scholarly journals Release of myosin II from the membrane-cytoskeleton of Dictyostelium discoideum mediated by heavy-chain phosphorylation at the foci within the cortical actin network

1992 ◽  
Vol 117 (6) ◽  
pp. 1231-1239 ◽  
Author(s):  
S Yumura ◽  
T Kitanishi-Yumura
Keyword(s):  
Heavy Chain ◽  
Myosin Ii ◽  
Cortical Region ◽  
Actin Network ◽  
Cortical Actin ◽  
Filamentous Form ◽  
Heavy Chains ◽  
Membrane Cytoskeleton ◽  
Myosin Filaments ◽  
Gamma S

Membrane-cytoskeletons were prepared from Dictyostelium amebas, and networks of actin and myosin II filaments were visualized on the exposed cytoplasmic surfaces of the cell membranes by fluorescence staining (Yumura, S., and T. Kitanishi-Yumura. 1990. Cell Struct. Funct. 15:355-364). Addition of ATP caused contraction of the cytoskeleton with aggregation of part of actin into several foci within the network, but most of myosin II was released via the foci. However, in the presence of 10 mM MgCl2, which stabilized myosin II filaments, myosin II remained at the foci. Ultrastructural examination revealed that, after contraction, only traces of monomeric myosin II remained at the foci. By contrast, myosin II filaments remained in the foci in the presence of 10 mM MgCl2. These observations suggest that myosin II was released not in a filamentous form but in a monomeric form. Using [gamma 32P]ATP, we found that the heavy chains of myosin II released from membrane-cytoskeletons were phosphorylated, and this phosphorylation resulted in disassembly of myosin filaments. Using ITP (a substrate for myosin II ATPase) and/or ATP gamma S (a substrate for myosin II heavy-chain kinase [MHCK]), we demonstrated that phosphorylation of myosin heavy chains occurred at the foci within the actin network, a result that suggests that MHCK was localized at the foci. These results together indicate that, during contraction, the heavy chains of myosin II that have moved toward the foci within the actin network are phosphorylated by a specific MHCK, with the resultant disassembly of filaments which are finally released from membrane-cytoskeletons. This series of reactions could represent the mechanism for the relocation of myosin II from the cortical region to the endoplasm.

A mechanism for the intracellular localization of myosin II filaments in the Dictyostelium amoeba

1993 ◽  
Vol 105 (1) ◽  
pp. 233-242 ◽  
Author(s):  
S. Yumura ◽  
T. Kitanishi-Yumura
Keyword(s):  
Intracellular Ph ◽  
Heavy Chain ◽  
Intracellular Localization ◽  
Myosin Ii ◽  
Cortical Region ◽  
Actin Network ◽  
Vegetative Cells ◽  
Heavy Chains ◽  
Ph Values ◽  
Intracellular Concentrations

When ATP is added to membrane-cytoskeletons prepared from Dictyostelium amoebae by the method described previously (S. Yumura and T. Kitanishi-Yumura, Cell Struct. Funct. 15, 355–364, 1990), myosin II is released from the membrane-cytoskeletons after contraction. Simultaneously, the heavy chains of myosin II are phosphorylated by a putative myosin II heavy-chain kinase, at foci within the actin network, with the resultant disassembly of filaments. In this study, we examined factors that control the release of myosin II from the membrane-cytoskeletons, on the assumption that inhibition of the release of myosin II keeps the myosin II in the cortical region, and is responsible for the localization of myosin II in the cortical region. The release of myosin II is inhibited at pH values below 6.5. This effect is not due to the inhibition of heavy-chain phosphorylation but is due to the suppression of disassembly of the filaments. In the membrane-cytoskeletons of aggregating cells, the release of myosin II is inhibited by Ca2+, and this effect is enhanced by pretreatment with calmodulin. In the membrane-cytoskeletons of vegetative cells, the release of myosin II is inhibited by pretreatment with calmodulin, and this effect is Ca2+-independent. The inhibition of the release of myosin II by Ca2+ and/or calmodulin is due to the inhibition of heavy-chain phosphorylation, and calmodulin is associated with the foci within the actin network. These results represent a possible mechanism for the intracellular localization of myosin II via regulation of the release of myosin from the cortical region by changes in intracellular pH and/or intracellular concentrations of Ca2+.

scholarly journals Dia- and Rok-dependent enrichment of capping proteins in a cortical region

