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Three-dimensional patterns and redistribution of myosin II and actin in mitotic Dictyostelium cells - PubMed

  • ️Wed Jan 01 1997

Three-dimensional patterns and redistribution of myosin II and actin in mitotic Dictyostelium cells

R Neujahr et al. J Cell Biol. 1997.

Abstract

Myosin II is not essential for cytokinesis in cells of Dictyostelium discoideum that are anchored on a substrate (Neujahr, R., C. Heizer, and G. Gerisch. 1997. J. Cell Sci. 110:123-137), in contrast to its importance for cell division in suspension (DeLozanne, A., and J.A. Spudich. 1987. Science. 236:1086-1091; Knecht, D.A., and W.F. Loomis. 1987. Science. 236: 1081-1085.). These differences have prompted us to investigate the three-dimensional distribution of myosin II in cells dividing under one of three conditions: (a) in shaken suspension, (b) in a fluid layer on a solid substrate surface, and (c) under mechanical stress applied by compressing the cells. Under the first and second conditions outlined above, myosin II does not form patterns that suggest a contractile ring is established in the furrow. Most of the myosin II is concentrated in the regions that flank the furrow on both sides towards the poles of the dividing cell. It is only when cells are compressed that myosin II extensively accumulates in the cleavage furrow, as has been previously described (Fukui, Y., T.J. Lynch, H. Brzeska, and E.D. Korn. 1989. Nature. 341:328-331), i.e., this massive accumulation is a response to the mechanical stress. Evidence is provided that the stress-associated translocation of myosin II to the cell cortex is a result of the dephosphorylation of its heavy chains. F-actin is localized in the dividing cells in a distinctly different pattern from that of myosin II. The F-actin is shown to accumulate primarily in protrusions at the two poles that ultimately form the leading edges of the daughter cells. This distribution changes dynamically as visualized in living cells with a green fluorescent protein-actin fusion.

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Figures

Figure 8
Figure 8

Distribution of F-actin in cells dividing in a fluid layer on a glass surface (A) or under an agar overlay (B). The microtubule antibody label in A1 and the GFP–tubulin label in B1 indicate the cells were in telophase. A2–A5 and B2–B5 show TRITC-phalloidin labeling in a series of confocal sections. Distances from the substrate are indicated in micrometers. Three-dimensional distributions of F-actin are shown in 6–9. These images are constructed in parallel to those of Figs. 3 and 4, which show the distribution of myosin II. In the uncompressed cell (A) as well as the compressed one (B) F-actin is most strongly enriched in polar regions. Bars, 5 μm for 1–5. In the other panels, dimensions are given on the coordinates in micrometers.

Figure 3
Figure 3

Myosin II distribution at two early stages of cytokinesis on a glass surface. Fixed cells were labeled with antibodies for myosin II and α-tubulin as for Fig. 1. The α-tubulin label shown in A1 and B1 visualizes the microtubule asters and elongated spindle diagnostic of mitotic cells in late anaphase or telophase. (A2–A5 and B2–B5) show the myosin II label in series of confocal sections from bottom to top of the cells. Numbers are distances in micrometers from the substrate surface. The color code is from dark red (low fluorescence intensity) to light yellow (high intensity), as indicated on the linear scales. For A6–A9 and B6–B9, three-dimensional distributions of myosin II were derived from serial confocal sections. In A6 and B6, the cells are depicted as unopened, and only myosin II located in the cortical region of the cells is visualized, similar to Fig. 1. In A7 and B7, the cells are shown unroofed by a sagittal section, A8 and B8 are cross sections through the center of the cleavage furrow, and A9 and B9 are frontal sections through the midline of these cells. The boundaries of cross sections through the cleavage furrow are marked by a white outline. Fluorescence intensities in the sections are color-coded from dark blue to red as marked on the linear scales. Numbers at the left end of the scales indicate the extracellular background values represented in black. Intensity values >100 were only exceptionally obtained, and the red color at the right end of the bars was attributed to these voxels. For details of ray tracing and color coding, see Materials and Methods. Dimensions are given on the coordinates in micrometers. Bars, 5 μm.

Figure 4
Figure 4

Myosin II distribution at two late stages of cytokinesis on a glass surface. This figure is organized in the same way as Fig. 3. A1 and B1 show labeled α-tubulin, A2–A5 and B2–B5 show myosin II label in four confocal sections from bottom to top of the cells. In A6–A9 and B6–B9, the distribution of myosin II is three-dimensionally reconstructed and visualized from the outside of the cells (A6 and B6) or with the cells optically sectioned in a sagittal plane (A7 and B7), through the cleavage furrow (A8 and B8), or frontally through their midline (A9 and B9). Bars, 5 μm.

