Insect myosins

The expression patterns of two novel unconventional myosins from Drosophila, have been characterized: myosin-IA (MIA) and myosin-IB (MIB). The appearance and distribution of both proteins during embryogenesis is correlated with the formation of a brush border within the alimentary canal as documented at the ultrastructural level. MIA and MIB, both found predominantly at the basolateral domain of immature enterocytes, exhibit increased expression at the apical domain of differentiated enterocytes co-incident with microvillus assembly. Colocalization of MIA and MIB to larval and adult gut by confocal microscopy demonstrates distinct but overlapping subcellular distributions of these two proteins. In the larval brush border, MIA is enriched in the subapical terminal web domain whereas MIB is found predominantly in the apical microvillar domain. In the adult gut, MIA and MIB both exhibit a microvillar component as MIA attains a more apical position in addition to its previous terminal web locale. MIB is also found in egg chambers during oogenesis, at both the basolateral and apical surfaces of the somatic follicle cells. MIA and MIB both demonstrate ATP-dependent extraction from the larval brush border cytoskeleton and exogenous F-actin, biochemical properties characteristic of functional myosins-I (Morgen, 1995).

95F myosin of Drosophila is a class IV myosin that exhibits tissue specific expression. The amino-terminal two thirds of Drosophila 95F myosin heavy chain (95F MHC) comprises a head domain that is 29-33% identical (60-65% similar) to other myosin heads, and contains ATP-binding, actin-binding and calmodulin/myosin light chain-binding motifs. The carboxy-terminal tail has no significant similarity to other known myosin tails, but does contain an approximately 100-amino acid region that is predicted to form an alpha-helical coiled-coil. The expression profile of the 95F MHC gene is complex. Transcripts are alternatively spliced and encode at least three protein isoforms; in addition, a fourth isoform is detected on Western blots. Transcripts and protein are present throughout the life cycle, with peak expression occurring during mid-embryogenesis and adulthood. Immunolocalization in early embryos demonstrates that the protein is primarily located in a punctate pattern throughout the peripheral cytoplasm. Most cells maintain a low level of 95F myosin expression throughout embryogenesis, but specific tissues appear to contain higher levels of the protein (Kellerman, 1992).

The 95F myosin, a class IV unconventional myosin, associates with particles in the cytoplasm of the Drosophila syncytial blastoderm and is required for the ATP- and F-actin-dependent translocation of these particles. The particles undergo a cell cycle-dependent redistribution from domains that surround each nucleus in interphase to transient membrane invaginations that provide a barrier between adjacent spindles during mitosis. When 95F myosin function is inhibited by antibody injection, profound defects in syncytial blastoderm organization occur. This disorganization is seen as aberrant nuclear morphology and position and is suggestive of failures in cytoskeletal function. Nuclear defects correlate with gross defects in the actin cytoskeleton, including indistinct actin caps and furrows, missing actin structures, abnormal spacing of caps, and abnormally spaced furrows. Three-dimensional examination of embryos injected with anti-95F myosin antibody reveals that actin furrows do not invaginate as deeply into the embryo as do normal furrows. These furrows do not separate adjacent mitoses, since microtubules cross over them. These inappropriate microtubule interactions lead to aberrant nuclear divisions and to the nuclear defects observed. It is proposed that 95F myosin function is required to generate normal actin-based transient membrane furrows. The motor activity of 95F myosin itself and/or components within the particles transported to the furrows by 95F myosin may be required for normal furrows to form (Mermall, 1995).

The Drosophila ninaC gene encodes for a class III myosin consisting of two head-specific proteins (132 kDa and 174 kDa). Their predicted amino acid sequences indicate that they may have both myosin I and kinase properties. The proteins are detected only in photoreceptor cells, but within this cell type, are detected in all classes of the compound eye photoreceptors. Within the photoreceptors, they are found in the rhabdomeral microvilli and the cytoplasm adjacent to the rhabdomeres. This distribution coincides with that shown previously for actin filaments. Immunolabelling of tissue from the ninaC P221 mutant, which lacks the 174 kDa protein, and two mutants whose rhabdomeres degenerate, suggests that the 132 kDa protein is present primarily in the cytoplasm adjacent to the rhabdomeres, and that the 174 kDa protein is concentrated in the rhabdomeres. This ultrastructural analysis shows that the axial cytoskeleton of the rhabdomeral microvilli (which contains filamentous actin) is absent in both the null and P221 mutants. In the photoreceptor cell cytoplasm, the number of multivesicular bodies in the null mutant, but not the P221 mutant, is 3-fold greater in comparison with wild-type (Hicks, 1992).

Phototransduction in Drosophila occurs through inositol lipid signaling that results in Ca2+ mobilization. The physiological roles of calmodulin (CaM) (See Drosophila Calmodulin) were studied in light adaptation and in regulation of the inward current that is brought about by depletion of cellular Ca2+ stores. Three resources providing decreased Ca-CaM content in photoreceptors were analysed: (1) transgenic Drosophila P[ninaCDeltaB] flies that have CaM-deficient photoreceptors; (2) the peptide inhibitor M5 that binds to Ca-CaM and prevents its action, and (3) Ca2+-free medium that prevents Ca2+ influx and thereby diminishes the generation of Ca-CaM. Several effects have been noted due to decrease in Ca-CaM level:

  1. Fluorescence of Ca2+ indicator reveals an enhanced light-induced Ca2+ release from internal stores.
  2. Measurements of the light-induced current in P[ninaCDeltaB] cells show a reduced light adaptation.
  3. Internal dialysis of M5 initially enhances excitation and subsequently disrupts the light-induced current.
  4. An inward dark current appears after depletion of the Ca2+ stores with ryanodine and caffeine.
Importantly, application of Ca-CaM into the photoreceptor cells prevents all of the above effects. It is proposed that negative feedback of Ca-CaM on Ca2+ release from ryanodine-sensitive stores mediates light adaptation, is essential for light excitation, and keeps the store-operated inward current under a tight control (Arnon, 1997).

