Phosphorylation at threonine-18 in addition to phosphorylation at serine-19 on myosin-II regulatory light chain is a mitosis-specific event.
BACKGROUND
Cell division is an inevitable and vitally indispensable event in cell life, when the nucleus and cytoskeleton undergo profound reorganization. Cytoplasmic division (cytokinesis) is known to occur immediately after the end of nuclear division, when the nuclear envelope breaks down, and chromosomes condense and segregate, but its driving mechanism remains enigmatic. Myosin, particularly myosin-II, is thought to be required for cytokinesis as a force-generating element, the activity of which is mainly regulated through phosphorylations on its 20-kDa regulatory light chains (RLCs).


METHODS
Multiparameter flow cytometric analysis was performed on fixed HeLa S3 cells (suspension culture cells) sequentially stained with the polyclonal antibody (termed PP1) against both phosphorylated sites (serine-19 and threonine-18) on the RLC, and with propidium iodide for DNA. "Positive" cells were sorted, followed by their microscopic examination. Fluorescence microscopy was employed to visualize the cell-cycle-dependent distribution of immunolabeled diphosphorylated RLCs in both HeLa S3 and adherent HeLa cells.


CONCLUSIONS
Doubly phosphorylated myosin RLCs were highly expressed in mitotic cells, suggesting the positive regulatory role of diphosphorylation in the redistribution of RLCs between daughter cells and then in cytokinesis. The increased immunofluorescence signal from the phosphorylated forms of RLC, together with flow cytometry, provides a clue with which to investigate the mechanisms governing the function of nonmuscle myosins during various cell motile events, including cytokinesis.
Gerashchenko
Bogdan I
BI
Department of Biological Science, Graduate School of Science, Hiroshima University, 1-3-31 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-8526, Japan.
Ueda
Kozue
K
Hino
Mizuki
M
Hosoya
Hiroshi
H
D000595:Amino Acid Sequence / Q000502:physiology; D000918:Antibody Specificity / Q000276:immunology; D002453:Cell Cycle / Q000502:physiology; D002455:Cell Division / Q000502:physiology; D003599:Cytoskeleton; D004247:DNA / Q000032:analysis; D005455:Fluorescent Antibody Technique; D006367:HeLa Cells; D006801:Humans; D008938:Mitosis / Q000502:physiology*; D018994:Myosin Light Chains / Q000378:metabolism*; D024462:Myosin Type II / Q000378:metabolism*; D010766:Phosphorylation; D012694:Serine / Q000378:metabolism; D013912:Threonine / Q000378:metabolism
D018994:Myosin Light Chains; D013912:Threonine; D012694:Serine; D004247:DNA; D024462:Myosin Type II