TARGETED DISRUPTION OF THE DICTYOSTELIUM RMLC GENE PRODUCES CELLS DEFECTIVE IN CYTOKINESIS AND DEVELOPMENT

Citation
Px. Chen et al., TARGETED DISRUPTION OF THE DICTYOSTELIUM RMLC GENE PRODUCES CELLS DEFECTIVE IN CYTOKINESIS AND DEVELOPMENT, The Journal of cell biology, 127(6), 1994, pp. 1933-1944
Citations number
59
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Cell Biology
Journal title
ISSN journal
0021-9525
Volume
127
Issue
6
Year of publication
1994
Part
2
Pages
1933 - 1944
Database
ISI
SICI code
0021-9525(1994)127:6<1933:TDOTDR>2.0.ZU;2-U
Abstract
Conventional myosin has two different light chains bound to the neck r egion of the molecule. It has been suggested that the light chains con tribute to myosin function by providing structural support to the neck region, therefore amplifying the conformational changes in the head f ollowing ATP hydrolysis (Rayment et al., 1993). The regulatory light c hain is also believed to be important in regulating the actin-activate d 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 o f the RMLC-coding region, leading to cells devoid of both the RMLC tra nscript 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; Manstei n et al., 1989), suggest the RMLC subunit is required for normal cytok inesis and cell motility. In contrast to the myosin heavy chain mutant s, however, the mlcR cells are able to cap cell surface receptors foll owing 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 v aried depending upon whether the cells were cultured in suspension or on a solid substrate. In vitro, purified RMLC- myosin assembled to for m thick filaments comparable to wildtype myosin, but the filaments the n exhibited abnormal disassembly properties. These results indicate th at in vivo RMLC is necessary for myosin function.