Myosin biology and biophysics

The myosin head consists of a globular catalytic domain that binds actin and hydrolyzes ATP. The neck domain consists of essential and regulatory light chains bound to a long alpha-helical portion of the heavy chain. The swinging neck-level model assumes that a swinging motion of the neck relative to the catalytic domain is the origin of movement. This model predicts that the step size, and consequently the sliding velocity, are linearly related to the length of the neck. This has been analyzed by characterizing a series of mutant Dictyostelium myosins that have different neck lengths. The 2xELCBS mutant has an extra binding site for essential light chain. The delta RLCBS mutant myosin has an internal deletion that removes the regulatory light chain binding site. The delta BLCBS mutant lacks both light chain binding sites. Wild-type myosin and these mutant myosins were subjected to the sliding filament in vitro motility assay. As expected, mutants with shorter necks move slower than wild-type myosin in vitro. Most significantly, a mutant with a longer neck moves faster than the wild type, and the sliding velocities of these myosins are linearly related to the neck length, as predicted by the swinging neck-lever model. A simple extrapolation to zero speed predicts that the fulcrum point is in the vicinity of the SH1-SH2 region in the catalytic domain (Uyeda, 1996).

Conventional myosin plays a key role in the cytoskeletal reorganization necessary for cytokinesis, migration, and morphological changes associated with development in nonmuscle cells. A fusion protein has been prepared between the green fluorescent protein (GFP) and the Dictyostelium discoideum myosin heavy chain (GFP-myosin). Expression of GFP-myosin rescues all myosin null cell defects. Additionally, GFP-myosin purified from these cells exhibits the same ATPase activities and in vitro motility as wild-type myosin. GFP-myosin is concentrated in the cleavage furrow during cytokinesis and in the posterior cortex of migrating cells. Surprisingly, GFP-myosin concentration increases transiently in the tips of retracting pseudopods. Contrary to previous thinking, this suggests that conventional myosin may play an important role in the dynamics of pseudopods as well as filopodia, lamellipodia, and other cellular protrusions (Moores, 1996).

Daughter cells with distinct fates can arise through intrinsically asymmetrical divisions. Prior to such divisions, factors crucial for determining cell fates become asymmetrically localized in the mother cell. In C. elegans, PAR proteins are required for the early asymmetrical divisions that establish embryonic polarity; PAR proteins are asymmetrically localized in early blastomeres, although the mechanism of their distribution is not known. Nonmuscle myosin II heavy chain (designated NMY-2) is identified by means of its interaction with the PAR-1 protein, a putative Ser/Thr protein kinase. Furthermore, injections of nmy-2 antisense RNA into ovaries of adult worms cause embryonic partitioning defects and lead to mislocalization of PAR proteins. It is therefore concluded that the NMY-2 is required for establishing cellular polarity in C. elegans embryos (Guo, 1996).

The assembly of myosin II molecules into a filament requires the electrostatic interaction of a domain localized toward the carboxyl-terminus of the myosin II tail. The smallest Dictyostelium myosin II fragment both necessary and sufficient for self-association consists of 294 amino acids that contain four clusters of positively charged and negatively charged residues. Fragments of the same length but which lack one of these positively or negatively charged clusters are incapable of self-assembly. This assembly domain is also found in myosin II from other species. Such charged clusters are found in a similar location in rabbit myosin II and are also essential for filament formation (Shoffner, 1996).

Myosin II heavy chains bind to phosphatidylserine (PS) liposomes via their COOH terminal regions and protein kinase C (PKC) phosphorylates the PS-bound heavy chains. Binding of fragments from the COOH termini of macrophage and brain heavy chain isoforms to PS or phosphatidylinositol liposomes increases turbidity. With mixed PS/phosphatidylcholine (PC) liposomes, at least 70 mol % PS was required for heavy chain binding. A similar level of PS was required for phosphorylation of fragments by PKC, indicating that binding of tail regions to PS is a prerequisite for phosphorylation by PKC. PKC phosphorylates one isoform with Vmax values 4-5 times higher than those of another, but the Km values for the two substrates are similar. The apparent Km values for PS liposomes (Klipid) were also similar for phosphorylation of either isoforms. Mixing PS with PC increased the Klipid and reduced the Vmax values but did not alter the Km values for the substrates. Assembly of one isoform, is significantly inhibited by the phosphorylation, indicating that nonmuscle myosin assembly can be regulated, in an isoform specific manner, via phosphorylation of heavy chains by PKC (Murakami, 1995).

Myosin is involved in postmitotic cell spreading. Butanedione monoxime (BDM), a known inhibitor of muscle myosin II, inhibits nonmuscle myosin II and myosin V adenosine triphosphatases. BDM reversibly inhibits PtK2 postmitotic cell spreading. Listeria motility is not affected by this drug. Electron microscopy studies show that some actin filaments in spreading edges are part of actin bundles that are also found in long, thin, structures that are connected to spreading edges and substrate (retraction fibers), and that 90% of this actin is oriented with barbed ends in the direction of spreading. The remaining actin in spreading edges has a more random orientation and spatial arrangement. Myosin II is associated with actin polymer in spreading cell edges, but not retraction fibers. Myosin II is excluded from lamellipodia that protrude from the cell edge at the end of spreading (Cramer, 1995).