Electron microscopy of the midgut of the 5th instar Manduca sexta (tobacco hornworm) larvae reveals enterocytes with an apical brush border surface comparable to that in the vertebrate intestine, with both microvillar (MV) and terminal web (TW) domains, the latter defined by a zone of organelle exclusion directly beneath the MV. In the larval dragon fly, the MV contain a bundle of actin filaments, as determined by staining with rhodamine phalloidin and heavy meromyosin decoration. Two-dimensional gel analysis reveals the presence of multiple isoelectric variants of actin with the major isoform corresponding to the non-muscle actin isoform II, expressed in Drosophila. Like the vertebrate BB, the Manduca BB can be isolated intact from enterocytes by mechanical shear. Immunochemical analysis of isolated BB fractions or whole homogenates of midgut reveal proteins of appropriate molecular weight immunoreactive with antibodies to the MV core proteins: BB myosin I, villin and fimbrin, and the TW components: spectrin, myosin II and tropomyosin. Immunocytochemical localization of a subset of these proteins at the light microscopic (spectrin) and electron microscopic (actin, villin, spectrin, myosin II, and tropomyosin) level reveals that the molecular architecture of the Manduca BB cytoskeleton is homologous to that found in vertebrates (Bonfanti, 1992).

To investigate the molecular functions of the regions encoded by alternative exons from the single Drosophila myosin heavy chain gene, the first kinetic measurements have been made of two muscle myosin isoforms that differ in all alternative regions. Myosin was purified from the indirect flight muscles of wild-type and transgenic flies expressing a major embryonic isoform (Emb18). The in vitro actin sliding velocity on the flight muscle isoform is among the fastest reported for a type II myosin and is 9-fold faster than with the embryonic isoform. With smooth muscle tropomyosin bound to actin, the actin sliding velocity on the embryonic isoform increases 6-fold, whereas that on the flight muscle myosin slightly decreases. No difference in the step sizes of Drosophila and rabbit skeletal myosins were found using optical tweezers, suggesting that the slower in vitro velocity with the embryonic isoform is due to altered kinetics. Basal ATPase rates for flight muscle myosin are higher than those of embryonic and rabbit myosin. These differences explain why the embryonic myosin cannot functionally substitute in vivo for the native flight muscle isoform, and demonstrate that one or more of the five myosin heavy chain alternative exons must influence Drosophila myosin kinetics (Swank, 2001).

At least one of the five MHC regions encoded by alternative exons must be responsible for the observed differences in actomyosin kinetics between the IFM and Emb18 isoforms. The remainder of the molecule is identical between these two isoforms because it arises from constitutive exons of the same gene, and because the adult tailpiece was engineered onto Emb18. Based on mapping the four motor domain alternative exons (exons 3, 7, 9, and 11) onto the three-dimensional chicken skeletal myosin head, it is believed that exon 7 may be responsible for the measured differences in ATPase rates because it encodes part of the lip of the nucleotide site and is part of 'switch one.' Exons 11 and/or 3 could contribute to the differences in actin sliding velocity, since they are located near the pivot point of the lever arm and have been proposed to affect step size. However, no difference in step size was found in this study; therefore, neither exons 3a and 3b nor exons 11e and 11c regulate step size. The possibility that exon 11a, 11b, or 11d expressed in one or more of the other myosin isoforms may affect step size cannot be excluded. Exon 9 encodes the 'rigid relay loop' that may be involved in propagating signals from the nucleotide site to the actin binding site and lever arm region. The final alternative exon, 15, encodes the rod hinge region. Although it has been shown that myosin S-1 head fragment is sufficient for in vitro motility, exon 15 could be involved in modulating function in vivo (Swank, 2001).

None of the four alternative exon regions in the myosin head corresponds to the loop 1 and loop 2 regions that have been investigated as possible sources of functional variation between myosins from different muscle types. The seven-amino acid insert, which determines differences in ATPase rate and in vitro motility velocity between the tonic and phasic smooth muscle isoforms, is not within a homologous region encoded by a Drosophila alternative exon. Thus, multiple mechanisms appear to have evolved to modify myosin function (Swank, 2001).

Yeast Myosin II

The myo2 gene of S. pombe, which encodes a type II myosin heavy chain, was cloned by virtue of its ability to promote diploidization in fission yeast cells. The myo2 gene encodes 1,526 amino acids in a single open reading frame. Myo2p shows homology to the head domains and the coiled-coil tail of the conventional type II myosin heavy chain and carries putative binding sites for ATP and actin. It also carries the IQ motif, which is a presumed binding site for the myosin light chain. However, Myo2p apparently carries only one IQ motif, while its counterparts in other species have two. There are nine proline residues, which should break alpha-helix, in the COOH-terminal coiled-coil region of Myo2p. Thus, Myo2p is rather unusual as a type II myosin heavy chain. Disruption of myo2 inhibits cell proliferation. myo2Delta cells show normal punctate distribution of interphase actin, but they produce irregular actin rings and septa and are impaired in cell separation. Overproduction of Myo2p is also lethal, apparently blocking actin relocation. Nuclear division proceeds without actin ring formation and cytokinesis in cells overexpressing Myo2p, gives rise to multinucleated cells with dumbbell morphology. Analysis using tagged Myo2p reveal that Myo2p colocalizes with actin in the contractile ring, suggesting that Myo2p is a component of the ring and responsible for its contraction. Genetic evidence suggests that the acto-myosin system may interact with the Ras pathway, which regulates mating and the maintenance of cell morphology in S. pombe (Kitayama, 1997).