2021 ◽  
Author(s):  
Anja Schmidt ◽  
Long Li ◽  
Zhiyi Lv ◽  
Shuling Yan ◽  
Jörg Großhans
Keyword(s):  
Myosin Ii ◽  
Rho Kinase ◽  
Cortical Region ◽  
Protein Enrichment ◽  
Cortical Actin ◽  
Capping Protein ◽  
Capping Proteins ◽  
Muscle Myosin ◽  
Drosophila Embryos

Rho signaling with its major targets the formin Dia, Rho kinase (Rok) and non-muscle myosin II control turnover, amount and contractility of actomyosin. Much less investigated has been a potential function for the distribution of F-actin plus and minus ends. In syncytial Drosophila embryos Rho1 signaling is high between actin caps, i. e. the cortical intercap region. Capping protein binds to free plus ends of F-actin to prevent elongation of the filament. Capping protein has served as a marker to visualize the distribution of F-actin plus ends in cells and in vitro. Here, we probed the distribution of plus ends with capping protein in syncytial Drosophila embryos. We found that Capping proteins are specifically enriched in the intercap region similar to Dia and MyoII but distinct from overall F-actin. The intercap enrichment of Capping protein was impaired in dia mutants and embryos, in which Rok and MyoII activation was inhibited. Our observations reveal that Dia and Rok/MyoII control Capping protein enrichment and support a model that Dia and Rok/MyoII control the organization of cortical actin cytoskeleton downstream of Rho1 signaling.

scholarly journals Distinct intracellular fates of membrane and secretory immunoglobulin heavy chains in a pre-B cell line.

1991 ◽  
Vol 115 (3) ◽  
pp. 619-624 ◽  
Author(s):  
A K Bachhawat ◽  
S Pillai
Keyword(s):  
B Cell ◽  
Cell Line ◽  
Ammonium Chloride ◽  
Heavy Chain ◽  
Heavy Chains ◽  
Golgi Compartment ◽  
Resistant Form ◽  
Gamma S ◽  
Intracellular Fate ◽  
Secretory Immunoglobulin

The intracellular fates of membrane and secretory immunoglobulin heavy chains were examined in a pre-B cell line that has switched to the gamma isotype. The membrane form of the heavy chain (gamma m) was rapidly degraded while the secretory form (gamma s) was retained intracellularly in association with BiP. The degradation of gamma m could not be inhibited by ammonium chloride, chloroquine, or monensin suggesting that it occurred in a nonlysosomal compartment. The inability to detect any Endo H-resistant form of gamma m before its degradation suggested that degradation occurs before entry into the Golgi compartment. Degradation of gamma m could be inhibited by incubation at 24 degrees C. In a derivative of this cell line expressing a transfected kappa gene, gamma s formed disulfide linked tetramers with kappa and was secreted, while gamma m, although associated with kappa, continued to be rapidly degraded. These observations suggest that membrane and secretory heavy chain proteins are retained by distinct intracellular mechanisms. Although masking of the CH1 domain abrogates gamma s retention, this domain does not influence the intracellular fate of gamma m.

scholarly journals Identification of three phosphorylation sites on each heavy chain of Acanthamoeba myosin II.

1981 ◽  
Vol 256 (24) ◽  
pp. 12811-12816 ◽  
Author(s):  
G.P. Côté ◽  
J.H. Collins ◽  
E.D. Korn
Keyword(s):  
Heavy Chain ◽  
Myosin Ii ◽  
Phosphorylation Sites

scholarly journals Novel Myosin Heavy Chain Kinase Involved in Disassembly of Myosin II Filaments and Efficient Cleavage in MitoticDictyostelium Cells

2002 ◽  
Vol 13 (12) ◽  
pp. 4333-4342 ◽  
Author(s):  
Akira Nagasaki ◽  
Go Itoh ◽  
Shigehiko Yumura ◽  
Taro Q.P. Uyeda
Keyword(s):  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Myosin Ii ◽  
Kinase Domain ◽  
Cleavage Furrow ◽  
Wild Type ◽  
Daughter Cells ◽  
Cleavage Process ◽  
Interphase Cells ◽  
Myosin Heavy Chain Kinase

We have cloned a full-length cDNA encoding a novel myosin II heavy chain kinase (mhckC) from Dictyostelium. Like other members of the myosin heavy chain kinase family, themhckC gene product, MHCK C, has a kinase domain in its N-terminal half and six WD repeats in the C-terminal half. GFP-MHCK C fusion protein localized to the cortex of interphase cells, to the cleavage furrow of mitotic cells, and to the posterior of migrating cells. These distributions of GFP-MHCK C always corresponded with that of myosin II filaments and were not observed in myosin II-null cells, where GFP-MHCK C was diffusely distributed in the cytoplasm. Thus, localization of MHCK C seems to be myosin II-dependent. Cells lacking the mhckC gene exhibited excessive aggregation of myosin II filaments in the cleavage furrows and in the posteriors of the daughter cells once cleavage was complete. The cleavage process of these cells took longer than that of wild-type cells. Taken together, these findings suggest MHCK C drives the disassembly of myosin II filaments for efficient cytokinesis and recycling of myosin II that occurs during cytokinesis.

scholarly journals The effect of heavy chain phosphorylation and solution conditions on the assembly of Acanthamoeba myosin-II.

