Role Of Myosin Research Articles

Myosin functions inXenopus retinal ganglion cell growth cone motilityin vivo

The role of myosins in Xenopus retinal ganglion cell growth cone motility in the optic tract was studied using two pharmacologic inhibitors with different specificities. 2,3-Butanedione monoxime (BDM) disrupts myosin-actin interactions of all myosins, and ML-7 specifically inhibits activation of myosin II. Both inhibitors caused growth cones to assume a collapsed morphology and decreased growth cone speed. Similar effects were observed in vitro. Interestingly, the effects of the two inhibitors, while similar, were clearly distinguishable, raising the possibility that different myosins may have different functional roles in growth cone motility. BDM caused growth cones to withdraw lamellipodia and some filopodia and eventually to freeze, whereas ML-7 caused total collapse and retraction. Concentrations of BDM and ML-7 that had no effect when applied independently stopped growth cones when applied simultaneously, suggesting that these inhibitors act synergistically on myosin function, thus providing evidence of specificity. These results imply that normal growth cone motility in the molecularly and spatially complex environment of the living brain requires myosin function.

The predominant defect in dilute melanocytes is in melanosome distribution and not cell shape, supporting a role for myosin V in melanosome transport.

Mice with mutations at the dilute locus, which encodes the heavy chain of a type V unconventional myosin, exhibit a reduction in coat colour intensity. This defect is thought to be caused by the absence in dilute melanocytes of the extensive dendritic arbor through which these cells normally deliver pigment-laden melanosomes to keratinocytes. The data on which this conclusion has been based can also be explained, however, by a defect in the outward transport of melanosomes within melanocytes of normal shape. To resolve this question, we compared the shape and pigment distribution within melanocytes present in primary cultures prepared from the epidermis of C57BL/6J pups that were either wild type (D/D) at dilute or homozygous for the dilute null allele d120J. These same comparisons were also performed on melanocytes in situ, where antibodies to the membrane tyrosine kinase receptor cKIT were used to visualize melanocyte cell shape independent of pigment distribution. Wild type melanocytes were found to be dendritic and to have melanosomes distributed throughout their dendrites both in vitro and in situ. Mutant melanocytes were also found to be dendritic in both cases, but their melanosomes were highly concentrated in the cell body and largely excluded from dendrites. We conclude, therefore, that the predominant defect in dilute melanocytes is in melanosome distribution, not cell shape. These results argue that the myosin V isoform encoded by the dilute locus functions in dendritic extensions to move melanosomes from their site of formation within the cell body to their site of intercellular transfer at dendritic tips. This conclusion is consistent with our recent demonstration by immunolocalization that the dilute myosin V isoform associates with melanosomes in mouse melanocytes.

Platelet actuation

This review article describes the different receptors, second-messengers and mechanisms involved in platelet activation. Several platelet agonists have well-defined receptors at the platelet membrane of which a number are single polypeptides with 7 hydrophobic transmembrane domains. These receptors are connected, via GTP regulatory proteins, with cytoplasmic second-messenger-generating enzymes. Phospholipase C and adenylate cyclase are the two best-known enzymes, generating inositol triphosphate (IP 3) and diacyl glycerol from phosphatidylinositol biphosphate and cyclic AMP from ATP respectively. The intraplatelet free calcium level, which is critical for the activation status of the platelet, is increased by IP 3 and is lowered in the presence of rising cyclic AMP concentrations. Shape-change occurs with small increases in intraplatelet calcium, while aggregation and secretion of granules take place at higher calcium levels. The role of myosin and actin filaments and of transmembrane glycoproteins is further discussed.

Role of myosin in neurotransmitter release: Functional studies at synapses formed in culture

To determine the functional role of presynaptic proteins in the neurotransmitter release, I have employed cholinergic synapses formed between superior cervical ganglion neurons in culture. These synapses expressed proteins characteristic of mature synapses: immunofluorescence staining showed the presence of synaptophysin, synaptotagmin, VAMP/synaptobrevin-2, syntaxin and neurexin. The function of these proteins seems to be similar to that of mature synapses because botulinum neurotoxins A, E and C 1 inhibited neurotransmitter release evoked by presynaptic action potentials. With this preparation, I have obtained evidence supporting roles for myosin II and myosin light chain kinase in neurotransmitter secretion. Acetylcholine release was inhibited by introduction of antibody against myosin II or inhibitors of myosin light chain kinase. This evidence suggests a model in which myosin light chain kinase phosphorylates myosin, and the resultant change in actin-myosin interactions is involved in some steps of neurotransmitter release.