Different isoforms of non-muscle myosin II have different distributions in vivo, even within individual cells. In order to understand how these different distributions arise, the distribution and dynamics of non-muscle myosins IIA and myosin IIB were examined in cultured cells. Cultured bovine aortic endothelia contain both myosins IIA and IIB. Both isoforms distribute along stress fibers, in linear or punctate aggregates within lamellipodia, and diffusely around the nucleus. However, the A isoform is preferentially located toward the leading edge of migrating cells, when compared with myosin IIB by double immunofluorescence staining. Conversely, the B isoform is enriched in structures at the cells' trailing edges. When fluorescent analogs of the two isoforms are co-injected into living cells, the injected myosins distribute with the same disparate localizations as endogenous myosins IIA and IIB. This indicates that the ability of the myosins to ‘sort’ within the cytoplasm is intrinsic to the proteins themselves, and not a result of localized synthesis or degradation. Furthermore, time-lapse imaging of injected analogs in living cells reveals differences in the rates at which the two isoforms rearrange during cell movement. The A isoform appears in newly formed structures more rapidly than the B isoform, and is also lost more rapidly when structures disassemble. These observations suggest that the different localizations of myosins IIA and IIB reflect different rates at which the isoforms transit through assembly, movement and disassembly within the cell. The relative proportions of different myosin II isoforms within a particular cell type may determine the lifetimes of various myosin II-based structures in that cell (Kolega,1998)

There are two isoforms of the vertebrate nonmuscle myosin heavy chain, MHC-A and MHC-B, that are encoded by two separate genes. The velocity of movement of actin filaments by MHC-A is 3.3-fold faster than that by MHC-B. Likewise, the Vmax of the actin-activated Mg(2+)-ATPase activity of MHC-A is 2.6-fold greater than that of MHC-B. There are distinct localizations for MHC-A and MHC-B. In interphase cells, MHC-B is present in the cell cortex and diffusely arranged in the cytoplasm. In highly polarized, rapidly migrating interphase cells, the lamellipodium is dramatically enriched for MHC-B, suggesting a possible involvement of MHC-B based contractions in leading edge extension and/or retraction. In contrast, MHC-A is absent from the cell periphery and is arranged in a fibrillar staining pattern in the cytoplasm. The two myosin heavy chain isoforms also have distinct localizations throughout mitosis. During prophase, the MHC-B redistributes to the nuclear membrane, and then resumes its interphase localization by metaphase. MHC-A, while diffuse within the cytoplasm at all stages of mitosis, also localizes to the mitotic spindle in two different cultured cell lines as well as in Xenopus blastomeres. During telophase both isoforms colocalize to the contractile ring. The different subcellular localizations of MHC-A and MHC-B, together with the data demonstrating that these myosins have markedly different enzymatic activities, strongly suggests that they have different functions (Kelley, 1996).

Recent work has identified additional, alternatively spliced isoforms of MHC-B cDNA with inserted sequences of 30 nucleotides (chicken and human) or 48 nucleotides (Xenopus) at a site corresponding to the ATP binding region in the MHC protein. There is a consensus sequence for phosphorylation by cyclin-p34cdc2 (cdc2) kinase (See CyclinB for information about cyclin-cdc2 function) suggesting that MHC is regulated in a cell cycle dependent manner. In cultured Xenopus XTC cells, two inserted MHC-B isoforms and a non-inserted MHC-A isoform have been identified. Cdc2 kinase catalyzes the phosphorylation of both inserted MHC-B isoforms but not MHC-A. The phosphorylated residue is Ser-214, the cdc2 kinase consensus site within the insert near the ATP binding region. The same site is phosphorylated in intact cells during log phase of growth and in cell-free lysates of Xenopus eggs stabilized in second meiotic metaphase but not interphase. Moreover, Ser-214 phosphorylation is detected during maturation of Xenopus oocytes when the cdc2 kinase-containing maturation-promoting factor is activated, but not in G2 interphase-arrested oocytes. These results demonstrate that MHC-B phosphorylation is tightly regulated by cdc2 kinase during meiotic cell cycles. Furthermore, MHC-A and MHC-B isoforms are differentially phosphorylated at these stages, suggesting that they may serve different functions in these cells (Kelley, 1995).

The promoter and flanking region of human nonmuscle myosin heavy chain (MHC)-A has been isolated. The sequence of this region shows many features typical of a housekeeping gene; there is no TATA element and no functional CAAT box. The GC content is high, having an average GC content of 74% in the 600 base pairs (bp) surrounding the transcriptional start sites, and multiple GC boxes (putative Sp1 binding sites) are present. A number of nucleotide sites are utilized for the initiation of transcription. Analysis of 5' and 3' deletion mutants in the promoter region defines the core promoter as extending from nucleotide -112 to +61, where +1 is a major transcriptional start site. An essential sequence for core promoter activity resides in the 36-bp region from -77 to -112 which includes a single potential AP-2 binding site and a single potential Sp1 binding site. The region just downstream from the transcriptional start site (between +62 and +257) is involved in cell type-specific activation of nonmuscle MHC-A gene expression. The increase in transcription due to this proximal downstream region is cell type dependent. This 196-bp region, which consists of 100 bp from exon 1 and 96 bp from intron 1, functions in a position- and orientation-dependent manner. This proximal downstream region appears to activate gene expression in cells via both pretranslational (transcription and/or mRNA stability) and translational mechanisms (Kawamoto, 1994).

Unique isoforms of nonmuscle myosin heavy chain II-B (MHC-B) are expressed in chicken and human neuronal cells. These isoforms, which appear to be generated by alternative splicing of pre-mRNA, differ from the MHC-B isoform present in a large number of nonmuscle cells in that they contain inserted cassettes of amino acids near the ATP binding region and/or near the actin binding region. The insert near the ATP binding region begins after amino acid 211 and consists of either 10 or 16 amino acids. The insert near the actin binding region begins after amino acid 621 and consists of 21 amino acids. In the developing chicken brain, mRNA encoding the 10-amino acid insert gradually increases after embryonic day 4, peaks in the 10-14-day embryo, and then declines. In contrast, the mRNA encoding the 21-amino acid insert appears just before birth and is abundantly expressed in the adult chicken cerebellum. There is a marked species difference between the distribution of the inserted isoforms in adult tissues. Employing human retinoblastoma (Y-79) and neuroblastoma (SK-N-SH) cell lines, an increase in expression of mRNA encoding the 10-amino acid inserted isoform is seen following treatment by a number of agonists or by serum deprivation. In each case, expression of the inserted MHC-B isoform correlates with cell differentiation (neuronal phenotype) and inhibition of cell division. Using a rat pheochromocytoma cell line (PC12), it was found that prior to treatment with nerve growth factor (NGF), there is no evidence for either inserted isoform, although noninserted MHC-B is present. NGF treatment results in the appearance of mRNA encoding MHC-B containing the 10-amino acid insert, concomitant with neurite outgrowth (Itoh, 1995).