Saccharomyces cerevisiae protein, Cyk1p, exhibits sequence similarity to the mammalian IQGAPs. IQGAPs represesent a new family of proteins implicated in connecting Cdc42 and calmodulin signaling to the remodeling of the actin cytoskeleton. IQGAP contain a calponin homology domain and interact directly with calmodulin as well as the activited form of Cdc42. IQGAP1 has been shown to form a homodimer and bundle actin filaments with high affinity in vitro. Gene disruption of Cyk1p results in a failure in cytokinesis without affecting other events in the cell cycle. Cyk1p is diffused throughout most of the cell cycle but localizes to a ring structure at the mother-bud junction after the initiation of anaphase. This ring contains filamentous actin and Myo1p, a myosin II homolog. In vivo observation with green fluorescent protein-tagged Myo1p shows that the ring decreases drastically in size during cell division and therefore may be contractile. These results indicate that cytokinesis in budding yeast is likely to involve an actomyosin-based contractile ring. The assembly of this ring occurs in temporally distinct steps: Myo1p localizes to a ring that overlaps the septins at the G1-S transition slightly before bud emergence; Cyk1p and actin then accumulate in this ring after the activation of the Cdc15 pathway late in mitosis. The localization of myosin is abolished by a mutation in Cdc12p, implicating a role for the septin filaments (see Drosophila Peanut) in the assembly of the actomyosin ring. The accumulation of actin in the cytokinetic ring is not observed in cells depleted of Cyk1p, suggesting that Cyk1p plays a role in the recruitment of actin filaments, perhaps through a filament-binding activity similar to that demonstrated for mammalian IQGAPs (Lippincott, 1998).

Yeast SRO7 was identified as a multicopy suppressor of a defect in Rho3p, a small GTPase that maintains cell polarity. Sro7p (closest Drosophila homolog CG17762) and Sro77p, a homologue of Sro7p, possess domains homologous to the protein that are encoded by the Drosophila tumor suppressor gene lethal (2) giant larvae. sro7Delta sro77Delta double mutants show a partial defect of organization of the polarized actin cytoskeleton and a cold-sensitive growth phenotype. A human counterpart of l(2)gl could suppress the sro7Delta sro77Delta defect. Similar to the l(2)gl protein, Sro7p forms a complex with Myo1p, a type II myosin. These results indicate that Sro7p and Sro77p are the yeast counterparts of the l(2)gl protein. Genetic analysis revealed that deletion of SRO7 and SRO77 shows reciprocal suppression with deletion of MYO1 (i.e., the sro7Delta sro77Delta defect is suppressed by myo1Delta and vice versa). In addition, SRO7 shows genetic interactions with MYO2, encoding an essential type V myosin: Overexpression of SRO7 suppresses a defect in MYO2 and, conversely, overexpression of MYO2 suppresses the cold-sensitive phenotype of sro7Delta sro77Delta mutants. These results indicate that Sro7 function is closely related to both Myo1p and Myo2p. A model is proposed in which Sro7 function is involved in the targeting of the myosin proteins to their intrinsic pathways (Kagami, 1998).

As in many eukaryotic cells, fission yeast cytokinesis depends on the assembly of an actin ring. myp2(+), a myosin-II in Schizosaccharomyces pombe, is conditionally required for cytokinesis. myp2(+), the second myosin-II identified in S. pombe, does not completely overlap in function with myo2(+). The catalytic domain of Myp2p is highly homologous to known myosin-IIs, and phylogenetic analysis places Myp2p in the myosin-II family. The Myp2p sequence contains well-conserved ATP- and actin-binding motifs, as well as two IQ motifs. However, the tail sequence is unusual, since it is predicted to form two long coiled-coils separated by a stretch of sequence containing 19 prolines. Disruption of myp2(+) is not lethal but under nutrient limiting conditions cells lacking myp2(+) function are multiseptated, elongated, and branched, indicative of a defect in cytokinesis. The presence of salt enhances these morphological defects. Deltamyp2 cells are cold sensitive in high salt, failing to form colonies at 17°C. Thus, myp2(+) is required under conditions of stress, possibly linking extracellular growth conditions to efficient cytokinesis and cell growth. GFP-Myp2p localizes to a ring in the middle of late mitotic cells, consistent with a role in cytokinesis. Double mutants of Deltamyp2 have been constructed with temperature-sensitive mutant strains defective in cytokinesis. Synthetic lethal interactions are observed between Deltamyp2 and three alleles of cdc11ts (early septation mutants), as well as more modest synthetic interactions with cdc14ts (an early septation mutant) and cdc16ts (a gene that regulates septum formation), implicating myp2(+) function for efficient cytokinesis under normal conditions (Bezanilla, 1997).

An actomyosin-based contractile ring provides the forces necessary for cell cleavage in several organisms. Myosin II is an essential component of the actomyosin ring and has also been detected as a 'spot' in interphase Schizosaccharomyces pombe cells. It is currently unknown if this myosin II-containing spot is important for cytokinesis. In this study, this myosin II-containing spot has been characterized using a combination of genetic and cell biological analyses. Whereas myosin II at the actomyosin ring undergoes rapid turnover, myosin II at the spot does not. Maintenance of the myosin II-containing spot is independent of F-actin function. Interestingly, maintenance of this myosin II spot in interphase requires the function of Rng3p, a UCS domain-containing protein, the Caenorhabditis elegans homolog of which has recently been shown to be a cochaperone for myosin II assembly. Disassembly of the spot in interphase prevents actomyosin ring formation in the subsequent mitosis, implying that the spot might represent a progenitor that is important for assembly of the actomyosin ring. Given that mitosis represents a short period of the fission yeast cell cycle, organization of this progenitor structure in interphase might ensure proper assembly of the actomyosin ring and successful cell division (Wong, 2002).