Molecular genetic truncation analysis of filament assembly and phosphorylation domains of Dictyostelium myosin heavy chain

Conventional myosin ('myosin II') is a major component of the cytoskeleton in a wide variety of eukaryotic cells, ranging from lower amoebae to mammalian fibroblasts and neutrophils. Gene targeting technologies available in the Dictyostelium discoideum system have provided the first genetic proof that this molecular motor protein is essential for normal cytokinesis, capping of cell surface receptors, normal chemotactic cell locomotion and morphogenetic shape changes during development. Although the roles of myosin in a variety of cell functions are becoming clear, the mechanisms that regulate myosin assembly into functional bipolar filaments within cells are poorly understood. Dictyostelium is currently the only system where mutant forms of myosin can be engineered in vitro, then expressed in their native context in cells that are devoid of the wild-type isoform. We have utilized this technology in combination with nested truncation and deletion analysis to map domains of the myosin tail necessary for in vivo and in vitro filament assembly, and for normal myosin heavy chain (MHC) phosphorylation. This analysis defines a region of 35 amino acids within the tail that is critical for filament formation both for purified myosin molecules and for myosin within the in vivo setting. Phosphorylation analysis of these mutants in intact cytoskeletons demonstrates that the carboxy-terminal tip of the myosin heavy chain is required for complete phosphorylation of the myosin tail.

Analysis of the mechanism for acetylcholine release at the synapse formed between rat sympathetic neurons in culture.

Superior cervical ganglion neurons (SCGNs) were isolated from 7-day-old rat SCG and cultured in MEM containing horse serum, fetal calf serum, and nerve growth factor. In this culture condition, it is well known that the SCGNs form cholinergic synapse. In 3-4 weeks cultured neurons, immunofluorescent staining for synaptophysin, a small synaptic vesicle associated protein, showed the presence of synaptophysin as small dots on the surface of the soma. Postsynaptic potentials could be recorded in 50-80% of the neurons responding to evoked action potentials elicited in neighboring neurons. Because of its relatively large cell size and the short distance to the terminal, this synapse is a useful model for studying the mechanisms of acetylcholine (ACh) release by introducing substances such as antibodies or selective inhibitors into the presynaptic neuron by means of the whole-cell clamp technique. In this model synapse we tested the possible role of myosin in ACh release. The distribution of myosin was studied by the immunofluorescent staining technique. Myosin was recognized by the anti-myosin II IgG at the same synaptic terminals that showed the presence of synaptophysin with its antibody. The functional blockade of myosin by the antibody itself, and that of myosin light chain kinase (MLCK) by a pseudosubstrate inhibitor of MLCK, SM-1, or by a selective inhibitor of MLCK, wortmannin, induced depression of synaptic transmission in a dose-dependent manner. These indicate that phosphorylation of myosin by MLCK may be necessary for ACh release mechanisms.

Isolation and characterization of a sea urchin zygote cortex that supports in vitro contraction and reactivation of furrowing.

The isolation of the cortex of the sea urchin blastomere by detergent lysis was explored with the aim of analyzing components important in the structure and function of the cortical cytoskeleton, and their relationship to such phenomena as contraction. Buffered EGTA medium supplemented with isotonic glycerol and with magnesium, at a level close to the reported internal cellular concentration, yields stable cytoskeletal cortices that retain their spherical shape. Cortices prepared this way contain actin, myosin, fascin and spectrin, components normally associated with the cortical cytoskeleton in a similar distribution to that in intact zygotes. They retain the organized cortical filamentous structure, including the actin-fascin bundles that form cores of microvilli. ATP and NaCl caused changes in cortical shape, described as either contraction or expansion, respectively. Spectrin, but not myosin, was partially extracted by NaCl, resulting in expansion of the cortex that suggests a role for spectrin in maintenance of cortical structure. ATP (but not ADP nor ATP gamma S), which caused the partial removal of myosin and spectrin, led to the contraction of the cortex, consistent with a role for myosin in cortical tension. In cortices isolated from dividing eggs, the zygotes retained their cleavage furrows and ATP induced continuation of furrow progression. This preparation appears to be a useful in vitro model for cytokinesis.

