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 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.

Figure 1

Electron micrographs of purified Myo2 prepared by rapid freezing on mica, fracturing, deep etching and rotary shadowing. A. Electron micrographs of rotary-shadowed Myo2 prepared under dephosphorylation conditions in 500 mM KCl, 10 mM imidazole pH 7.0. The first four molecules from the left end measure around 65 nm. The 8 molecules on the right have extensions beyond 65 nm varying in shape and orientation. Panel height is 150 nm. B. Anaglyph of electron micrographs of a field of rotary-shadowed Myo2 prepared under dephosphorylation conditions in 50 mM KCl, 2 mM MgCl₂, 10 mM imidazole pH 7.0. Scale bar, 100 nm. C. Distribution of Myo2p tail lengths. Myo2 purified under dephosphorylation conditions was prepared in 500 mM KCl, 10 mM imidazole pH 7.0.

Figure 2

FRAP of live cells.(A) Time series of images of cells from FRAP experiments. Each image is of 4 μm thick confocal section, including all the fluorescence in the cell. Left, cell expressing mEGFP; middle, interphase cell expressing mEGFP-Myo2; right, mitotic cell expressing mEGFP-Myo2. Red box indicates area of cells that was photobleached. (B) Averaged time courses of the recovery of fluorescence in the bleached zone (black lines): Left, mEGFP (n = 9 cells), middle, mEGFP-Myo2 in interphase (n = 8 cells) and right, mEGFP-Myo2 in mitotic phase (n = 8 cells). The grey areas show the standard error of the mean. Yellow lines are the time courses of recovery simulated by Vcell with diffusion coefficients of 22 μm²/s (mEGFP), 3.0 μm²/s (mEGFP-Myo2 in interphase) and 2.5 μm²/s (mEGFP-Myo2 in mitosis). Red lines are time courses of recovery calculated by Vcell using diffusion coefficients from FCS experiments. (C) Time course of fluorescence fluctuations in the contractile ring in a cell expressing mEGFP-Myo2, after photobleaching the tip of the cell. (D) Vcell model of time course of the recovery from photobleaching of mEGFP in the first frame (0.1 s). The second frame is time 0.2 s. The third frame is time 2.5 s. These images are sections through the mid plane of the cell. The pseudo-color scale gives the concentrations in μM used in the Vcell model based on the normalized fluorescence intensity in FRAP experiments.

Figure 3

Fluorescence correlation spectroscopy on mEGFP and mEGFP-Myo2 in live cells and cell extracts. (A) Cytoplasm of live cells: (□) cell expressing mEGFP; (○) interphase cell expressing mEGFP-Myo2; and (●) mitotic cell expressing mEGFP-Myo2. (B) Cell extracts. Asynchronous cells were lysed by bead beating at 4°C after mixing 1:1 with 10 mM imidazole pH 7.0, 50 to 400 mM KCl, 1 mM MgCl₂, 1 mM EGTA, 1% sucrose, 1 mM DTT, EDTA-free Protease Inhibitor tablet- cOmplete (catalog #11873580001, Roche) 1 tablet/50 ml buffer and 2 mM ATP. After clarification at 100,000 g for 20 min, samples of supernatant were diluted 10-fold in the same buffer; (□) mEGFP in cell lysate with 50 mM KCl; mEGFP-Myo2 in interphase cell lysates with (○) 50 mM KCl, and (●) 400mM KCl. Inset shows the autocorrelation curve for 10 nM Alexa 488 dye (Molecular Probes, Eugene) dissolved in dimethylformamide and diluted to calibrate the confocal volume. (C) Photon counting histograms of (□) a cell expressing mEGFP (left); (○) an interphase cell expressing mEGFP-Myo2 (middle); and (●) a mitotic cell expressing mEGFP-Myo2 (right). Symbols are the raw data. The black lines are curves fit to this data. (D) Cytosolic molecular brightness plotted as counts per second per molecule (CPSM) for 10 cells expressing mEGFP, 6 interphase cells expressing mEGFP-Myo2 and 7 mitotic cells expressing mEGFP-Myo2. Means ± SDs.