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 A novel positive selection for identifying cold-sensitive myosin II mutants in Dictyostelium.

Genetics ◽  
1995 ◽  
Vol 140 (2) ◽  
pp. 505-515 ◽  
Author(s):  
B Patterson ◽  
J A Spudich
Keyword(s):  
Positive Selection ◽  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Time Scale ◽  
Myosin Ii ◽  
Rapid Onset ◽  
Chemical Mutagenesis ◽  
Wild Type ◽  
Cold Sensitive ◽  
Selection For

Abstract We developed a positive selection for myosin heavy chain mutants in Dictyostelium. This selection is based on the fact that brief exposure to azide causes wild-type cells to release from the substrate, whereas myosin null cells remain adherent. This procedure assays myosin function on a time scale of minutes and has therefore allowed us to select rapid-onset cold-sensitive mutants after random chemical mutagenesis of Dictyostelium cells. We developed a rapid technique for determining which mutations lie in sequences of the myosin gene that encode the head (motor) domain and localized 27 of 34 mutants to this domain. We recovered the appropriate sequences from five of the mutants and demonstrated that they retain their cold-sensitive properties when expressed from extrachromosomal plasmids.

scholarly journals Myosin heavy-chain kinase A from Dictyostelium possesses a novel actin-binding domain that cross-links actin filaments

2006 ◽  
Vol 395 (2) ◽  
pp. 373-383 ◽  
Author(s):  
Misty Russ ◽  
Daniel Croft ◽  
Omar Ali ◽  
Raquel Martinez ◽  
Paul A. Steimle
Keyword(s):  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Actin Filaments ◽  
Myosin Ii ◽  
Coiled Coil ◽  
Actin Binding ◽  
Coiled Coil Domain ◽  
Cross Links ◽  
Myosin Heavy Chain Kinase ◽  
Kinase A

Myosin heavy-chain kinase A (MHCK A) catalyses the disassembly of myosin II filaments in Dictyostelium cells via myosin II heavy-chain phosphorylation. MHCK A possesses a ‘coiled-coil’-enriched domain that mediates the oligomerization, cellular localization and actin-binding activities of the kinase. F-actin (filamentous actin) binding by the coiled-coil domain leads to a 40-fold increase in MHCK A activity. In the present study we examined the actin-binding characteristics of the coiled-coil domain as a means of identifying mechanisms by which MHCK A-mediated disassembly of myosin II filaments can be regulated in the cell. Co-sedimentation assays revealed that the coiled-coil domain of MHCK A binds co-operatively to F-actin with an apparent KD of approx. 0.5 μM and a stoichiometry of approx. 5:1 [actin/C(1–498)]. Further analyses indicate that the coiled-coil domain binds along the length of the actin filament and possesses at least two actin-binding regions. Quite surprisingly, we found that the coiled-coil domain cross-links actin filaments into bundles, indicating that MHCK A can affect the cytoskeleton in two important ways: (1) by driving myosin II-filament disassembly via myosin II heavy-chain phosphorylation, and (2) by cross-linking/bundling actin filaments. This discovery, along with other supporting data, suggests a model in which MHCK A-mediated bundling of actin filaments plays a central role in the recruitment and activation of the kinase at specific sites in the cell. Ultimately this provides a means for achieving the robust and highly localized disruption of myosin II filaments that facilitates polarized changes in cell shape during processes such as chemotaxis, cytokinesis and multicellular development.

scholarly journals Crystal Structure of the  -Kinase Domain of Dictyostelium Myosin Heavy Chain Kinase A

2010 ◽  
Vol 3 (111) ◽  
pp. ra17-ra17 ◽  
Author(s):  
Q. Ye ◽  
S. W. Crawley ◽  
Y. Yang ◽  
G. P. Cote ◽  
Z. Jia
Keyword(s):  
Crystal Structure ◽  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Kinase Domain ◽  
Dictyostelium Myosin ◽  
Myosin Heavy Chain Kinase ◽  
Kinase A

scholarly journals WD Repeat Domain of Dictyostelium Myosin Heavy Chain Kinase C Functions in both Substrate Targeting and Cellular Localization

2009 ◽  
Vol 9 (2) ◽  
pp. 344-349
Author(s):  
Atiya Franklin ◽  
Linzi Hyatt ◽  
Alyssa Chowdhury ◽  
Paul A. Steimle
Keyword(s):  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Cellular Localization ◽  
Myosin Ii ◽  
Content Type ◽  
Kinase C ◽  
Dictyostelium Myosin ◽  
Repeat Domain ◽  
Myosin Heavy Chain Kinase ◽  
Wd Repeat

ABSTRACT Myosin II disassembly in Dictyostelium discoideum is regulated by three structurally related myosin heavy chain kinases (myosin II heavy chain kinase A [MHCK-A], -B, and -C). We show that the WD repeat domain of MHCK-C is unique in that it mediates both substrate targeting and subcellular localization, revealing a target for regulation that is distinct from those of the other MHCKs.

