar filaments is required for many functions of Dictyostelium myosin II and there may be no specific sequence required for localization of myosin to the cleavage furrow.

Myosin II redistribution during rear retraction and the role of filament assembly and disassembly.

The literature to date suggests a role for myosin II in rear retraction, including evidence that myosin undergoes a characteristic 'C'-to-spot redistribution at the cell posterior which is associated with retraction. Here we investigate the mechanism of both retraction and the'C'-to-spot using Dictyostelium cells containing mutant forms of myosin that affect its polymerization. 3 x Asp-myosin forms few if any filaments. When 3 x Asp cells are added to a wild-type mound, the mutant cells move directionally, but rear retraction is markedly delayed,demonstrating that myosin II filaments are essential for efficient retraction. In addition, using a GFP-tagged 3 x Asp-myosin, we observed a posterior spot pattern associated with retraction,but no cortical 'C' pattern preceding it. This suggests that filamentous myosin is required to produce the 'C', and that its failure to form results in defective rear retraction. In contrast, an alternate mutant myosin that forms filaments constitutively, 3 x Ala-myosin, forms 'Cs' and then spot patterns at the posterior, but in the interim the spots do not disintegrate. This suggests that spot dissolution occurs by filament depolymerization. In summary our data demonstrate a role for myosin II and the 'C'-to-spot in efficient rear retraction, and define filament assembly as critical for formation of the 'C' and filament disassembly as critical for dissolution of the spot.