Myosin II and vesicular transport

The participation of nonmuscle myosins in the transport of organelles and vesicular carriers along actin filaments has been documented. In contrast, there is no evidence for the involvement of myosins in the production of vesicles involved in membrane traffic. The putative TGN coat protein p200 is myosin II. The recruitment of myosin II to Golgi membranes is dependent on actin and is regulated by G proteins. Using an assay that studies the release of transport vesicles from the TGN in vitro, functional evidence is provided that p200/myosin is involved in the assembly of basolateral transport vesicles carrying vesicular stomatitis virus G protein (VSVG) from the TGN of polarized MDCK cells. The 50% reduced efficiency in VSVG vesicle release from the TGN in vitro after depletion of p200/myosin II can be reestablished to control levels by the addition of purified nonmuscle myosin II. Several inhibitors of the actin-stimulated ATPase activity of myosin specifically inhibit the release of VSVG-containing vesicles from the TGN (Musch, 1997).

The zebrafish egg provides a useful experimental system to study events of fertilization, including exocytosis. Cortical granules are: (1) released nonsynchronously over the egg surface and (2) mobilized to the plasma membrane in two phases, depending upon vesicle size and location. Measurements of the timing and extent of exocytosis reveal a steady release of small granules during the first 30 seconds of egg activation. This is followed by an explosive discharge of large granules, beginning at 30 seconds and continuing for 1-2 minutes. Cortical granule translocation and fusion with the plasma membrane is followed by the concurrent expansion of a fusion pore and release of granule contents. A dramatic rearrangement of the egg surface follows exocytosis. Cortical crypts (sites of evacuated granules) display a purse-string-like contraction, resulting in their gradual flattening and disappearance from the egg surface. The hypothesis was tested that subplasmalemmal filamentous (F-) actin acts as a physical barrier to secretion and is locally disassembled prior to granule release. Experimental results show a reduction of rhodamine-phalloidin and antimyosin staining at putative sites of secretion, acceleration of the timing and extent of granule release in eggs pretreated with cytochalasin D, and dose-dependent inhibition of exocytosis in permeabilized eggs preincubated with phalloidin. An increase in assembled actin is detected during the period of exocytosis. Localization studies show that F-actin and myosin-II codistribute with an inward-moving, membrane-delimited zone of cytoplasm that circumscribes cortical crypts during their transformation. Furthermore, cortical crypts display a distinct delay in transformation when incubated continuously with cytochalasin D following egg activation. It is proposed that closure of cortical crypts is driven by a contractile ring whose forces depend on dynamic actin filaments and perhaps actomyosin interactions (Becker, 1999).

Myosin II and mitosis

The role of conventional myosin II in cytokinesis in Dictyostelium cells was investigated by examining cells under both adhesive and nonadhesive conditions. On an adhesive surface, both wild-type and myosin-null cells undergo the normal processes of mitotic rounding, cell elongation, polar ruffling, furrow ingression, and separation of daughter cells. When cells are denied adhesion through culturing in suspension or on a hydrophobic surface, wild-type cells undergo these same processes. However, cells lacking myosin round up and polar ruffle, but fail to elongate, furrow, or divide. These differences show that cell division can be driven by two mechanisms, termed Cytokinesis A, which requires myosin, and Cytokinesis B, which is cell adhesion dependent. Cytokinesis A is myosin-dependent active furrowing and is the sole means of division under circumstances where cells cannot adhere to a surface, such as in suspension or on a hydrophobic surface. Cytokinesis B is myosin independent and possibly results from the traction forces generated by polar pseudopods exerting force on an an adhesive surface. Cells were examined that express a myosin whose two light chain-binding sites had been deleted (DeltaBLCBS-myosin). Although this myosin is a slower motor than wild-type myosin and has constitutively high activity due to the abolition of regulation by light-chain phosphorylation, cells expressing DeltaBLCBS-myosin divide in suspension. DeltaBLCBS-myosin undergoes relatively normal spatial and temporal changes in localization during mitosis. The rate of furrow progression in cells expressing a DeltaBLCBS-myosin is similar to that in wild-type cells (Zang, 1997).

Myosin II generates force for the division of eukaryotic cells. Still unknown is the molecular basis of the spatial and temporal localization of myosin II to the cleavage furrow, although models often imply that interaction between myosin II and actin filaments is essential. Examined in this study was the localization of a chimeric protein consisting of the green fluorescent protein fused to the N terminus of truncated myosin II heavy chain in Dictyostelium cells. This chimera is missing the myosin II motor domain, and it does not bind actin filaments. Surprisingly, it still localizes to the cleavage furrow region during cytokinesis. These results indicate that myosin II localization during cytokinesis occurs through a mechanism that does not require it to be the force-generating element or to interact with actin filaments directly (Zang, 1998).