Myosins are motor proteins involved in processes like cell motility, vesicle transport, or cytokinesis. In a variety of organisms, a novel group of proteins forming the UCS (UNC-45/CRO1/SHE4) domain-containing family are essential for proper myosin function. The Saccharomyces cerevisae UCS domain protein She4p is involved in two myosin-requiring events, endocytosis and mRNA localization. In contrast to UCS domain proteins from other organisms that interact with class II myosins, She4p associates with yeast class I and class V myosins. She4p binds to motor domains of class V myosin Myo4p and class I myosin Myo5p, and this binding depends on She4p's UCS domain. In vivo, She4p is essential for the function and localization of Myo3p, Myo4p, and Myo5p (but not of Myo2p) and for colocalization of class I myosins with cortical actin patches. In vitro, She4p stimulates binding of Myo5p to filamentous actin. Wild-type She4p, but not a mutant lacking the UCS domain, accumulates in a cap-like structure at the bud tip. This localization requires Myo2p and actin, suggesting a Myo2-dependent mechanism by which She4p is targeted to the bud cap. Localization of She4p is essential for proper positioning and myosin-actin association of cortical Myo5p. These results suggest that She4p is a novel myosin motor domain binding protein and operates as a localized regulator of myosin function of class I and likely class V myosins (Wesche, 2003).

In eukaryotes, cytokinesis generally involves an actomyosin ring, the contraction of which promotes daughter cell segregation. Assembly of the contractile ring is tightly controlled in space and time. In the fission yeast, contractile ring components are first organized by the anillin-like protein Mid1 into medial cortical nodes. These nodes then coalesce laterally into a functional contractile ring. Although Mid1 is present at the medial cortex throughout G2, recruitment of contractile ring components to nodes starts only at mitotic onset, indicating that this event is cell-cycle regulated. Polo kinases are key temporal coordinators of mitosis and cytokinesis, and the Polo-like kinase Plo1 is known to activate Mid1 nuclear export at mitotic onset, coupling division plane specification to nuclear positio. This study provide evidence that yeasts Plo1 also triggers the recruitment of contractile ring components into medial cortical nodes. Plo1 binds at least two independent sites on Mid1, including a consensus site phosphorylated by Cdc2. Plo1 phosphorylates several residues within the first 100 amino acids of Mid1, which directly interact with the IQGAP Rng2, and influences the timing of myosin II recruitment. Plo1 thereby facilitates contractile ring assembly at mitotic onset (Almonacid, 2011).

Dictyostelium Myosin II

Dictyostelium cells that lack a functional myosin II heavy chain are motile and capable of aggregation, but fail to undergo further multicellular development. A Dictyostelium mutant expressing a cold-sensitive myosin heavy chain has been used to examine the requirement for myosin throughout the course of development. The loss of myosin function upon cooling is rapid and reversible. Temperature-shift experiments reveal that myosin is essential during two different stages of development. During aggregation, myosin function appears to be necessary for cells to sort correctly in a way that allows further development to occur. During the final stage of development, it is required for the formation of a complete stalk and the raising of the spore head. Development between these stages, however, proceeds normally in the absence of myosin function. Aggregates at non-permissive temperature undergo an aberrant form of development resulting in a ball of cells. These structures contain defective spores and a miniature stalk (Springer, 1994).

Cold-sensitive myosin mutants represent powerful tools for dissecting discrete deficiencies in myosin function. Biochemical characterization of two such mutants, G680V and G691C, has allowed the identification of separate facets of myosin motor function perturbed by each alteration. Compared with wild type, the G680V myosin exhibits a substantially enhanced affinity for several nucleotides, decreased ATPase activity, and overoccupancy or creation of a novel strongly actin-binding state. The properties of the novel strong binding state are consistent with a partial arrest or pausing at the onset of the mechanical stroke. However, the G691C mutant exhibits an elevated basal ATPase indicative of premature phosphate release. By releasing phosphate without a requirement for actin binding, the G691C can bypass the part of the cycle involving the mechanical stroke. The two mutants, despite having alterations in glycine residues separated by only 11 residues, have dramatically different consequences on the mechanochemical cycle (Patterson, 1997).

The myosin II heavy chain (MHC)-specific protein kinase C (MHC-PKC) isolated from Dictyostelium discoideum has been implicated in the regulation of myosin II assembly in response to the chemoattractant, cAMP. Elimination of MHC-PKC results in the abolishment of MHC phosphorylation in response to cAMP. Cells devoid of MHC-PKC exhibit substantial myosin II overassembly, as well as aberrant cell polarization, chemotaxis, and morphological differentiation. Cells overexpressing the MHC-PKC contain highly phosphorylated MHC, exhibit impaired myosin II localization with no apparent cell polarization and chemotaxis. MHC-PKC phosphorylates MHC in response to cAMP and plays an important role in the regulation of myosin II localization during chemotaxis (Abu-Elneel, 1996).

Cellular slime mold (Dictyostelium) cells lacking myosin II are impaired in multicellular motility. These results are extended by determining whether myosin contractile function is necessary for normal multicellular motility and shape control. Myosin from mutants lacking the essential (mlcE2) myosin light chain retains the ability to form bipolar filaments that bind actin, but shows no measurable in vitro or in vivo contractile function. The contractile function is necessary for cell shape control since mlcE2 cells, like myosin heavy-chain null mutants (mhcA2), are defective in their ability to control their three-dimensional shape. When mixed with wild-type cells in chimeric aggregation streams, the mlcE2 cells are able to move normally, unlike mhcA2 cells, which accumulate at the edges of the stream and become distorted by their interactions with wild-type cells. When mhcA2 cells are mixed with mlcE2 streams, the mhcA2 cells are excluded. The normal behavior of the mlcE2 cells in this assay suggests that myosin II, in the absence of motor function, is sufficient to allow movement in this constrained, multicellular environment. It is hypothesized that myosin II is a major contributor to cortical integrity even in the absence of contractile function (Xu, 2001).