Chapter 10 Contributions of multiple forms of myosin to nerve outgrowth

Publisher Summary This chapter focuses on the role of myosins in growth cone locomotion, axon assembly and transport. Myosins appear likely to have multiple functional roles in neurons. At least seven families of myosin have been identified in vertebrate cells. Although it has not yet been determined whether representatives from each of these families are expressed in nervous tissue, myosins I, II and V are abundant in neurons and therefore are likely to be involved in growth cone locomotion and axon assembly. Myosin V forms dimers but does not form filaments. Homozygous myosin V deletion mutations in mice result in lethal convulsions indicating that myosin V could be crucial to nervous system function. It is important to understand these roles and the results of their interactions in living cells to fully explain the mechanisms that underlie growth cone locomotion and axonal assembly. An understanding of the mechanisms underlying axon growth will help guide the development of strategies designed to facilitate axonal regeneration following nerve injury.

Role of myosin in the stimulatory effect of caldesmon on the interaction between actin, myosin, and ATP.

We have previously shown that caldesmon at low concentrations stimulates the interaction between actin, myosin, and ATP, while at high concentrations it inhibits the interaction [Ishikawa, R., Okagaki, T., Higashi-Fujime, S., & Kohama, K. (1991) J. Biol. Chem. 266, 21784-21790]. When the effect of caldesmon at low concentrations was monitored by measuring myosin ATPase activity in the absence of actin, the effect was slightly but significantly stimulatory; and at higher concentrations no inhibitory effect was observed. Therefore, we related the stimulatory effect with the myosin-binding property of caldesmon. In the presence of actin, a low concentration of caldesmon was not enough to evince the stimulatory effect: myosin concentration must also be low. This is because the stimulatory effect was obscured when myosin concentration was elevated. Ca(2+)-calmodulin abolished the stimulatory effect of caldesmon. However, the concentration of calmodulin required to abolish the stimulation was higher than that required to abolish the inhibition.

O-16 - Role of myosin in transmitter release at rat sympatheric ganglion.

O-16 - Role of myosin in transmitter release at rat sympatheric ganglion.

The role of myosin I and II in cell motility.

It has been recognized since the turn of the century that cell motility by non-muscle cells requires virtually continuous restructuring of the cytoskeleton (see refs [1-4]). It is also clear that cell motility requires a mechanism for converting chemical energy into mechanical work. The proteins actin and myosin, two important constituents of the cytoskeleton, have been postulated to act as the chemicomechanical transducer in motile cells. Central to their role as a force generating mechanism in motile cells is the ability of myosin (a) to hydrolyze ATP when it interacts with actin and (b) to form filaments. Recent studies on mammalian cells and on the cellular slime mold Dictyostelium discoideum have shed light and at the same time raised questions regarding the involvement of myosin in cell motility. Moreover, they have demonstrated the presence of two types of myosins, called myosin II and myosin I, that have unique biochemical and regulatory properties and that may play different roles in mediating cell motility. In this chapter we will discuss the properties of these two myosins and then describe what is known about their involvement in Dictyostelium and mammalian cell motility.

Myosin in hepatocytes is essential for bile canalicular contraction

Active dynamic contraction of bile canaliculi has been observed in cultured doublet hepatocytes using time-lapse cinephotomicrography. This contractile movement plays an important role in normal bile formation. The mechanism of bile canalicular contraction has been proved to involve the Ca(2+)-calmodulin system and pericanalicular actin filaments. However, the role of myosin in this system is still unknown. In this study, using the newly synthesized myosin light-chain kinase inhibitor ML-9, we found that the treatment of cultured doublet hepatocytes with ML-9 inhibited canalicular contraction. This inhibitory effect suggests that myosin is involved in this complex cellular function and that the integrity of the actin-myosin system, as well as the Ca(2+)-calmodulin system is essential for normal bile canalicular contraction.

