Phosphorylation at threonine-18 in addition to phosphorylation at serine-19 on myosin-II regulatory light chain is a mitosis-specific event

Bogdan I. Gerashchenko, Kozue Ueda, Mizuki Hino, Hiroshi Hosoya

Research output: Contribution to journalArticlepeer-review

10 Citations (Scopus)

Abstract

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). Materials and 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. Results and 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 mechnisms governing the function of nonmuscle myosins during various cell motile events, including cytokinesis.

Original languageEnglish
Pages (from-to)150-157
Number of pages8
JournalCytometry
Volume47
Issue number3
DOIs
Publication statusPublished - 2002 Mar 1
Externally publishedYes

Keywords

  • Cell sorting
  • Cytokinesis
  • Diphosphorylation
  • Flow cytometry
  • Immunofluorescence
  • Mitotic cells
  • Myosin RLC
  • PP1 antibody

ASJC Scopus subject areas

  • Pathology and Forensic Medicine
  • Biophysics
  • Hematology
  • Endocrinology
  • Cell Biology

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