To explore the role of nonmuscle myosin II isoforms during mouse gametogenesis, fertilization, and early development, localization and microinjection studies were performed using monospecific antibodies to myosin IIA and IIB isotypes. Each myosin II antibody recognizes a 205-kDa protein in oocytes, but not mature sperm. Myosin IIA and IIB demonstrate differential expression during meiotic maturation and following fertilization: only the IIA isoform detects metaphase spindles or accumulates in the mitotic cleavage furrow. In the unfertilized oocyte, both myosin isoforms are polarized in the cortex directly overlying the metaphase-arrested second meiotic spindle. Cortical polarization is altered after spindle disassembly with Colcemid: the scattered meiotic chromosomes initiate myosin IIA and microfilament assemble in the vicinity of each chromosome mass. During sperm incorporation, both myosin II isotypes concentrate in the second polar body cleavage furrow and the sperm incorporation cone. In functional experiments, the microinjection of myosin IIA antibody disrupts meiotic maturation to metaphase II arrest, probably through depletion of spindle-associated myosin IIA protein and antibody binding to chromosome surfaces. Conversely, the microinjection of myosin IIB antibody blocks microfilament-directed chromosome scattering in Colcemid-treated mature oocytes, suggesting a role in mediating chromosome-cortical actomyosin interactions. Neither myosin II antibody, alone or coinjected, blocks second polar body formation, in vitro fertilization, or cytokinesis. Finally, microinjection of a nonphosphorylatable 20-kDa regulatory myosin light chain specifically blocks sperm incorporation cone disassembly and impedes cell cycle progression, suggesting that interference with myosin II phosphorylation influences fertilization. Thus, conventional myosins break cortical symmetry in oocytes by participating in eccentric meiotic spindle positioning, sperm incorporation cone dynamics, and cytokinesis. Although murine sperm do not express myosin II, different myosin II isotypes may have distinct roles during early embryonic development (Simerly, 1998).

During mitosis the sites of myosin phosphorylation are switched between the inhibitory sites, Ser 1/2, and the activation sites, Ser 19/Thr 18, suggesting a regulatory role of myosin phosphorylation in cell division. To explore the function of myosin phosphatase in cell division, the possibility that myosin phosphatase activity may be altered during cell division was examined. The myosin phosphatase targeting subunit (MYPT) undergoes mitosis-specific phosphorylation and the phosphorylation is reversed during cytokinesis. MYPT phosphorylated either in vivo or in vitro in the mitosis-specific way shows higher binding to myosin II (two- to three-fold) as compared to MYPT from cells in interphase. Furthermore, the activity of myosin phosphatase is increased more than twice and it is suggested this reflects the increased affinity of myosin binding. These results indicate the presence of a unique positive regulatory mechanism for myosin phosphatase in cell division. The activation of myosin phosphatase during mitosis would enhance dephosphorylation of the myosin regulatory light chain, thereby leading to the disassembly of stress fibers during prophase. The mitosis-specific effect of phosphorylation is lost on exit from mitosis, and the resultant increase in myosin phosphorylation may act as a signal to activate cytokinesis (Totsukawa, 1999).

The role of myosin II in mitosis is generally thought to be restricted to cytokinesis. New evidence is presented that cortical myosin II is also required for spindle assembly in cells. Drug- or RNAi-mediated disruption of myosin II in cells interferes with normal spindle assembly and positioning. Time-lapse movies reveal that these treatments block the separation and positioning of duplicated centrosomes after nuclear envelope breakdown (NEBD), thereby preventing the migration of the microtubule asters to opposite sides of chromosomes. Immobilization of cortical movement with tetravalent lectins produces similar spindle defects to myosin II disruption and suggests that myosin II activity is required within the cortex. Latex beads bound to the cell surface move in a myosin II-dependent manner in the direction of the separating asters. It was proposed that after NEBD, completion of centrosome separation and positioning around chromosomes depends on astral microtubule connections to a moving cell cortex (Rosenblatt, 2004).

Cell division after mitosis is mediated by ingression of an actomyosin-based contractile ring. The active, GTP-bound form of the small GTPase RhoA is a key regulator of contractile-ring formation. RhoA concentrates at the equatorial cell cortex at the site of the nascent cleavage furrow. During cytokinesis, RhoA is activated by its RhoGEF, ECT2. Once activated, RhoA promotes nucleation, elongation, and sliding of actin filaments through the coordinated activation of both formin proteins and myosin II motors. Anillin is a 124 kDa protein that is highly concentrated in the cleavage furrow in numerous animal cells in a pattern that resembles that of RhoA. Although anillin contains conserved N-terminal actin and myosin binding domains and a PH domain at the C terminus, its mechanism of action during cytokinesis remains unclear. This study shows that human anillin contains a conserved C-terminal domain that is essential for its function and localization. This domain shares homology with the RhoA binding protein Rhotekin and directly interacts with RhoA. Further, anillin is required to maintain active myosin in the equatorial plane during cytokinesis, suggesting it functions as a scaffold protein to link RhoA with the ring components actin and myosin. Although furrows can form and initiate ingression in the absence of anillin, furrows cannot form in anillin-depleted cells in which the central spindle is also disrupted, revealing that anillin can also act at an early stage of cytokinesis (Piekny, 2008).

Various organisms might employ distinct mechanisms to recruit anillin, which executes its conserved function as a scaffold protein in the contractile ring. In both S. pombe and animal cells, anillin-like proteins are early markers of the nascent cleavage furrow. In most systems, anillin is not required for furrow formation. C. elegans have three anillin-like proteins, and ani-1 is required for contractile events in the early embryo, such as membrane ruffling and pseudocleavage, but is not strictly required for cytokinesis. Drosophila anillin is required for cellularization in early embryos and promotes late cytokinetic events in S2 cells. Fission yeast express two anillin-related proteins, Mid1p and Mid2p, which regulate proper positioning of the division plane and septation, respectively. Whereas in human cells anillin localizes via its C-terminal Rho binding domain, in S. pombe mid1p localization involves a C-terminal amphipathic helix. Interestingly, the AHD is not well conserved in mid1p, nor is there compelling evidence that RhoA triggers contractile-ring formation in S. pombe (Piekny, 2008).

