Targeted disruption of the Dictyostelium RMLC gene produces cells defective in cytokinesis and development.

Chen, Pengxin; Ostrow, Bruce D.; Tafuri, Sherrie R.; Chisholm, Rex L.

doi:10.1083/jcb.127.6.1933

Cited by 101 publications

(103 citation statements)

References 57 publications

(90 reference statements)

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“…Thus, one apparent function of light chains is the stabilization of this structure. Consistent with this structural analysis, myosins lacking the RLCs have a tendency to aggregate through the neck region (33,34,35). Our results show that the ability of ELC mutants to rescue the cytokinesis defect of mlcE Ϫ cells correlates with their ability to bind to heavy chain, establishing that a stoichiometric binding of ELC is necessary in vivo for normal myosin function.…”

Section: Discussionsupporting

confidence: 82%

“…It has been reported that myosin lacking the RLCs has a tendency to aggregate (33,34,35), presumably mediated by the exposed, hydrophobic ␣-helix that serves as binding sites for myosin light chains (13,14). An important question is whether the poor cellular myosin function observed with the weak binding mutants ⌬N28, ⌬N11, ⌬C15 is due to a defect in enzymatic activity or aggregation of a significant amount of the myosin lacking bound ELC.…”

Section: Abilities Of Mutant Elcs To Rescue the Cytokinesis Defect Ofmentioning

confidence: 99%

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Both the Amino and Carboxyl Termini of Dictyostelium Myosin Essential Light Chain Are Required for Binding to Myosin Heavy Chain

Chen

Chisholm

1995

Journal of Biological Chemistry

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Dictyostelium myosin deficient in the essential light chain (ELC) does not function normally either in vivo or in vitro (Pollenz, R. S., Chen, T. L., Trivinos-Lagos, L., and Chisholm, R. L. (1992) Cell 69, 951-962). Since normal myosin function requires association of ELC, we investigated the domains of ELC that are necessary for binding to the myosin heavy chain (MHC). Deleting the NH 2 -terminal 11 or 28 amino acid residues (⌬N11 or ⌬N28) or the COOH-terminal 15 amino acid residues (⌬C15) abolished binding of the ELC to the MHC when the mutants were expressed in wild-type (WT) cells. In contrast, the ELC carrying deletion or insertion of four amino acid residues (D4 or I4) in the central linker segment bound the MHC in WT cells, although less efficient competition with WT ELC suggested that the affinity for the MHC is reduced. When these mutants were expressed in ELC-minus (mlcE ؊ ) cells, where the binding to the heavy chain is not dependent on efficient competition with the endogenous ELC, ⌬N28 and ⌬N11 bound to the MHC at 15% of WT levels and ⌬C15 did not bind to a significant degree. I4 and D4, however, bound with normal stoichiometry. These data indicate that residues at both termini of the ELC are required for association with the MHC, while the central linker domain appears to be less critical for binding. When the mutants were analyzed for their ability to complement the cytokinesis defect displayed by mlcE ؊ cells, a correlation to the level of ELC carried by the MHC was observed, indicating that a stoichiometric ELC-MHC association is necessary for normal myosin function in vivo.Myosin is a mechanochemical enzyme that generates force during muscle contraction and has a fundamental role in a variety of cell movements (for review, see Ref. 2). Conventional myosin is a hexameric protein composed of a pair of heavy chains (MHC) 1 and two pairs of light chains, called essential light chains (ELC) and regulatory light chains (RLC). Both light chains bind to the neck of myosin (3, 4). We have shown that ELC-deficient Dictyostelium cell lines are defective in cytokinesis, and myosin purified from these cells does not show significant actin-activated ATPase activity (1). Since normal myosin function requires association of the ELC, we set out to map the domains of the ELC that are critical for ELC-MHC interaction.Sequence comparisons of ELCs from various sources show that the COOH-terminal half of the molecule is more conserved than the NH 2 -terminal half (5, 6) and that it may represent the site required for association with MHC. Skeletal muscle alkali light chain A1 in which the COOH-terminal 14 residues were chemically removed did not bind the S1 heavy chain, although the conformation remained largely unchanged (7). Transfection studies also showed that a COOH-terminal truncation of the alkali light chain A1 failed to colocalize with acto-myosin structures in cultured myocytes (8).The ELC belongs to the calmodulin-troponin gene family, which contains four helix-loop-helix structures, also known as EF-hand...