Cells must exert force against the substrate to migrate. The vectors (both the direction and the magnitude) of the traction force generated by Dictyostelium cells was studied using an improved non-wrinkling silicone substrate. During migration, the cells show two 'alternate' phases of locomotory behavior, an extension phase and a retraction phase. In accordance with these phases, two alternate patterns were identified in the traction force. During the extension phase, the cell exerts a 'pulling force' toward the cell body in the anterior and the posterior regions and a 'pushing force' in the side of the cell (pattern 1). During the retraction phase, the cell exerts a 'pushing force' in the anterior region, although the force disappears in the side and the posterior regions of the cell (pattern 2). Myosin II heavy chain null cells show a single pattern in their traction force comparable to 'pattern 1', although they still have the alternate biphasic locomotory behavior similar to the wild-type cells. Therefore, the generation of 'pushing force' in the anterior and the cancellation of the traction force in the side and the posterior during the retraction phase are deficient in myosin knock-out mutant cells, suggesting that these activities depend on myosin II via the posterior contraction. Considering all these results, it is hypothesized that there is a highly coordinated, biphasic mechanism of cell migration in Dictyostelium (Uchida, 2003).

Contractile networks are fundamental to many cellular functions, particularly cytokinesis and cell motility. Contractile networks depend on myosin-II mechanochemistry to generate sliding force on the actin polymers. However, to be contractile, the networks must also be crosslinked by crosslinking proteins, and to change the shape of the cell, the network must be linked to the plasma membrane. Discerning how this integrated network operates is essential for understanding cytokinesis contractility and shape control. This study analyzed the cytoskeletal network that drives furrow ingression in Dictyostelium. The actin polymers are assembled into a meshwork and myosin-II does not assemble into a discrete ring in the Dictyostelium cleavage furrow of adherent cells. Myosin-II generates regional mechanics by increasing cleavage furrow stiffness and slows furrow ingression during late cytokinesis as compared to myoII nulls. Actin crosslinkers dynacortin and fimbrin similarly slow furrow ingression and contribute to cell mechanics in a myosin-II-dependent manner. By using FRAP, it was shown that the actin crosslinkers have slower kinetics in the cleavage furrow cortex than in the pole, that their kinetics differ between wild-type and myoII null cells, and that the protein dynamics of each crosslinker correlate with its impact on cortical mechanics. These observations suggest that myosin-II along with actin crosslinkers establish local cortical tension and elasticity, allowing for contractility independent of a circumferential cytoskeletal array. Furthermore, myosin-II and actin crosslinkers may influence each other as they modulate the dynamics and mechanics of cell-shape change (Reichl, 2008).

C. elegans Myosin II

Molecular and physiological studies of cells implicate interactions between the cytoskeleton and the intracellular calcium signaling machinery as an important mechanism for the regulation of calcium signaling. However, little is known about the functions of such mechanisms in animals. A key component of the calcium signaling network is the intracellular release of calcium in response to the production of the second messenger inositol 1,4,5-trisphosphate (IP3), mediated by the IP3 receptor (IP3R). C. elegans IP3Rs, encoded by the gene itr-1, interact directly with myosin II. The interactions between two myosin proteins, UNC-54 and MYO-1, and ITR-1 were identified in a yeast two-hybrid screen and subsequently confirmed in vivo and in vitro. The interaction sites on both the IP3R and MYO-1 have been defined. To test the effect of disrupting the interaction in vivo interacting fragments of both proteins were overexpressed in C. elegans. This decreases the animal's ability to upregulate pharyngeal pumping in response to food. This is a known IP3-mediated process. Other IP3-mediated processes, e.g., defecation, were unaffected. Thus it appears that interactions between IP3Rs and myosin are required for maintaining the specificity of IP3 signaling in C. elegans and probably more generally (Walker, 2002).

Rho-binding kinase and the myosin phosphatase targeting subunit regulate nonmuscle contractile events in higher eukaryotes. Genetic evidence indicates that the C. elegans homologs regulate embryonic morphogenesis by controlling the actin-mediated epidermal cell shape changes that transform the spherical embryo into a long, thin worm. LET-502/Rho-binding kinase triggers elongation while MEL-11/myosin phosphatase targeting subunit inhibits this contractile event. Mutations are described in the nonmuscle myosin heavy chain gene nmy-1 that were isolated as suppressors of the mel-11 hypercontraction phenotype. However, a nmy-1 null allele displays elongation defects less severe than mutations in let-502 or in the single nonmuscle myosin light chain gene mlc-4. This results because nmy-1 is partially redundant with another nonmuscle myosin heavy chain, nmy-2, which was previously known only for its role in anterior/posterior polarity and cytokinesis in the early embryo. At the onset of elongation, NMY-1 forms filamentous-like structures similar to actin, and LET-502 is interspersed with these structures, where it may trigger contraction. MEL-11, which inhibits elongation, is initially cytoplasmic. In response to LET-502 activity, MEL-11 becomes sequestered away from the contractile apparatus, to the plasma membrane, when elongation commences. Upon completion of morphogenesis, MEL-11 again appears in the cytoplasm where it may halt actin/myosin contraction (Piekny, 2003).