Figure 1
Figure 1

Stages of cytokinesis (A–E) in suspended cells of Dictyostelium discoideum. Cells growing in shaken suspension with liquid medium were fixed and labeled with antibodies for myosin II and α-tubulin. Mitotic cleavage stages identified by their microtubule organization were subjected to confocal imaging. (Left) Three-dimensional reconstructions based on the immunolabel of myosin II. Since myosin II is distributed within the entire cytoplasmic space, it could be used to visualize the shape of the dividing cells. Low to high concentrations of myosin II in the cell cortex are color-coded from dark blue to red. It should be noted that the ray tracing technique used reflects primarily the distribution of myosin II in those cortical regions of the cells that are faced towards the observer. In other regions of the three-dimensional images, the colors are shifted towards blue. For details of the imaging technique applied we refer to Materials and Methods. (Right) Cell shape as revealed by differential interference contrast microscopy. The dark spot in A is most likely an engulfed contaminating particle. What appears as an extension on the right in D is in fact another cell close to the dividing one. Bars, 10 μm.

Figure 7
Figure 7

Myosin II distribution at early (A and B) and late (C and D) stages of cytokinesis in compressed cells. Before fixation, the cells had been compressed by agar overlay for either 6 min (A and C) or 15 min (B and D). A1 and C1 show microtubule labeling to indicate the stage of cytokinesis. In A2–A5 and C2–C5, and B1–B5 and D1–D5, myosin II label is shown in series of confocal sections. Numbers in these panels indicate distances in micrometers from the substrate surface. The three-dimensional myosin II distributions in 6–9 are reconstructed in a way comparable to those of the uncompressed cells shown in Figs. 3 and 4. Bars, 5 μm for 1–5. In the other panels, dimensions are given in micrometers on the coordinates.

Figure 2
Figure 2

Shape changes of substrate attached D. discoideum cells. (A) Scanning electron micrographs of an early (left) and late (right) cleavage stage. Formation of the cleavage furrow from the top and lateral surfaces of the cells is recognizable as well as anchorage of the cells on the substrate by extensions of their polar regions. (B) Pairs of phase-contrast (left) and RICM images (right) of a cell undergoing cytokinesis. The numbers are seconds required for the cell to proceed from a rounded state to the end of cleavage. The transit from initiation of the furrow (between 40 and 70 s) to the final stage of cleavage takes only 2 min. The dark areas in the RICM images indicate that the cell has been in contact with the substrate not only with the extensions at its poles, but over most of its basal surface including the furrow region. Interference fringes at the 140- and 170-s stages indicate the detachment from the substrate of a strand that still connects the daughter cells. Bars, 10 μm.

Figure 5
Figure 5

Mechanical stress–induced translocation of myosin II in interphase (A) and during mitosis (B and C). In A, interphase cells were fixed at short intervals after compression to demonstrate redistribution of myosin II in the flattened cells, thereby excluding influences of optical conditions that vary between uncompressed or compressed cells. For B and C, cells attached to a glass surface were incubated under a layer of fluid (left) as for Figs. 3 and 4 or were compressed under an agar layer for 6 min (middle) or 15 min (right). After these treatments, cells were fixed and antibody labeled for myosin II. Interphase cells (A) and examples of early (B) and late (C) stages of cytokinesis are shown in phase contrast (upper panels) and immunofluorescence images (lower panels). Bar, 5 μm.

Figure 6
Figure 6

Phosphorylation dependence of the mechanical stress–induced translocation of myosin II. Wild-type AX2 cells (left) are compared with cells producing myosin II heavy chains in which three phosphorylatable threonine residues are replaced by alanine (right). For both strains, interphase cells are shown either uncompressed or compressed for 15 min by an agar overlay. The cells were either fixed by the picric acid/formaldehyde standard procedure used in the present paper (A) or in cold formaldehyde-acetone (B) (Egelhoff et al., 1991). The fixed cells were labeled with anti–myosin II antibody and viewed by conventional fluorescence microscopy. Bar, 10 μm.

Figure 9
Figure 9

Myosin II (A) and actin (B and C) dynamics visualized by GFP fusion proteins during cytokinesis of cells compressed by agar overlay. The time series of A reveals translocation of cortical myosin II into the cleavage furrow. The series of B and C exemplify changes in the distribution of GFP–actin in two cells dividing under an agar overlay. Fluorescence intensities were recorded in the confocal mode. For the cells shown in A and B, fluorescence images (top) are shown in parallel with phase-contrast images (bottom). To avoid sensitization by fluorescent compounds taken up from nutrient medium, cells were cultivated on bacteria. On the bottom of each frame, times after beginning of the record are given in minutes and seconds. Bars, 5 μm.

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