Integrity of the cytoskeletal system is important for phagocytosis by Kupffer cells

The mechanism of phagocytosis by Kupffer cells is believed to involve the Ca2(+)-calmodulin system. However, the role of myosin in this system is still unknown. In this study, we found that myosin light chain kinase inhibitor (ML-9) inhibited phagocytosis by cultured Kupffer cells using polystyrene beads, a time-lapse VTR system and fluorescent staining techniques. The inhibitory effects of ML-9 suggest that myosin may be involved in this complex cellular function and that the integrity of the cytoskeletal system is essential for normal phagocytosis.

Dictyostelium discoideum contains a gene encoding a myosin I heavy chain.

We have cloned and completely sequenced a gene encoding the heavy chain of Dictyostelium myosin I. Like the myosin I molecules from Acanthamoeba, the Dictyostelium myosin I heavy chain is composed of a globular head domain fused to a 45-kDa glycine-, proline-, and alanine-rich carboxyl-terminal domain, rather than the coiled-coil rod domain of conventional myosins. Comparisons of the Dictyostelium myosin I heavy-chain amino acid sequence with those of the Acanthamoeba myosins I reveal that they are highly similar throughout, including the unconventional carboxyl-terminal domains. The Dictyostelium myosin I gene is expressed in growing cells as a 3600-nucleotide mRNA. Measurements of the steady-state level of this mRNA at different times during starvation-induced aggregation and development are consistent with a role for myosin I in chemotaxis and aggregation. Generation of Dictyostelium cells lacking myosin I by gene disruption and/or antisense RNA production should provide a way to test directly the role of this nonfilamentous myosin in cell motility. These experiments will be simplified by the fact that Southern blot analyses of Dictyostelium genomic DNA are consistent with there being a single myosin I heavy-chain gene.

Comparative structure of the protease-sensitive regions of the subfragment-1 heavy chain from smooth and skeletal myosins.

The heavy chain fragments generated by restricted proteolysis of the smooth chicken gizzard myosin subfragment-1 (S-1) with trypsin, Staphylococcus aureus V8 protease, and chymotrypsin were isolated and submitted to partial amino acid sequencing. The comparison between the smooth and striated muscle myosin sequences permitted the unambiguous structural characterization of the two protease-vulnerable segments joining the three putative domain-like regions of the smooth head heavy chain. The smooth carboxyl-terminal connector is a serine-rich region located around positions 632-640 of the rabbit skeletal sequence and would represent the "A" site that is conformationally sensitive to the myosin 10 S-6 transition and to its interaction with actin (Ikebe, M., and Hartshorne, D. J. (1986) Biochemistry 25, 6177-6185). A third site which undergoes a nucleotide-dependent chymotryptic cleavage which inactivates the Mg2+-ATPase (Okamoto, Y., and Sekine, T. (1981) J. Biochem. (Tokyo) 90, 833-842, 843-849) was identified at Trp-31/Ser-32. It is vicinal to Lys-34 that is monomethylated in the skeletal heavy chain but not at all in the smooth sequence. However, the two trimethyl lysine residues present in the skeletal sequence are conserved in the same regions of the smooth S-1 and may play a general functional role in myosin. The smooth central 50-kDa segment could be selectively destroyed by a mild tryptic digestion in the absence of any unfolding agent, with a concomitant inhibition of the ATPase activities. This feature is in line with the proposed domain structure of the S-1 heavy chain and also suggests a relationship between the specific biochemical properties of the smooth S-1 and the particular conformation of its 50-kDa region.

Antisense RNA inactivation of myosin heavy chain gene expression in Dictyostelium discoideum.