Myosin II regulation during C. elegans embryonic elongation: LET-502/ROCK, MRCK-1 and PAK-1, three kinases with different roles

Myosin II plays a central role in epithelial morphogenesis; however, its role has mainly been examined in processes involving a single cell type. This study analyzed the structure, spatial requirement and regulation of myosin II during C. elegans embryonic elongation, a process that involves distinct epidermal cells and muscles. Novel GFP probes were developed to visualize the dynamics of actomyosin remodeling; it was found that the assembly of myosin II filaments, but not actin microfilaments, depends on the myosin regulatory light chain (MLC-4) and essential light chain (MLC-5). To determine how myosin II regulates embryonic elongation, mlc-4 mutants were rescued with various constructs and found that MLC-4 is essential in a subset of epidermal cells. Phosphorylation of two evolutionary conserved MLC-4 serine and threonine residues is important for myosin II activity and organization. In an RNAi screen for potential myosin regulatory light chain kinases, it was found that the ROCK, PAK and MRCK homologs act redundantly. The combined loss of ROCK and PAK, or ROCK and MRCK, completely prevented embryonic elongation, but a constitutively active form of MLC-4 could only rescue a lack of MRCK. This result, together with systematic genetic epistasis tests with a myosin phosphatase mutation, suggests that ROCK and MRCK regulate MLC-4 and the myosin phosphatase. Moreover, it is suggested that ROCK and PAK regulate at least one other target essential for elongation, in addition to MLC-4 (Gally, 2009).

Non-junctional E-Cadherin clusters regulate the actomyosin cortex in the C. elegans zygote

Classical cadherins (see Drosophila Shotgun) are well known for their essential function in mediating cell-cell adhesion via their extra-cellular cadherin domains and intra-cellular connections to the actin cytoskeleton. There is evidence, however, of adhesion-independent cadherin clusters existing outside of cell-cell junctions. What function, if any, these clusters have is not known. HMR-1, the sole classical cadherin in Caenorhabditis elegans, plays essential roles during gastrulation, blastomere polarity establishment, and epidermal morphogenesis. To elucidate the physiological roles of non-junctional cadherin, HMR-1 was analyzed in the C. elegans zygote, which is devoid of neighbors. Non-junctional clusters of HMR-1 form during the one-cell polarization stage and associate with F-actin at the cortex during episodes of cortical flow. Non-junctional HMR-1 clusters downregulate RHO-1 (see Drosophila Rho1) activity and inhibit accumulation of non-muscle myosin II (NMY-2; see Drosophila Zipper ) at the anterior cortex. HMR-1 clusters were found to impede cortical flows and play a role in preserving the integrity of the actomyosin cortex, preventing it from splitting in two. Importantly, an inverse relationship was uncovered between the amount of HMR-1 at the cell surface and the rate of cytokinesis. The effect of HMR-1 clusters on cytokinesis is independent of their effect on NMY-2 levels, and is also independent of their extra-cellular domains. Thus, in addition to their canonical role in inter-cellular adhesion, HMR-1 clusters regulate RHO-1 activity and NMY-2 level at the cell surface, reinforce the stability of the actomyosin cortex, and resist its movement to influence cell-shape dynamics (Padmanabhan, 2016).

Vertebrate myosin types

Smooth muscle and nonmuscle myosin are distinct from one another. Myosin heavy chain-A and -B isoforms show a differential expression in a variety of human adult and fetal tissues and cells. When extracts of human adult aorta are subjected to gel electrophoresis, two distinct Coomassie Blue-stained bands and a fused band are seen migrating at approximately 200 kDa. These bands can be detected with four different specific antibodies recognizing the two different smooth muscle myosin heavy chain isoforms (204 kDa and 200 kDa) and the two different nonmuscle myosin heavy chain isoforms (A and B). Thus there are four different myosin isoforms in adult and fetal aortas (Phillips, 1995).

A comparision was made of the physical properties and actin-binding characteristics of several nonmuscle and striated muscle tropomyosins, and the effects of these isoforms were examined on the interactions of actin with two structurally distinct classes of myosin: striated muscle myosin-II and brush border (BB) myosin-I. Bacterially expressed nonmuscle tropomyosins bind to F-actin with the expected stoichiometry and with affinities comparable to that of a tissue produced alpha-tropomyosin, although the striated muscle tropomyosin has a lower affinity for F-actin than a tissue-purified striated muscle alpha tropomyosin. The isoforms also protect F-actin from severing by villin. The in vitro sliding of actin filaments translocated by muscle myosin-II increases 25-65% in the presence of one isoform. Four other isoforms have no detectable effect on myosin-II motility. The actin-activated MgATPase activity of brush border myosin-I is inhibited 75-90% by all of the tropomyosin isoforms tested. BB myosin-I motility (50 nm/s) is completely inhibited by both the 248 and 284 amino acid tropomyosins. These results demonstrate that bacterially produced tropomyosins can differentially regulate myosin enzymology and mechanochemistry, and suggest a role for tropomyosin in the coordinated regulation of myosin isoforms in vivo (Fanning, 1995).

Myosin interaction with actin

Despite a high degree of conservation in the amino-acid sequence of the 130K motor domain (head region) of diverse myosins, there are large differences in the enzymatic and motile activities of myosins from diverse species and cell types. However, the degree of conservation is not uniform throughout the head sequence; therefore, one reasonable hypothesis is that the functional differences between myosins derive from the poorly conserved areas. The most prominent divergent region occurs at the 50K/20K junction, a region of the molecule sensitive to proteolytic digestion and a binding site for actin. Chimaeras of this region of myosin were constructed by substituting the 9-amino-acid Dictyostelium junction region with those from myosins from other species. The actin-activated ATPase correlates well with the activity of the myosin from which the junction region was derived. These results suggest that this region, likely to be part of the myosin head that interacts directly with actin, is important in determining the enzymatic activity of myosin (Uyeda, 1994).

Since it has not been possible to crystallize the actomyosin complex, the x-ray structures of the individual proteins together with data obtained by fiber diffraction and electron microscopy have been used to build detailed models of filamentous actin (f-actin) and the actomyosin rigor complex. In the f-actin model, a single monomer uses 10 surface loops and two alpha-helices to make sometimes complicated interactions with its four neighbors. In the myosin molecule, both the essential and regulatory light chains show considerable structural homology to calmodulin. General principles are evident in their mode of attachment to the target alpha-helix of the myosin heavy chain. The essential light chain also makes contacts with other parts of the heavy chain and with the regulatory light chain. The actomyosin rigor interface is extensive, involving interaction of a single myosin head with regions on two adjacent actin monomers. A number of hydrophobic residues on the interacting apposing faces of actin and myosin contribute to the main binding site. This site is flanked on three sides by charged myosin surface loops that form predominantly ionic interactions with adjacent regions of actin. Hydrogen bonding is likely to play a significant role in actin-actin and actin-myosin interactions since many of the contacts involve loops (Milligan, 1996).