1989 ◽  
Vol 109 (4) ◽  
pp. 1529-1535 ◽  
Author(s):  
J H Sinard ◽  
T D Pollard
Keyword(s):  
Light Scattering ◽  
Ionic Strength ◽  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Critical Concentration ◽  
Myosin Ii ◽  
Acidic Ph ◽  
Thick Filaments ◽  
Low Ionic Strength

At low ionic strength, Acanthamoeba myosin-II polymerizes into bipolar minifilaments, consisting of eight molecules, that scatter about three times as much light as monomers. With this light scattering assay, we show that the critical concentration for assembly in 50-mM KCl is less than 5 nM. Phosphorylation of the myosin heavy chain over the range of 0.7 to 3.7 P per molecule has no effect on its KCl dependent assembly properties: the structure of the filaments, the extent of assembly, and the critical concentration for assembly are the same. Sucrose at a concentration above a few percent inhibits polymerization. Millimolar concentrations of MgCl2 induce the lateral aggregation of fully formed minifilaments into thick filaments. Compared with dephosphorylated minifilaments, minifilaments of phosphorylated myosin have a lower tendency to aggregate laterally and require higher concentrations of MgCl2 for maximal light scattering. Acidic pH also induces lateral aggregation, whereas basic pH leads to depolymerization of the myosin-II minifilaments. Under polymerizing conditions, millimolar concentrations of ATP only slightly decrease the light scattering of either phosphorylated or dephosphorylated myosin-II. Barring further modulation of assembly by unknown proteins, both phosphorylated and dephosphorylated myosin-II are expected to be in the form of minifilaments under the ionic conditions existing within Acanthamoeba.

scholarly journals Inhibition of acanthamoeba actomyosin-II ATPase activity and mechanochemical function by specific monoclonal antibodies.

1984 ◽  
Vol 99 (3) ◽  
pp. 1024-1033 ◽  
Author(s):  
D P Kiehart ◽  
T D Pollard
Keyword(s):  
Monoclonal Antibodies ◽  
Atpase Activity ◽  
Conformational Changes ◽  
Binding Sites ◽  
Polyclonal Antibodies ◽  
Myosin Ii ◽  
Antigenic Site ◽  
Actin Binding ◽  
Filamentous Form ◽  
Antibody Effects

Monoclonal and polyclonal antibodies that bind to myosin-II were tested for their ability to inhibit myosin ATPase activity, actomyosin ATPase activity, and contraction of cytoplasmic extracts. Numerous antibodies specifically inhibit the actin activated Mg++-ATPase activity of myosin-II in a dose-dependent fashion, but none blocked the ATPase activity of myosin alone. Control antibodies that do not bind to myosin-II and several specific antibodies that do bind have no effect on the actomyosin-II ATPase activity. In most cases, the saturation of a single antigenic site on the myosin-II heavy chain is sufficient for maximal inhibition of function. Numerous monoclonal antibodies also block the contraction of gelled extracts of Acanthamoeba cytoplasm. No polyclonal antibodies tested inhibited ATPase activity or gel contraction. As expected, most antibodies that block actin-activated ATPase activity also block gel contraction. Exceptions were three antibodies M2.2, -15, and -17, that appear to uncouple the ATPase activity from gel contraction: they block gel contraction without influencing ATPase activity. The mechanisms of inhibition of myosin function depends on the location of the antibody-binding sites. Those inhibitory antibodies that bind to the myosin-II heads presumably block actin binding or essential conformational changes in the myosin heads. A subset of the antibodies that bind to the proximal end of the myosin-II tail inhibit actomyosin-II ATPase activity and gel contraction. Although this part of the molecule is presumably some distance from the ATP and actin-binding sites, these antibody effects suggest that structural domains in this region are directly involved with or coupled to catalysis and energy transduction. A subset of the antibodies that bind to the tip of the myosin-II tail appear to inhibit ATPase activity and contraction through their inhibition of filament formation. They provide strong evidence for a substantial enhancement of the ATPase activity of myosin molecules in filamentous form and suggest that the myosin filaments may be required for cell motility.

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