Ran mediates chromatin signaling, requiring cortical myosin II assembly, controls cortical polarity during polar body extrusion in mouse oocytes

The molecular basis for asymmetric meiotic divisions in mammalian oocytes that give rise to mature eggs and polar bodies remains poorly understood. Asymmetrically positioned meiotic chromosomes provide the cue for cortical polarity in mouse oocytes. This study shows that the chromatin-induced cortical response can be fully reconstituted by injecting DNA-coated beads into metaphase II-arrested eggs. The injected DNA beads induce a cortical actin cap, surrounded by a myosin II ring, in a manner that depends on the number of beads and their distance from the cortex. The Ran GTPase plays a critical role in this process, because dominant-negative and constitutively active Ran mutants disrupt DNA-induced cortical polarization. The Ran-mediated signaling to the cortex is independent of the spindle but requires cortical myosin II assembly. It is hypothesized that a RanGTP gradient serves as a molecular ruler to interpret the asymmetric position of the meiotic chromatin (Deng, 2007).

It appears that a unique characteristic of the mouse female meiotic system is that cortical polarity is cued by an internal asymmetry coming from the position of the DNA. Although it remains unclear whether any in vivo predetermined cortical cues exist to bias the movement of the meiotic chromatin, these experiments demonstrate that the egg is capable of establishing cortical polarity in any orientation in response to the DNA cue. It is interesting to note that DNA beads placed near the center of the oocyte failed to induce any cortical actomyosin assembly but were only effective within 20 µm of the plasma membrane. This distance-dependent signal propagation explains why oocytes with a defect in chromosome migration fail to undergo polar body extrusion. An intrinsic dependence of cortical actomyosin assembly on asymmetrically positioned chromosomes helps to ensure that polar body extrusion occurs in a highly restricted cortex overlying the chromosomes, therefore minimizing the loss of oocyte cytoplasm (Deng, 2007).

Because neither actin nor microtubules are required for chromatin-induced myoII cortical assembly, propagation of the signal through the cytoplasm is unlikely to be mediated through cytoskeleton-based transport. The distance dependence in the DNA bead-induced cortical response suggests that the signal decays rapidly as the distance from the chromatin increases, with a signaling range of up to 20 µm. This is consistent with the spatial range of the RanGTP gradient measured in Xenopus oocytes and somatic cells. Signal decay through Ran GTP hydrolysis could provide a convenient molecular ruler that ensures the assembly of actin and myosin occurs only when the chromosomes are within a certain distance of the cortex (Deng, 2007).

Involvement of a RanGTP gradient in mediating DNA signal to the cortex is consistent with the quantitative observation that the actin caps became narrower as bead distance to the nearest cortex increased. Similarly, a smaller gradient, for example, that generated by a single DNA bead, would be expected to result in a narrower actin cap, which in fact was observed. It is interesting to note that injection of the constitutively active RanQ69L at a high concentration, which could flatten the endogenous RanGTP gradient, inhibited DNA-induced cortical polarity as opposed to inducing multiple caps. This may suggest that some other factors critical for cortical cytoskeleton assembly exist in limited quantities and may become dispersed due to the global increase in active Ran concentration. Additionally, it was found that neither RanGTP- nor RCC1-coated beads were sufficient to induce cortical polarity or spindle assembly in mouse oocytes, suggesting that whereas these proteins are essential for chromatin signaling, chromatin may play additional roles during these processes (Deng, 2007).

Surprisingly, activation of myoII, which is regulated by MLCK, is required for the cortical accumulation of both actin and the PAR-3 polarity protein in response to the chromatin signal, suggesting that myoII activation may be a critical step downstream of the Ran signal. Although RanT24N did not inhibit global activation of MAP kinase, it appears that RanGTP is required for concentrating MAPK kinase activity to the vicinity of the chromosomes, which could result in local activation of MLCK and stimulation of myoII assembly. The function of myoII during this process may be distinct from the role of myoII in asymmetrically dividing C. elegans zygotes. In this mitotic system, myoII is proposed to concentrate polarity determinants to the anterior cortical domain through its actin-based motor activity, whereas the polarity function of myoII in mouse oocytes may not require its motor activity. MyoII may instead play a scaffolding role in tethering actin filaments and the PAR-3/aPKC polarity complex (Deng, 2007).

The tumor suppressor Lgl1 regulates NMII-A cellular distribution and focal adhesion morphology to optimize cell migration

The Drosophila tumor suppressor Lethal (2) giant larvae (Lgl) regulates the apical-basal polarity in epithelia and asymmetric cell division. However, little is known about the role of Lgl in cell polarity in migrating cells. This study shows direct physiological interactions between the mammalian homologue of Lgl (Lgl1) and the nonmuscle myosin II isoform A (NMII-A). Lgl1 and NMII-A form a complex in vivo, and data is provided that Lgl1 inhibits NMII-A filament assembly in vitro. Furthermore, depletion of Lgl1 results in the unexpected presence of NMII-A in the cell leading edge, a region that is not usually occupied by this protein, suggesting that Lgl1 regulates the cellular localization of NMII-A. Finally, it was shown that depletion of Lgl1 affects the size and number of focal adhesions, as well as cell polarity, membrane dynamics, and the rate of migrating cells. Collectively these findings indicate that Lgl1 regulates the polarity of migrating cells by controlling the assembly state of NMII-A, its cellular localization, and focal adhesion assembly (Dahan, 2012).

The tumor suppressor Lgl1 forms discrete complexes with NMII-A, and Par6α-aPKCζ that are affected by Lgl1 phosphorylation

Non-muscle myosin IIA (NMII-A) and the tumor suppressor Lgl1 play a central role in the polarization of migrating cells. Mammalian Lgl1 interacts directly with NMII-A, inhibiting its ability to assemble into filaments in vitro. Lgl1 also regulates the cellular localization of NMII-A, the maturation of focal adhesions and cell migration. In Drosophila, phosphorylation of Lgl affects its association with the cytoskeleton. This study shows that phosphorylation of mammalian Lgl1 by aPKCζ prevents its interaction with NMII-A both in vitro and in vivo, and affects its inhibition on NMII-A filament assembly. Phosphorylation of Lgl1 affects its cellular localization and is important for the cellular organization of the acto-NMII cytoskeleton. It was further shown that Lgl1 forms two distinct complexes in vivo, Lgl1-NMIIA and Lgl1-Par6α-aPKCζ, and that the complexes formation is affected by the phosphorylation state of Lgl1. The complex Lgl1-Par6α-aPKCζ resides in the leading edge of the cell. Finally, it was shown that aPKCzeta and NMII-A compete to bind directly to Lgl1 via the same domain. These results provide new insights into the mechanism regulating the interaction between Lgl1, NMII-A, Par6α, and aPKCζ in polarized migrating cells (Dahan, 2013).

