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Cytoskeleton (Hoboken). 2018 Apr;75(4):164-173. doi: 10.1002/cm.21425. Epub 2017 Dec 14.

Fission yeast Myo2: Molecular organization and diffusion in the cytoplasm.

Author information

1
Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut 06520-8103.
2
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06520-8103.
3
Section on Integrative Biophysics, Eunice Kennedy Shriver National Institute of Child Health and Human Development, NIH, Bethesda, Maryland 20892-1855.
4
Department of Cell Biology, Yale University, New Haven, Connecticut 06520-8103.

Abstract

Myosin-II is required for the assembly and constriction of cytokinetic contractile rings in fungi and animals. We used electron microscopy, fluorescence recovery after photobleaching (FRAP), and fluorescence correlation spectroscopy (FCS) to characterize the physical properties of Myo2 from fission yeast Schizosaccharomyces pombe. By electron microscopy, Myo2 has two heads and a coiled-coiled tail like myosin-II from other species. The first 65 nm of the tail is a stiff rod, followed by a flexible, less-ordered region up to 30 nm long. Myo2 sediments as a 7 S molecule in high salt, but aggregates rather than forming minifilaments at lower salt concentrations; this is unaffected by heavy chain phosphorylation. We used FRAP and FCS to observe the dynamics of Myo2 in live S. pombe cells and in cell extracts at different salt concentrations; both show that Myo2 with an N-terminal mEGFP tag has a diffusion coefficient of ∼ 3 µm2 s-1 in the cytoplasm of live cells during interphase and mitosis. Photon counting histogram analysis of the FCS data confirmed that Myo2 diffuses as doubled-headed molecules in the cytoplasm. FCS measurements on diluted cell extracts showed that mEGFP-Myo2 has a diffusion coefficient of ∼ 30 µm2 s-1 in 50 to 400 mM KCl concentrations.

KEYWORDS:

contractile ring; electron microscopy; myosin-II

PMID:
29205883
PMCID:
PMC5899921
[Available on 2019-04-01]
DOI:
10.1002/cm.21425

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