The role of myosin in the contraction of striated muscle cells is well known, but its importance in nonmuscle cells is not yet clear. The function of myosin in Dictyostelium discoideum has been investigated by isolating cells which specifically lack myosin heavy chain (MHC A) protein. Cells were transformed with a vector encoding RNA complementary to mhcA messenger RNA (antisense RNA). Stable transformants have a dramatic reduction in the amount of MHC A protein, grow slowly, and generate giant multinucleated progeny, indicating an impairment in cytokinesis. Surprisingly, the cells adhere to surfaces, extend pseudopods and are capable of ameboid locomotion. The developmental sequence that is initiated by starving cells is severely impaired by the lack of myosin. The cells are unable to form multicellular aggregates normally and do not undergo subsequent morphogenesis. By changing the food source from liquid medium to bacteria, expression of the endogenous mhcA messenger RNA can be increased relative to expression of antisense RNA. When grown in this way, the transformed cells accumulate MHC A protein, remain mononucleate, and proceed through development normally.

Dynamic viscoelastic changes of surimi (minced fish meat) during thermal gelation.

The present study was undertaken to clarify the continuous viscoelasticity changes of surimi during heating. A low frequency parallel plate type dynamic viscoelasticity measuring instru-went was used, with a programmable temperature control unit. The change in storage modulus of rigidity could be divided into at least eight characteristic regions. Four structure-forming regions and four critical or unstable regions appeared alternatively on increasing the temperature from 5 to 80°C. The mechanisms of setting and modori phenomena were investigated in con-nection with these regions. The role of myosin, which is the major component of meat gel, and the rlles of additives (sugars and polyphosphates) on gelation were also characterized. The results generally agreed with the results obtained by other measurements, and showed that the present methodology is very useful for determining the continuous viscoelastic changes of food gels during processing. The present methodology can be applied in the food industry in planning the combinations of additives and in searching for suitable heating conditions.

Actin-myosin interaction: The role of myosin in determining the actin pattern in self-assembled ‘hybrid’ contractile units

Self-assembly of actin-myosin filamentous complexes was assayed by polymerizing rabbit G-ADP actin on formed filaments of lobster myosin. The resulting contractile units indicate a 12-member actin orbital rather than the six-member orbital obtained previously using rabbit myosin and actin. Furthermore, the pattern of actin distribution surrounding the myosin filament is similar to that of the lobster tonic muscle sarcomere rather than the trigonal actin position characteristic of vertebrate muscle. The results show that the pattern and mode of actin complexing is determined by the specific myosin and the arrangement of the cross-bridges on the organized filament.

Effects of muscle proteins on the interaction between actin and an actin-depolymerizing protein from starfish oocytes.

The effects of myosin, heavy meromyosin (HMM), alpha-actinin, tropomyosin, and calmodulin on the interaction between an actin-depolymerizing protein (depactin) from starfish oocytes and rabbit skeletal actin were investigated. 1. Alpha-actinin or tropomyosin did not affect the inhibitory effect of depactin on salt-induced polymerization of actin and did not induce actin polymerization when added to a mixture of actin and depactin in a buffer solution which was designed to keep actin in F-form. Myosin or HMM allowed polymerization of actin under similar conditions except that ATP was not included. Addition of ATP, inorganic pyrophosphate, adenyl-5'-yl imidodiphosphate, or ADP abolished the effect of myosin. 2. None of these proteins could keep actin in a polymerized form when depactin was added to the mixture of actin and these proteins in the presence of ATP. In the absence of ATP, however, myosin protected actin against depactin. 3. The association constant between actin and depactin was estimated from competitive binding experiments using HMM to be around 4.5 X 10(6) M-1 assuming the association constant between actin and HMM to be 3 X 10(9) M-1 (Greene, L.E. & Eisenberg, E. (1980) J. Biol. Chem. 255, 549-555). 4. Actin did not activate Mg-ATPase activity of HMM in the presence of depactin. From the above results, the mode of interaction of myosin with actin in the presence of depactin and the possible role of myosin in actin assembly in the cell are discussed.

Isolation and characterization of myosin from the adrenal medulla

A protein with adenosine triphosphatase activity was isolated from bovine adrenal medulla and subsequently purified by ammonium sulfate precipitation and agarose gel filtration using a discontinuous two-buffer system. Characterization of this protein by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate, by assay of the activity of Ca 2+, K +-EDTA and Mg 2+ dependent adenosine triphosphatases by amino acid analysis and by electron microscopy has shown that the adrenal medullary myosin closely resembles those myosins isolated from muscle and other non-muscle cells. The possible roles of myosin in the adrenal medulla are discussed.