A model is proposed for the role of Lgl1-NMIIA and Lgl1-Par6α-aPKCζ in establishing front-rear polarization in migrating cells. In migrating polarized cells Lgl1 resides at the cell’s leading edge in a complex with Par6α-aPKCζ, and it is this complex which defines the leading edge of the cell. In the lamellipodium Lgl1 binds to NMII-A but not to aPKCζ, inhibiting NMII-A filament assembly. These events allow the cell to polymerize F-actin to move the cell forward. According to this model Lgl1 is absent from the rear part of the cell, allowing NMII-A to assemble into filaments to enable cell retraction (Dahan, 2013).

Myosin II regulates extension, growth and patterning in the mammalian cochlear duct

The sensory epithelium of the mammalian cochlea comprises mechanosensory hair cells that are arranged into four ordered rows extending along the length of the cochlear spiral. The factors that regulate the alignment of these rows are unknown. Results presented in this study demonstrate that cellular patterning within the cochlea, including the formation of ordered rows of hair cells, arises through morphological remodeling that is consistent with the mediolateral component of convergent extension. Non-muscle myosin II is shown to be expressed in a pattern that is consistent with an active role in cellular remodeling within the cochlea, and genetic or pharmacological inhibition of myosin II results in defects in cellular patterning that are consistent with a disruption in convergence and extension. These results identify the first molecule, myosin II, which directly regulates cellular patterning and alignment within the cochlear sensory epithelium. The results also provide insights into the cellular mechanisms that are required for the formation of highly ordered cellular patterns (Yamamoto, 2009).

zic2a and zic5 are required during neurulation for apical F-actin and active myosin II localization and junction integrity

During neurulation, vertebrate embryos form a neural tube (NT), the rudiment of the central nervous system. In mammals and birds, a key step in cranial NT morphogenesis is dorsolateral hinge-point (DLHP) bending, which requires an apical actomyosin network. The mechanism of DLHP formation is poorly understood, although several essential genes have been identified, among them Zic2, which encodes a zinc-finger transcription factor. DLHP formation in the zebrafish midbrain was found to requires actomyosin and Zic function. Given this conservation, the zebrafish was used to study how genes encoding Zic proteins regulate DLHP formation. It was demonstrated that the ventral zic2a expression border predicts DLHP position. Using morpholino (MO) knockdown, it was shown that zic2a and zic5 are required for apical F-actin and active myosin II localization and junction integrity. Furthermore, myosin II activity can function upstream of junction integrity during DLHP formation, and canonical Wnt signaling, an activator of zic gene transcription, is necessary for apical active myosin II localization, junction integrity and DLHP formation. It is concluded that zic genes act downstream of Wnt signaling to control cytoskeletal organization, and possibly adhesion, during neurulation. This study identifies zic2a and zic5 as crucial players in the genetic network linking patterned gene expression to morphogenetic changes during neurulation, and strengthens the utility of the zebrafish midbrain as a NT morphogenesis model (Nyholm, 2009).

Myosin II motors and F-actin dynamics drive the coordinated movement of the centrosome and soma during CNS glial-guided neuronal migration

Lamination of cortical regions of the vertebrate brain depends on glial-guided neuronal migration. The conserved polarity protein Par6alpha localizes to the centrosome and coordinates forward movement of the centrosome and soma in migrating neurons. The cytoskeletal components that produce this unique form of cell polarity and their relationship to polarity signaling cascades are unknown. This study shows that F-actin and Myosin II motors are enriched in the neuronal leading process and that Myosin II activity is necessary for leading process actin dynamics. Inhibition of Myosin II decreased the speed of centrosome and somal movement, whereas Myosin II activation increased coordinated movement. Ectopic expression or silencing of Par6alpha inhibited Myosin II motors by decreasing Myosin light-chain phosphorylation. These findings suggest leading-process Myosin II may function to 'pull' the centrosome and soma forward during glial-guided migration by a mechanism involving the conserved polarity protein Par6alpha (Solecki, 2009).

Myosin II, axon growth cone dynamics and dendrites

Growth cones are required for the forward advancement and navigation of growing axons. Modulation of growth cone shape and reorientation of the neurite are responsible for the change of outgrowth direction that underlies navigation. Change of shape involves the reordering of the cytoskeleton. Reorientation of the neurite requires the generation of tension, which is supplied by the ability of the growth cone to crawl on a substrate. The specific molecular mechanisms responsible for these activities are unknown but are thought to involve actomyosin-generated force combined with linkage to the cell surface receptors that are responsible for adhesion. To test whether myosin IIB is responsible for the force generation, shape dynamics and filopodial-mediated traction force in growth cones from myosin IIB knock-out (KO) mice were quantified and compared with neurons from normal littermates. Growth cones from the KO mice spread less, showed alterations in shape dynamics and actin organization, and had reduced filopodial-mediated traction force. Although peak traction forces produced by filopodia of KO cones were decreased significantly, KO filopodia occasionally developed forces equivalent to those in the wild type. This indicates that other myosins participate in filopodial-dependent traction force. Therefore, myosin IIB is necessary for normal growth cone spreading and the modulation of shape and traction force but acts in combination with other myosins for some or all of these activities. These activities are essential for growth cone forward advancement, which is necessary for outgrowth. Thus outgrowth is slowed, but not eliminated, in neurons from the myosin IIB KO mice (Bridgman, 2001).

