The N-terminus of the long MLCK induces a disruption in normal spindle morphology and metaphase arrest

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Abstract

We have shown previously that only the long myosin light chain kinase (MLCK), which is the predominant MLCK isoform expressed in nonmuscle cells, localizes to the cleavage furrow. To further examine the in vivo localization of the long MLCK in HeLa cells and the mechanisms responsible for kinase targeting during the cell cycle, we examined the distribution of the endogenous kinase and constructed green fluorescent protein (GFP) fusions of long HeLa MLCK truncations. A GFP fusion containing the N-terminal IgG domain and the five DXR motifs localized to stress fibers during interphase and the cleavage furrow during mitosis. Although individual fusions of the five DXRs and IgG domain both independently localized to stress fibers, only the five DXRs demonstrated a cortical localization in mitotic cells. Thus, robust targeting of the long MLCK to the cleavage furrow required the five DXRs and additional sequences from the IgG domain. Expression of the IgG domain alone or with five DXRs increased the number of multinucleate cells tenfold, whereas expression of the five DXRs or GFP had no effect. Furthermore, expression of the IgG domain alone or with five DXRs disrupted normal spindle morphology during mitosis. Extended astral microtubules and increased bundling of kinetochore microtubules, and spindle pole fragmentation were detected in mitotic cells. These microtubule defects were associated with abnormalities in metaphase chromosome alignment and a subsequent metaphase arrest caused by activation of the spindle assembly checkpoint at the kinetochores of mono-oriented chromosomes. Together, these results suggest that MLCK has an unexpected regulatory function during mitosis.

Original languageEnglish (US)
Pages (from-to)1481-1493
Number of pages13
JournalJournal of Cell Science
Volume117
Issue number8
DOIs
StatePublished - Mar 15 2004

Fingerprint

Myosin-Light-Chain Kinase
Metaphase
Immunoglobulin G
Green Fluorescent Proteins
Mitosis
Microtubules
Kinetochores
Stress Fibers
Phosphotransferases
Chromosomes
Spindle Poles
M Phase Cell Cycle Checkpoints
Interphase
HeLa Cells
Cell Cycle
Protein Isoforms
Cell Count

Keywords

  • Cell division
  • Metaphase arrest
  • Microtubules
  • Mitosis
  • Myosin light chain kinase

ASJC Scopus subject areas

  • Cell Biology

Cite this

The N-terminus of the long MLCK induces a disruption in normal spindle morphology and metaphase arrest. / Dulianinova, Natalia; Patskovsky, Yury V.; Bresnick, Anne R.

In: Journal of Cell Science, Vol. 117, No. 8, 15.03.2004, p. 1481-1493.

Research output: Contribution to journalArticle

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