Fission yeast Myo2: Molecular organization and diffusion in the cytoplasm

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 µm s 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 µm s in 50 to 400 mM KCl concentrations.

Comparative Analysis of the Roles of Non-muscle Myosin-IIs in Cytokinesis in Budding Yeast, Fission Yeast, and Mammalian Cells

“…This is consistent with its unipolar organization in the cytokinesis nodes, as revealed by quantitative high-speed fluorescence photoactivation localization microscopy (FPALM) ( Figure 3B; Laplante et al, 2016). In addition, cytosolic Myo2 in interphase cells also exists as a non-filament form (Friend et al, 2018).…”

“…This was validated by rotary-shadowing EM showing that Myo2 forms a two-headed structure with an 85-90 nm rod tail Pollard et al, 2017;Friend et al, 2018). In contrast to myosin-IIs in animals and amoebas, purified Myo2 does not form filaments or mini-filaments under a wide range of salt concentrations, including physiological concentration (Pollard et al, 2017;Friend et al, 2018). This is consistent with its unipolar organization in the cytokinesis nodes, as revealed by quantitative high-speed fluorescence photoactivation localization microscopy (FPALM) ( Figure 3B; Laplante et al, 2016).…”

“…Myo2 has a short tail with 711 residues, which is predicted to contain α-helical rod followed by a flexible, less-ordered region due to the presence of proline residues within the last ∼150 residues (Figures 1, 3A). This was validated by rotary-shadowing EM showing that Myo2 forms a two-headed structure with an 85-90 nm rod tail Pollard et al, 2017;Friend et al, 2018). In contrast to myosin-IIs in animals and amoebas, purified Myo2 does not form filaments or mini-filaments under a wide range of salt concentrations, including physiological concentration (Pollard et al, 2017;Friend et al, 2018).…”

Molecular form and function of the cytokinetic ring

“…Unlike animal and amoeboid myosin-II, the two myosin-II heavy chains in S. pombe, Myo2 and Myp2, do not form filaments. The non-essential Myp2 is single-headed (Bezanilla and Pollard, 2000) whereas biochemical studies of the essential Myo2 indicate that it functions as a double-headed motor rather than a bipolar minifilament (Pollard et al, 2017;Friend et al, 2018) (Fig. 2A).…”

“…Under molecular crowding conditions, reconstitution of SCPR model of CR formation using 1 µm beads coated with FH1-FH2 fragments or myosin-II. Myosin-II is ∼90 nm (Friend et al, 2018) and is presented at ∼100 times its size relative to the bead. FH1-FH2 fragments are presented at ∼200× their size relative to the bead.…”