The myosin family of motor proteins is implicated in mediating actin-based growth cone motility, but the roles of many myosins remain unclear. Myosin 1c (M1c; formerly myosin I beta) has been implicated in the retention of lamellipodia. The role of myosin II (MII) in chick dorsal root ganglion neuronal growth cone motility and the contribution of M1c and MII to retrograde F-actin flow have been assessed using chromophore-assisted laser inactivation (CALI). CALI of MII reduces neurite outgrowth and growth cone area by 25%, suggesting a role for MII in lamellipodial expansion. Micro-CALI of MII causes a rapid reduction in local lamellipodial protrusion in growth cones with no effects on filopodial dynamics. This is the opposite of the effects of micro-CALI of M1c, which causes an increase in lamellipodial protrusion. Fiduciary beads were used to observe retrograde F-actin flow during the acute loss of M1c or MII. Micro-CALI of M1c reduced retrograde bead flow by 76%, whereas micro-CALI of MII or the MIIB isoform did not. Thus, M1c and MIIB serve opposite and nonredundant roles in regulating lamellipodial dynamics, and M1c activity is specifically required for retrograde F-actin flow (Diefenbach, 2002).

Growth cones of myosin-IIB-knockout mice have reduced outgrowth rates and traction force. There is a close relationship between traction force, retrograde flow and forward advance of growth cones. All three activities appear to be at least partially myosin dependent. Therefore, differences in retrograde flow rates between growth cones from myosin-IIB-knockout mice and their normal littermates were examined. By placing nerve-growth-factor-coated silica beads on the surface of growth cones with laser tweezers, or by tracking GFP-myosin IIA spots, it was found that the retrograde flow rate was increased more than two fold in the knockout growth cones compared with the wild type. These data suggest that both myosin IIA and IIB normally contribute to retrograde flow and the properties of the flow are strongly influenced by myosin IIB because of its location and abundance. However, in the absence of myosin IIB, myosin IIA takes over this function. The change in retrograde flow rate may reflect the difference in functional properties of these two myosins. Knockout growth cones also exhibit reduced stability of lamellipodia, possibly as a partial consequence of this increased retrograde flow rate. In addition, microtubules penetrate a shorter distance into filopodia, which suggests that the increase in flow rate may adversely affect the microtubule-dependent maturation of filopodia. Taken together these data support the idea that the forward advance of the growth cone is myosin II dependent and involves multiple myosin II isoforms (Brown, 2003).

Dendritic spines show rapid motility and plastic morphology, which may mediate information storage in the brain. It is presently believed that polymerization/depolymerization of actin is the primary determinant of spine motility and morphogenesis. This study shows myosin IIB, a molecular motor that binds and contracts actin filaments, is essential for normal spine morphology and dynamics and represents a distinct biophysical pathway to control spine size and shape. Myosin IIB is enriched in the postsynaptic density (PSD) of neurons. Pharmacologic or genetic inhibition of myosin IIB alters protrusive motility of spines, destabilizes their classical mushroom-head morphology, and impairs excitatory synaptic transmission. Thus, the structure and function of spines is regulated by an actin-based motor in addition to the polymerization state of actin (Ryu, 2006).

The cell biological processes underlying axon growth and guidance are still not well understood. An outstanding question is how a new segment of the axon shaft is formed in the wake of neuronal growth cone advance. For this to occur, the highly dynamic, splayed-out microtubule (MT) arrays characteristic of the growth cone must be consolidated (bundled together) to form the core of the axon shaft. MT-associated proteins stabilize bundled MTs, but how individual MTs are brought together for initial bundling is unknown. This study shows that laterally moving actin arcs, which are myosin II-driven contractile structures, interact with growing MTs and transport them from the sides of the growth cone into the central domain. Upon Myosin II inhibition, the movement of actin filaments and MTs immediately stopped and MTs unbundled. Thus, Myosin II-dependent compressive force is necessary for normal MT bundling in the growth cone neck (Burnette, 2008).

Actin arcs form from condensation of dendritic actin networks in the transition zone, where their myosin II-dependent contractility is essential for severing actin bundles associated with filopodium roots. Evidence presented in this study suggests that myosin II contractility also plays a key role in regulating the shape and structure of the C domain. Specifically, actin bundles derived from actin arcs interact with MTs on the sides of the growth cone and transport them into the central (C) domain. The resulting inward MT transport appears to place active constraints on C domain as follows. MTs are normally compressed during bundling. Constantly applied compression appears to be necessary for maintaining the structure of the growth cone neck, since MTs here spread out and unbundle after myosin II inhibition. This finding suggests that MTs in the C domain are not yet stably crosslinked. It is speculated that MTs held in close proximity by actin/myosin II contractility may facilitate crosslinking by MAPs in the growth cone neck. The latter process in turn promotes generation of the stable 'consolidated' MT arrays characteristic of the neurite shaft, which were found to be morphologically unaffected by myosin II inhibition (Burnette, 2008).

Myosin II has been widely implicated as an effector in neurite retraction. In addition, several repulsive cues thought to be involved in inhibition of nerve regeneration increase Myosin II activity through Rho-->ROCK signaling. Indeed, inhibition of this pathway is being explored in therapeutic contexts; for example, application of the pharmacological ROCK inhibitor Y-27632 or the Rho inhibitor C3 quantitatively improved nerve regeneration in animal models. Evidence has been presented that contractile actin bundles in the C domain mediate Rho/ROCK/Myosin II-dependent neurite retraction. In these studies, it was noted that MTs in the C domain were more splayed out after Rho Kinase (ROCK) inhibition, consistent with the role proposed here for Myosin II in MT transport and bundling. These and related observations suggest that Rho/ROCK inhibition, while permitting or even promoting axon growth, could also result in guidance deficits. Indeed, ROCK inhibition has two separate effects on stimulus evoked neurite growth. First, growth responses occur with a shorter delay, consistent with reduction of a barrier to neurite advance. Second, MT transport and bundling in the growth cone neck are compromised. These findings are consistent with ROCK and downstream myosin II activity regulating MT bundling during neurite shaft consolidation (Burnette, 2008).