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 LvsA, a Protein Related to the Mouse Beige Protein, Is Required for Cytokinesis in Dictyostelium

1999 ◽  
Vol 10 (12) ◽  
pp. 4429-4439 ◽  
Author(s):  
Eunice Kwak ◽  
Noel Gerald ◽  
Denis A. Larochelle ◽  
Kalpa K. Vithalani ◽  
Maria L. Niswonger ◽  
...  
Keyword(s):  
Sequence Analysis ◽  
Suspension Culture ◽  
Cell Lines ◽  
Heavy Chain ◽  
Large Volume ◽  
Myosin Ii ◽  
Cleavage Furrow ◽  
Membrane Processing ◽  
Chediak Higashi Syndrome ◽  
Unusual Phenotype

We isolated a Dictyostelium cytokinesis mutant with a defect in a novel locus called large volume sphere A (lvsA). lvsA mutants exhibit an unusual phenotype when attempting to undergo cytokinesis in suspension culture. Early in cytokinesis, they initiate furrow formation with concomitant myosin II localization at the cleavage furrow. However, the furrow is later disrupted by a bulge that forms in the middle of the cell. This bulge is bounded by furrows on both sides, which are often enriched in myosin II. The bulge can increase and decrease in size multiple times as the cell attempts to divide. Interestingly, this phenotype is similar to the cytokinesis failure of Dictyosteliumclathrin heavy-chain mutants. Furthermore, both cell lines cap ConA receptors but form only a C-shaped loose cap. Unlike clathrin mutants,lvsA mutants are not defective in endocytosis or development. The LvsA protein shares several domains in common with the molecules beige and Chediak–Higashi syndrome proteins that are important for lysosomal membrane traffic. Thus, on the basis of the sequence analysis of the LvsA protein and the phenotype of thelvsA mutants, we postulate that LvsA plays an important role in a membrane-processing pathway that is essential for cytokinesis.

Aberrant pattern formation in myosin heavy chain mutants of Dictyostelium

Development ◽  
1994 ◽  
Vol 120 (3) ◽  
pp. 591-601 ◽  
Author(s):  
D. Traynor ◽  
M. Tasaka ◽  
T. Takeuchi ◽  
J. Williams
Keyword(s):  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Cell Types ◽  
Culture Conditions ◽  
Wild Type ◽  
Multicellular Aggregate ◽  
A Cells ◽  
Air Water Interface ◽  
Shape Changes ◽  
Roller Tube Culture

In mutant Dictyostelium strains that fail to accumulate the myosin heavy chain (MHC A), development is relatively normal up to the tight aggregate stage but is arrested prior to formation of the apical tip (DeLozanne and Spudich 1987, Knecht and Loomis, 1987). We show that in aggregates formed by such MHC A deficient (MHC A-) strains the proportions of pstA and pstB cells, the two prestalk cell types, and of prespore cells are similar to those found during normal development but their distribution is radically different. During the initial stages of normal slug formation, pstA cells move to the tip, pstB cells accumulate in the base and prespore cells occupy the remainder of the aggregate. In the aggregates initially formed by MHC A- mutants pstA cells are present in a central core, pstB cells are present in the cortex and prespore cells lie sandwiched between them. Eventually, cells within the cortex differentiate into mature stalk cells but spores are never formed. Mixing experiments, in which MHC A- cells are allowed to co-aggregate with an excess of normal cells, show that MHC A- prestalk cells enter the aggregate relatively normally but are unable to enter the slug tip or to migrate into the stalk at culmination and that MHC A- prespore cells accumulate in the lower part of the spore head during culmination. Thus MHC A- cells appear to be able to move within the multicellular aggregate but are incapable of participating in normal morphogenesis. The structures formed by MHC A- cells are very similar to those of the agglomerates that form when wild-type cells are developed in roller-tube culture, conditions that result in loss of the polarity imparted by the presence of an air-water interface. We propose formation of such a structure by MHC A- cells to be a default response, caused by their inability to undertake the shape changes and intercalatory cell movements that are necessary to form and extend the tip.

scholarly journals Ifm(2)2 is a myosin heavy chain allele that disrupts myofibrillar assembly only in the indirect flight muscle of Drosophila melanogaster.

1988 ◽  
Vol 107 (6) ◽  
pp. 2613-2621 ◽  
Author(s):  
M Chun ◽  
S Falkenthal
Keyword(s):  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Flight Muscle ◽  
Microscopic Analysis ◽  
Thick Filament ◽  
Indirect Flight Muscle ◽  
Chain Gene ◽  
Wild Type ◽  
Electron Microscopic ◽  
Sarcomere Assembly

Using a combination of molecular and genetic techniques we demonstrate that Ifm(2)2 is an allele of the single-copy sarcomeric myosin heavy chain gene. Flies homozygous for this allele accumulate wild-type levels of mRNA and protein in tubular muscle of adults, but fail to accumulate detectable amounts of myosin heavy chain mRNA or protein in the indirect flight muscle. We propose that the mutation interferes with either transcription of the gene or splicing of the primary transcript in the indirect flight muscle and not in other muscle tissues. Biochemical and electron microscopic analysis of flies homozygous for this mutation has revealed that thick filament assembly is abolished in the indirect flight muscle resulting in the instability of wild-type thick filament proteins. In contrast, thin filament and Z disc assembly are marginally affected. We discuss a working hypothesis for sarcomere assembly and define and experimental approach to test the predictions of this proposed pathway for sarcomere assembly.

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