Targeting of myosin by Rho family GTPases

GTPases of the Rho family regulate actinomyosin-based contraction in non-muscle cells. Activation of Rho increases contractility, leading to cell rounding and neurite retraction in neuronal cell lines. Activation of Rac promotes cell spreading and interferes with Rho-mediated cell rounding. Activation of Rac may antagonize Rho by regulating phosphorylation of the myosin-II heavy chain. Stimulation of PC12 cells or N1E-115 neuroblastoma cells with bradykinin induces phosphorylation of threonine residues in the myosin-II heavy chain; this phosphorylation is Ca2+ dependent and regulated by Rac. Both bradykinin-mediated and constitutive activation of Rac promote cell spreading, accompanied by a loss of cortical myosin II. These results identify the myosin-II heavy chain as a new target of Rac-regulated kinase pathways, and implicate Rac as a Rho antagonist during myosin-II-dependent cell-shape changes (van Leeuwen, 1999).

The molecular events responsible for Rac-mediated cytoskeletal changes are not well understood, but they involve activation of serine/threonine-kinase pathways. To search for stimuli that induce Rac-dependent changes in neuronal cell lines, an immobilized fusion protein consisting of glutathione-S-transferase fused to Pak1 was used to measure activation of Rac in lysates of rat PC12 cells in response to several receptor agonists. The serine/threonine kinase Pak1 is a downstream effector of both Rac and Cdc42 that specifically binds these GTPases in their active (GTP-bound) states. Stimulation of PC12 cells with the neuropeptide bradykinin leads to activation of Rac without inducing a measurable change in Cdc42 activity. Ectopic expression of constitutively active Tiam1 (C1199Tiam1), a guanine-nucleotide-exchange factor for Rac, potently activates Rac and also increases the amount of GTP-bound Cdc42 to some extent in these cells (van Leeuwen, 1999).

Pak serine/threonine kinases are activated directly by GTP-bound Rac or Cdc42 and are thought to be important in the regulation of the actinomyosin cytoskeleton downstream of these GTPases. To investigate a role for this kinase family in MHC phosphorylation, dominant-negative Pak1(L83, L86, R299) was overexpressed in PC12 cells. This kinase-defective mutant no longer binds Rac or Cdc42, thus avoiding complicating effects resulting from titration of GTP-bound Rac or Cdc42. Similar to dominant-negative RacN17, Pak1(L83, L86, R299) interfers with bradykinin-induced MHC phosphorylation. Most cells expressing dominant-negative Pak1(L83, L86, R299) are round and do not spread in response to bradykinin. Moreover, myosin II in these cells remains associated with F-actin at the cell cortex even after stimulation with bradykinin (van Leeuwen, 1999).

The morphological consequences of Pak1 activation were investigated. Overexpression of either wild-type Pak1 or Pak1(E423), an activated variant of this kinase, induces cell spreading accompanied by some redistribution of myosin II, although the observed changes are very different from those produced by C1199Tiam1 or Rac1V12. The prominent lamellae, which do not appear to contain any myosin II, as observed in Tiam1- and RacV12-expressing cells, are not seen in the Pak1-overexpressing cells. Whereas dominant-negative Pak1 clearly inhibits bradykinin-induced MHC phosphorylation, overexpression of wild-type Pak1 or Pak1(E423) is not sufficient to promote MHC phosphorylation, in either the presence or the absence of bradykinin. It is speculated that these differences between the results of Rac and Pak activation either reflect improper localization of the overexpressed kinase or otherwise indicate that another member of this kinase family, and not Pak1, is involved in regulating MHC phosphorylation. A similar discrepancy was found in Rat-1 fibroblasts, where dominant-negative Pak1(L83, L86, R299) inhibits Ras transformation, whereas an activated kinase does not cooperate with either Ras or Raf in cell transformation. Indeed, instead of Pak1, Pak3 has been shown to activate Raf-1 downstream of Ras17. Together, these results indicate that a Pak-like kinase activity may regulate MHC phosphorylation and cell spreading. The identity of the kinase involved remains to be established (van Leeuwen, 1999).

MHC phosphorylation is Ca2+ dependent. Apparently, bradykinin, which signals through G-protein-coupled receptors, provides extra signals to increase MHC phosphorylation. Using pharmacological inhibitors, a determination was made of which bradykinin-induced second-messenger pathways cooperate with Rac to induce MHC phosphorylation. MHC phosphorylation is completely dependent on the influx of extracellular Ca2+. Chelation of Ca2+ from the tissue-culture medium with EGTA abolishes phosphorylation in response to bradykinin. MHC phosphorylation is also effectively blocked by the presence of an inhibitor of receptor-operated Ca2+-channels. Even the sustained increase in MHC phosphorylation observed in cells overexpressing Tiam1 is reduced to undetectable levels in cells that have been depleted of Ca2+ by pretreatment with a membrane-permeable Ca2+ chelator BAPTA-AM. Conversely, Ca2+ influx, artificially induced with the Ca2+ ionophore ionomycin, is sufficient to induce phosphorylation of the MHC. These results indicate that threonine phosphorylation of the myosin-II heavy chain involves a calcium-dependent kinase pathway, which appears to be regulated or sensitized by Rac (van Leeuwen, 1999).

