Regulatory light chain mutations affect myosin motor function and kinetics

Bernard M. Chaudoir, Patricia A. Kowalczyk, Rex L. Chisholm*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

16 Scopus citations

Abstract

The actin-based motor protein myosin II plays a critical role in many cellular processes in both muscle and non-muscle cells. Targeted disruption of the Dictyostelium regulatory light chain (RLC) caused defects in cytokinesis and multicellular morphogenesis. In contrast, a myosin heavy chain mutant lacking the RLC binding site, and therefore bound RLC, showed normal cytokinesis and development. One interpretation of these apparently contradictory results is that the phenotypic defects in the RLC null mutant results from mislocalization of myosin caused by aggregation of RLC null myosin. To distinguish this from the alternative explanation that the RLC can directly influence myosin activity, we expressed three RLC point mutations (E12T, G18K and N94A) in a Dictyostelium RLC null mutant. The position of these mutations corresponds to the position of mutations that have been shown to result in familial hypertrophic cardiomyopathy in humans. Analysis of purified Dictyostelium myosin showed that while these mutations did not affect binding of the RLC to the MHC, its phosphorylation by myosin light chain kinase or regulation of its activity by phosphorylation, they resulted in decreased myosin function. All three mutants showed impaired cytokinesis in suspension, and one produced defective fruiting bodies with short stalks and decreased spore formation. The abnormal myosin localization seen in the RLC null mutant was restored to wild-type localization by expression of all three RLC mutants. Although two of the mutant myosins had wild-type actin-activated ATPase, they produced in vitro motility rates half that of wild type. N94A myosin showed a fivefold decrease in actin-ATPase and a similar decrease in the rate at which it moved actin in vitro. These results indicate that the RLC can play a direct role in determining the force transmission and kinetic properties of myosin.

Original languageEnglish (US)
Pages (from-to)1611-1620
Number of pages10
JournalJournal of cell science
Volume112
Issue number10
StatePublished - 1999

Keywords

  • Cytokinesis
  • Dictyostelium discoideum
  • Motor function
  • Myosin
  • Myosin regulation
  • Regulatory light chain

ASJC Scopus subject areas

  • Cell Biology

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