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

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.

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

Molecular Biology of the Cell ◽

10.1091/mbc.e02-04-0228 ◽

2002 ◽

Vol 13 (12) ◽

pp. 4333-4342 ◽

Cited By ~ 18

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.

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Non-muscle myosin II heavy chain has a cryptic cell-adhesion domain

Biochemical Journal ◽

10.1042/bj3090569 ◽

1995 ◽

Vol 309 (2) ◽

pp. 569-574 ◽

Cited By ~ 1

Author(s):

F Grinnell ◽

C H Ho

Keyword(s):

Cell Adhesion ◽

Sequence Analysis ◽

Heavy Chain ◽

Myosin Ii ◽

Cross Reactivity ◽

Bhk Cells ◽

A Cell ◽

The Cross ◽

Muscle Myosin

We have discovered a cryptic cell-adhesion domain in non-muscle myosin II heavy chain. A 205 kDa cell-adhesion-promoting polypeptide (p205) was extracted from BHK cells by Nonidet P-40 or Dounce homogenization. Adhesion to p205 was specifically inhibited by the peptide Gly-Arg-Gly-Asp-Ser-Pro, indicating a role for the Arg-Gly-Asp cell-adhesion motif. Purified p205 was identified as non-muscle myosin II heavy chain, based on sequence analysis and on the cross-reactivity of p205 with anti-(bovine trachea myosin) antibodies. Further experiments showed that the heavy chain of purified myosin II has cell-adhesion-promoting activity in a cell-blotting assay, and cross-reacted with anti-p205 antibodies. Finally, the adhesion domain was located in the tail portion of myosin II heavy chain, where an Arg-Gly-Asp-containing sequence can be found.

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Faculty Opinions recommendation of Dictyostelium and Acanthamoeba myosin II assembly domains go to the cleavage furrow of Dictyostelium myosin II-null cells.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1013732.191599 ◽

2003 ◽

Author(s):

William Bement

Keyword(s):

Myosin Ii ◽

Cleavage Furrow ◽

Dictyostelium Myosin

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

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)42967-5 ◽

1981 ◽

Vol 256 (24) ◽

pp. 12811-12816 ◽

Cited By ~ 16

Author(s):

G.P. Côté ◽

J.H. Collins ◽

E.D. Korn

Keyword(s):

Heavy Chain ◽

Myosin Ii ◽

Phosphorylation Sites

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

The Journal of Cell Biology ◽

10.1083/jcb.109.4.1529 ◽

1989 ◽

Vol 109 (4) ◽

pp. 1529-1535 ◽

Cited By ~ 27

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.

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Rapid Generation of Single-Tumor Spheroids for High-Throughput Cell Function and Toxicity Analysis

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057106292763 ◽

2006 ◽

Vol 11 (8) ◽

pp. 922-932 ◽

Cited By ~ 321

Author(s):

Andrea Ivascu ◽

Manfred Kubbies

Keyword(s):

Tumor Cell ◽

Suspension Culture ◽

Cell Lines ◽

Cell Function ◽

Tumor Cell Lines ◽

Proliferating Cells ◽

Basement Membrane Extract ◽

Rapid Generation ◽

Size Heterogeneity

Spheroids are widely used in biology because they provide an in vitro 3-dimensional (3D) model to study proliferation, cell death, differentiation, and metabolism of cells in tumors and the response of tumors to radiotherapy and chemotherapy. The methods of generating spheroids are limited by size heterogeneity, long cultivation time, or mechanical accessibility for higher throughput fashion. The authors present a rapid method to generate single spheroids in suspension culture in individual wells. A defined number of cells ranging from 1000 to 20,000 were seeded into wells of poly-HEMA-coated, 96-well, round-or conical-bottom plates in standard medium and centrifuged for 10 min at 1000 g. This procedure generates single spheroids in each well within a 24-h culture time with homogeneous sizes, morphologies, and stratification of proliferating cells in the rim and dying cells in the core region. Because a large number of tumor cell lines form only loose aggregates when cultured in 3D, the authors also performed a screen for medium additives to achieve a switch from aggregate to spheroid morphology. Small quantities of the basement membrane extract Matrigel, added to the culture medium prior to centrifugation, most effectively induced compact spheroid formation. The compact spheroid morphology is evident as early as 24 h after centrifugation in a true suspension culture. Twenty tumor cell lines of different lineages have been used to successfully generate compact, single spheroids with homogenous size in 96-well plates and are easily accessible for subsequent functional analysis.

