Expression of light meromyosin in Dictyostelium blocks normal myosin II function.
The ability of myosin II to form filaments is essential for its function in vivo. This property of self association is localized in the light meromyosin (LMM) region of the myosin II molecules. To explore this property in more detail within the context of living cells, we expressed the LMM portion of the Dictyostelium myosin II heavy chain gene in wild-type Dictyostelium cells. We found that the LMM protein was expressed at high levels and that it folded properly into alpha-helical coiled-coiled molecules. The expressed LMM formed large cytoplasmic inclusions composed of entangled short filaments surrounded by networks of long tubular structures. Importantly, these abnormal structures sequestered the cell's native myosin II, completely removing it from its normal cytoplasmic distribution. As a result the cells expressing LMM displayed a myosin-null phenotype: they failed to undergo cytokinesis and became multinucleate, failed to form caps after treatment with Con A, and failed to complete their normal developmental cycle. Thus, expression of the LMM fragment in Dictyostelium completely abrogates myosin II function in vivo. The dominant-negative character of this phenotype holds promise as a general method to disrupt myosin II function in many cell types without the necessity of gene targeting.
Burns
C G
CG
Department of Cell Biology, Duke University Medical Center, Durham, North Carolina 27710, USA.
Reedy
M
M
Heuser
J
J
De Lozanne
A
A
D000595:Amino Acid Sequence; D000818:Animals; D003599:Cytoskeleton / Q000378:metabolism; D004023:Dictyostelium / Q000378:metabolism*; D005613:Freeze Etching; D006651:Histocytochemistry; D008854:Microscopy, Electron; D008856:Microscopy, Fluorescence; D018715:Microscopy, Video; D008969:Molecular Sequence Data; D015879:Myosin Subfragments / Q000096:biosynthesis / Q000378:metabolism* / Q000648:ultrastructure; D009218:Myosins / Q000378:metabolism* / Q000648:ultrastructure; D010446:Peptide Fragments / Q000096:biosynthesis / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000378:metabolism
D015879:Myosin Subfragments; D010446:Peptide Fragments; D011994:Recombinant Proteins; D009218:Myosins