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Section: Discussionsupporting

confidence: 82%

Section: Abilities Of Mutant Elcs To Rescue the Cytokinesis Defect Ofmentioning

confidence: 99%

Both the Amino and Carboxyl Termini of Dictyostelium Myosin Essential Light Chain Are Required for Binding to Myosin Heavy Chain

Chen

Chisholm

1995

Journal of Biological Chemistry

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“…The method is essentially Knecht and Loomis, 1987) essential light chain II (Pollenz et al, 1992) regulatory light chain II (Chen et al, 1994) Actin-binding proteins Profilin I and II (G actin sequestering) I (Haugwitz et al, 1994) Cortexillin I and II (bundling) I Liu et al, 1992) TalA (Talin-like) II (Niewohner et al, 1997) CluA (novel) I (Zhu et al, 1997) AmiA/PiaA (novel) I (Nagasaki et al, 1998) DtrA (novel) II (Ginger et al, 1998) Identified by forward genetics (tagging = restriction enzyme-mediated integration, REMI)…”

Section: Proteins Identified From Type I Mutantsmentioning

confidence: 99%

“…These include the null mutants of PAK-like protein kinase PAKa (Chung and Firtel, 1999), and Dictyostelium homologue of H-Ras, RasG (Tuxworth et al, 1997), as well as essential (Pollenz et al, 1992) and regulatory (Chen et al, 1994) light chains of myosin II. PAKa protein localized to cleavage furrow in cytokinesis and is involved in the assembly of myosin II, probably regulating myosin II heavy chain kinase (Chung and Firtel, 1999).…”

Section: Proteins Identified From Type II Mutantsmentioning

confidence: 99%

Advances in Cytokinesis Research. Identification of Proteins Involved in Cytokinesis of Dictyostelium.

Adachi

2001

Cell Struct. Funct.

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ABSTRACT. Dictyostelium is one of the model systems of choice for studying the cytokinesis of animal-type cells. Two types of cytokinesis mutants have been used to identify proteins involved in the cytokinesis of Dictyostelium: (1) type I, the mutant cells grow on substrates to produce giant multinucleate cells; (2) type II, the mutant cells divide nearly normally on substrates, but are unable to divide at all and get highly multinucleate in suspension culture. These two mutant types might correspond to the myosin II-independent and myosin II-including cytokinesis mechanisms, respectively.

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“…Previous studies have shown that myosin II is essential for several motile processes in this organism. Mutants defective in the myosin II heavy and light chains display a complete block in cytokinesis, capping of cell surface receptors, and development (6)(7)(8)(9). In addition, the lack of myosin heavy chain (MHC) leads to severe defects in cell motility (10,11).…”

mentioning

confidence: 99%

Single-headed myosin II acts as a dominant negative mutation in Dictyostelium.

Burns

Larochelle

Erickson

et al. 1995

Proc. Natl. Acad. Sci. U.S.A.

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Conventional myosin II is an essential protein for cytokinesis, capping of cell surface receptors, and development of Dictyostelium cells. Myosin II also plays an important role in the polarization and movement of cells. All conventional myosins are double-headed molecules but the significance of this structure is not understood since singleheaded myosin II can produce movement and force in vitro. We found that expression of the tail portion of myosin II in Dictyostelium led to the formation of single-headed myosin II in vivo. The resultant cells contain an approximately equal ratio of double-and single-headed myosin II molecules. Surprisingly, these cells were completely blocked in cytokinesis and capping of concanavalin A receptors although development into fruiting bodies was not impaired. We found that this phenotype is not due to defects in myosin light chain phosphorylation. These results show that single-headed myosin II cannot function properly in vivo and that it acts as a dominant negative mutation for myosin II function. These results suggest the possibility that cooperativity of myosin II heads is critical for force production in vivo.

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Targeted disruption of the Dictyostelium RMLC gene produces cells defective in cytokinesis and development.

Cited by 101 publications

References 57 publications

Both the Amino and Carboxyl Termini of Dictyostelium Myosin Essential Light Chain Are Required for Binding to Myosin Heavy Chain

Both the Amino and Carboxyl Termini of Dictyostelium Myosin Essential Light Chain Are Required for Binding to Myosin Heavy Chain

Advances in Cytokinesis Research. Identification of Proteins Involved in Cytokinesis of Dictyostelium.

Single-headed myosin II acts as a dominant negative mutation in Dictyostelium.

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