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Sequence analysis of mutations that affect the synthesis, assembly and enzymatic activity of the unc-54 myosin heavy chain of Caenorhabditis elegans

Journal of Molecular Biology ◽

10.1016/0022-2836(85)90170-6 ◽

1985 ◽

Vol 183 (4) ◽

pp. 543-551 ◽

Cited By ~ 74

Author(s):

Nick J. Dibb ◽

Daniel M. Brown ◽

Jonathan Karn ◽

Donald G. Moerman ◽

Suzanne L. Bolten ◽

...

Keyword(s):

Caenorhabditis Elegans ◽

Sequence Analysis ◽

Enzymatic Activity ◽

Myosin Heavy Chain ◽

Heavy Chain

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Factors Affecting the Selection of Callus Cell Lines and the Preparation of the Cell Suspension Culture of Artemisia annua L.

Plant Tissue Culture and Biotechnology ◽

10.3329/ptcb.v23i2.17507 ◽

2014 ◽

Vol 23 (2) ◽

pp. 157-163 ◽

Cited By ~ 2

Author(s):

Ch’ng Song Jin ◽

Chan Lai Keng

Keyword(s):

Suspension Culture ◽

Cell Suspension ◽

Cell Lines ◽

Cell Suspension Culture ◽

Artemisia Annua ◽

Leaf Explants ◽

In Vitro Production ◽

Callus Cell ◽

Alternative Mode ◽

Artemisia Annua L

Artemisinin, an important antimalarial drug against multidrug resistant strains of Plasmodium, can be produced in Artemisia annua L. Field production of artemisinin is affected by environmental condition and geographical location. In vitro production via cell suspension culture is an alternative mode and cell line selection is important to ensure sustainable production of biomass and artemisinin. Callus cell lines were derived from the leaf explants of five A. annua clones grown in two different locations in Vietnam. Thirty-four callus cell lines with consistent growth index (GI) were selected from these five clones and were categorized into fast (GI > 20), intermediate (GI 15 - 20) and slow (GI < 15) growing groups. The selected lines were found to have different morphology in term of colour and texture. The callus texture did affect the cell growth of A. annua in which the friable callus type showed faster, consistent and sustainable cell biomass production.D. O. I.http://dx.doi.org/10.3329/ptcb.v23i2.17507Plant Tissue Cult. & Biotech. 23(2): 157-163, 2013 (December)

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Lymphoid and other tissue-specific phenotypes of polyomavirus enhancer recombinants: positive and negative combinational effects on enhancer specificity and activity

Molecular and Cellular Biology ◽

10.1128/mcb.6.6.2068-2079.1986 ◽

1986 ◽

Vol 6 (6) ◽

pp. 2068-2079

Author(s):

B A Campbell ◽

L P Villarreal

Keyword(s):

Cell Lines ◽

Heavy Chain ◽

Murine Leukemia Virus ◽

Tissue Specificity ◽

Leukemia Virus ◽

Simian Virus 40 ◽

Simian Virus ◽

Lymphoid Cells ◽

B Lymphoid ◽

Murine Leukemia

Heterologous enhancer recombinants and deletions of the polyomavirus (Py) noncoding region were constructed and analyzed for tissue specificity of DNA replication and transcription in a number of lymphoid and other cell lines. The simian virus 40 72-base-pair repeat, mouse immunoglobulin heavy-chain enhancer, and Moloney murine leukemia virus enhancer were inserted into the PvuII-D locus (nucleotides 5128 through 5265) of Py. The ability of these recombinants and the parental PvuII-D deletion mutant to replicate in permissive 3T6 cells and MOP-6 cells as well as in nonpermissive mouse B lymphoid, T lymphoid, mastocyte, and embryonal carcinoma cells was determined. Wild-type Py DNA was not permissive for replication in most lymphoid cell lines, except one hybridoma line. Simply deleting the Py PvuII-D region, however, gave Py an expanded host range, allowing high-level replication in some T lymphoid and mastocytoma cell lines, indicating that this element can be a tissue-specific negative as well as positive element. Substitution of the murine leukemia virus enhancer for Py PvuII-D yielded a Py genome which retained the ability to replicate in 3T6 cells but also replicated well in B lymphoid cells. Substitution with the immunoglobulin heavy-chain enhancer allowed replication in B lymphoid cells but interfered with replication in 3T6 cells and mastocytomas. Surprisingly, substitution with the simian virus 40 72-base-pair enhancer repeat gave a recombinant which would not replicate in any cell line tried, including MOP-6 cells, even though other recombinants with this enhancer would replicate. Thus, we observed both cooperation and interference in these combinations between enhancer components and the Py genome and that these combined activities were cell specific. These results are presented as evidence that there may be a positional dependence, or syntax, for the recognition of genetic elements controlling Py tissue specificity.

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