Targeted disruption of the Dictyostelium RMLC gene produces cells defective in cytokinesis and development.
Conventional myosin has two different light chains bound to the neck region of the molecule. It has been suggested that the light chains contribute to myosin function by providing structural support to the neck region, therefore amplifying the conformational changes in the head following ATP hydrolysis (Rayment et al., 1993). The regulatory light chain is also believed to be important in regulating the actin-activated ATPase and myosin motor function as assayed by an in vitro motility assay (Griffith et al., 1987). Despite extensive in vitro biochemical study, little is known regarding RMLC function and its regulatory role in vivo. To better understand the importance and contribution of RMLC in vivo, we engineered Dictyostelium cell lines with a disrupted RMLC gene. Homologous recombination between the introduced gene disruption vector and the chromosomal RMLC locus (mlcR) resulted in disruption of the RMLC-coding region, leading to cells devoid of both the RMLC transcript and the 18-kD RMLC polypeptide. RMLC-deficient cells failed to divide in suspension, becoming large and multinucleate, and could not complete development following starvation. These results, similar to those from myosin heavy chain mutants (DeLozanne et al., 1987; Manstein et al., 1989), suggest the RMLC subunit is required for normal cytokinesis and cell motility. In contrast to the myosin heavy chain mutants, however, the mlcR cells are able to cap cell surface receptors following concanavilin A treatment. By immunofluorescence microscopy, RMLC null cells exhibited myosin localization patterns different from that of wild-type cells. The myosin localization in RMLC null cells also varied depending upon whether the cells were cultured in suspension or on a solid substrate. In vitro, purified RMLC- myosin assembled to form thick filaments comparable to wild-type myosin, but the filaments then exhibit abnormal disassembly properties. These results indicate that in vivo RMLC is necessary for myosin function.
Chen
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Department of Cell and Molecular Biology, Northwestern University Medical School, Chicago, Illinois 60611.
Ostrow
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Tafuri
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Chisholm
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D000199:Actins / Q000302:isolation & purification; D000818:Animals; D015153:Blotting, Western; D000252:Calcium-Transporting ATPases / Q000032:analysis; D002449:Cell Aggregation; D002455:Cell Division; D003599:Cytoskeleton / Q000201:enzymology; D016054:DNA, Protozoan / Q000235:genetics; D004274:DNA, Recombinant; D004023:Dictyostelium / Q000235:genetics / Q000254:growth & development*; D005455:Fluorescent Antibody Technique; D017125:Genes, Protozoan / Q000235:genetics*; D008856:Microscopy, Fluorescence; D016296:Mutagenesis; D009218:Myosins / Q000235:genetics* / Q000378:metabolism / Q000648:ultrastructure; D010641:Phenotype; D014170:Transformation, Genetic
D000199:Actins; D016054:DNA, Protozoan; D004274:DNA, Recombinant; D009218:Myosins; D000252:Calcium-Transporting ATPases