During embryogenesis the spinal cord shifts position along the anterior-posterior axis relative to adjacent tissues. How motor neurons whose cell bodies are located in the spinal cord while their axons reside in adjacent tissues compensate for such tissue shift is not well understood. Using live cell imaging in zebrafish, this study shows that as motor axons exit from the spinal cord and extend through extracellular matrix produced by adjacent notochord cells, these cells shift several cell diameters caudally. Despite this pronounced shift, individual motoneuron cell bodies stay aligned with their extending axons. This alignment requires myosin phosphatase activity (see Drosophila Pp1-87B) within motoneurons, and that mutations in the myosin phosphatase subunit mypt1 increase myosin (see Drosophila Zipper) phosphorylation causing a displacement between motoneuron cell bodies and their axons. Thus, this study demonstrates that spinal motoneurons fine-tune their position during axonogenesis and the myosin II regulatory network was identified as a key regulator (Bremer, 2016).

FGF signaling regulates cytoskeletal remodeling during epithelial morphogenesis

Changes in the cytoskeletal architecture underpin the dynamic changes in tissue shape that occur during development. It is clear that such changes must be coordinated so that individual cell behaviors are synchronized; however, the mechanisms by which morphogenesis is instructed and coordinated are unknown. After its induction in non-neural ectoderm, the inner ear undergoes morphogenesis, being transformed from a flat ectodermal disk on the surface of the embryo to a hollowed sphere embedded in the head. Evidence that this shape change relies on extrinsic signals subsequent to genetic specification. By using specific inhibitors, it was found that local fibroblast growth factor (FGF) signaling triggers a phosphorylation cascade that activates basal myosin II through the activation of phopholipase Cgamma. Myosin II exhibits a noncanonical activity that results in the local depletion of actin filaments. Significantly, the resulting apical actin enrichment drives morphogenesis of the inner ear. Thus, FGF signaling directly exerts profound cytoskeletal effects on otic cells, coordinating the morphogenesis of the inner ear. The iteration of this morphogenetic signaling system suggests that it is a more generally applicable mechanism in other epithelial tissues undergoing shape change (Sai, 2008).

Basal actin depolymerization in otic ectoderm suggested a mechanism by which FGF signals, emanating from basally apposed mesoderm and neural ectoderm, remodeled the cytoskeleton. Somatic stage 10 otic regions were cultured with SU5402, a specific inhibitor of FGF signaling, for 4 hr. Such treatment does not affect genetic specification of the otic ectoderm. In otic region explants treated with carrier only (DMSO), actin filaments were apically biased and morphogenesis occurred as normal. In contrast, SU5402-treated otic regions showed loss of apical actin enrichment and a failure to invaginate. Only basal application of FGF beads could direct morphogenesis in stage 10 otic ectoderm isolates. Additionally, immunolocalization of FGFR1, the major FGF receptor in the otic placode at these stages, showed a basal bias, suggesting FGF is transduced basally. Thus mediators of FGF signaling were investigated. FGF signaling occurs through two distinct pathways: a Ras-dependent MAP kinase pathway and via the phosphorylation of phospholipase C gamma (PLCγ). Double phosphorylated Erk1/2 was detected in stage 10 otic ectoderm nuclei, but treatment of the stage 10 otic region with the MEK1/2 inhibitor U0126 only slightly altered F-actin localization and did not significantly inhibit invagination. Phosphorylated (and therefore active) PLCγ was detected at the basal side of otic placode cells. The basal localization of pPLCγ suggested activation by basal FGF signals. This was confirmed by treatment of stage 10 otic regions with SU5402. Phospho-PLCγ immunoreactivity was diminished in such treated otic regions (Sai, 2008).

Activated PLCγ affects the membrane translocation of typical protein kinase C (PKC). PKCα was detected basally in the stage 10 otic placode, reminiscent of the distributions of FGFR1 and phosphorylated PLCγ. A specific PLCγ inhibitor, U73122, caused a diminution of juxtamembrane PKCα immunoreactivity. In contrast, the inactive isomer U73343, used as a negative control, did not affect PKC localization. Whether PLCγ, acting through PKCα, leads to the asymmetric distribution of F-actin in otic placode cells was investigated. Actin polarization and otic invagination was lost in stage 10 otic regions treated with U73122. These data strongly suggest that localized PLCγ activation by FGF causes depletion of actin fibers on the basal side of the otic placode (Sai, 2008).

The involvement of actin and myosin in the closure of the mammalian neural plate and during Drosophila mesodermal invagination has been well documented, and in these cases F-actin and myosin II have been shown to colocalize and are thought to drive apical constriction. This is true during later inner ear morphogenesis, but during the earliest morphogenetic steps described in this study, F-actin and active myosin II are on opposite sides of the otic cell. Indeed, such a reciprocal localization can be detected in the early neural tube, even though during later morphogenesis colocalization is observed. Similarly, during the earliest steps of Drosophila gastrulation, myosin II is initially basal and its localization appears to be concomitant with basal F-actin loss and apical actin enrichment in the invaginating mesoderm. The signaling mechanisms that underlie these steps are still unclear, but recent data suggest the involvement of FGF receptor signal transduction in the closure of the neural tube, independent of any effect on cell fate specification. Taken together, these observations raise the intriguing possibility that the coordination of tissue morphogenesis by receptor signaling, through the activation of myosin II, may describe a general mechanism that underpins the cytoskeletal changes associated with epithelial morphogenesis (Sai, 2008).

zipper : Biological Overview | Evolutionary Homologs part 1/3 | Evolutionary Homologs part 2/3 | Regulation | Developmental Biology | Effects of Mutation | References

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