The sorting of mRNA is a determinant of cell asymmetry. The cellular signals that direct specific RNA sequences to a particular cellular compartment are unknown. In fibroblasts, ß-actin mRNA has been shown to be localized toward the leading edge, where it plays a role in cell motility and asymmetry. A signaling pathway initiated by extracellular receptors acting through Rho GTPase and Rho-kinase regulates this spatial aspect of gene expression in fibroblasts by localizing ß-actin mRNA via actomyosin interactions. Consistent with the role of Rho as an activator of myosin, inhibition of myosin ATPase, myosin light chain kinase (MLCK), and the knockout of myosin II-B in mouse embryonic fibroblasts all inhibit ß-actin mRNA from localizing in response to growth factors. It is concluded that the sorting of ß-actin mRNA in fibroblasts requires a Rho mediated pathway operating through a myosin II-B-dependent step and it is postulated that polarized actin bundles direct the mRNA to the leading edge of the cell (Latham, 2001).

How could myosin II direct mRNA movement to the leading edge? It is postulated that the two-headed myosin filaments can translocate on polarized bundles of actin filaments (a.k.a., 'stress fibers') toward the leading edge associated with an mRNA complex that can bind the myosin. This hypothesis is supported by work demonstrating that, in moving fibroblasts, these actin bundles have a polarity with barbed ends increasingly directed toward the lamellipodium and thus could constrain activated myosin II-B movement only toward the leading edge. There is evidence that myosin activation and deactivation can be spatially regulated. Rho-kinase can lead to phosphorylation of the light chains internally near the nucleus, where myosin filament assembly, stress fiber formation, and motility occurs. PKC in the periphery of the cell can phosphorylate myosin II-B heavy chains and promote disassembly at the leading edge. This could transport the mRNA bound to the myosin toward the leading edge, where it would anchor to actin filaments when the myosin filament disassembles. Extracellular signals can act through the signaling mediators Rho and Rho-kinase in the regulation of ß-actin mRNA distribution via actomyosin interactions. In this way, spatially localized protein synthesis is a component of gene expression that can respond rapidly (2 min) to extracellular signals and immediately effect physiological changes within specific cellular compartments (Latham, 2001).

Phagocytosis through Fcgamma receptor (FcgammaR) or complement receptor 3 (CR) requires Arp2/3 complex-mediated actin polymerization, although each receptor uses a distinct signaling pathway. Rac and Cdc42 are required for actin and Arp2/3 complex recruitment during FcgammaR phagocytosis, while Rho controls actin assembly at complement receptor 3 (CR) phagosomes. To better understand the role of Rho in CR phagocytosis, the idea was tested that a known target of Rho, Rho-kinase (ROK), might control phagocytic cup formation and/or engulfment of particles. Inhibitors of ROK (dominant-negative ROK and Y-27632) and of the downstream target of ROK, myosin-II (ML7, BDM, and dominant-negative myosin-II), were used to test this idea. Inhibition of the Rho --> ROK --> myosin-II pathway causes a decreased accumulation of Arp2/3 complex and F-actin around bound particles, which leads to a reduction in CR-mediated phagocytic engulfment. FcgammaR-mediated phagocytosis, in contrast, is independent of Rho or ROK activity and is only dependent on myosin-II for particle internalization, not for actin cup formation. While myosins have been previously implicated in FcgammaR phagocytosis, this is the first demonstration of a role for myosin-II in CR phagocytosis (2002).

All vertebrates contain two nonmuscle myosin II heavy chains, A and B, which differ in tissue expression and subcellular distributions. To understand how these distinct distributions are controlled and what role they play in cell migration, myosin IIA and IIB were examined during wound healing by bovine aortic endothelial cells. Immunofluorescence has shown that myosin IIA skews toward the front of migrating cells, coincident with actin assembly at the leading edge, whereas myosin IIB accumulates in the rear 15-30 min later. Inhibition of myosin light-chain kinase, protein kinases A, C, and G, tyrosine kinase, MAP kinase, and PIP3 kinase does not affect this asymmetric redistribution of myosin isoforms. However, posterior accumulation of myosin IIB, but not anterior distribution of myosin IIA, is inhibited by dominant-negative rhoA and by the rho-kinase inhibitor, Y-27632, which also inhibits myosin light-chain phosphorylation. This inhibition is overcome by transfecting cells with constitutively active myosin light-chain kinase. These observations indicate that asymmetry of myosin IIB, but not IIA, is regulated by light-chain phosphorylation mediated by rho-dependent kinase. Blocking this pathway inhibits tail constriction and retraction, but does not affect protrusion, suggesting that myosin IIB functions in pulling the rear of the cell forward (Kolega, 2003).

During cytokinesis, regulatory signals are presumed to emanate from the mitotic spindle. However, what these signals are and how they lead to the spatiotemporal changes in the cortex structure, mechanics, and regional contractility are not well understood in any system. To investigate pathways that link the microtubule network to the cortical changes that promote cytokinesis, chemical genetics was used in Dictyostelium to identify genetic suppressors of nocodazole, a microtubule depolymerizer. 14-3-3 is enriched in the cortex, helps maintain steady-state microtubule length, contributes to normal cortical tension, modulates actin wave formation, and controls the symmetry and kinetics of cleavage furrow contractility during cytokinesis. Furthermore, 14-3-3 acts downstream of a Rac small GTPase (RacE), associates with myosin II heavy chain, and is needed to promote myosin II bipolar thick filament remodeling. It is concluded that 14-3-3 connects microtubules, Rac, and myosin II to control several aspects of cortical dynamics, mechanics, and cytokinesis cell shape change. Furthermore, 14-3-3 interacts directly with myosin II heavy chain to promote bipolar thick filament remodeling and distribution. Overall, 14-3-3 appears to integrate several critical cytoskeletal elements that drive two important processes-cytokinesis cell shape change and cell mechanics (Zhou, 2010).

Essential light chain of myosin

zipper Evolutionary Homologs continued: part 2/3 | part 3/3

Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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