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First published online 20 November 2007
doi: 10.1242/jcs.015875

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A novel mitosis-specific dynamic actin structure in Dictyostelium cells

Department of Biology, Faculty of Science, Yamaguchi University, Yamaguchi 753-8512, Japan

View larger version (66K): [in this window] [in a new window]   Fig. 1. The dynamics of actin in myosin-null (HS1) cells during cell division and interphase. HS1 cells expressing GFP-actin were observed during cell division (A) and interphase (B) by TIRF microscopy. (A) During cell division, actin localized mainly at both polar pseudopods (arrowheads) and at the centers of each daughter cell (circles). The latter structures are referred to as MiDASes in the present study. (B) In interphase, actin localized mainly in pseudopods (arrowheads) at the leading edge of a migrating cell, but any large actin-containing structures such as MiDASes could not be observed. (C) Trajectories of centroids of two MiDASes observed until the completion of cytokinesis. The arrowhead indicates the original position, and the arrows indicate the final positions of each MiDAS. (D) Time-course of the area of MiDASes in both daughter halves. The arrow shows the timing of complete scission of the cell (410 seconds), showing that they disappeared shortly after the completion of cytokinesis. A movie (supplementary material Movie 1) is available, showing that MiDASes move into each daughter half. (E) Typical MiDASes (circles) in wild-type cells; they only appeared transiently. Bars, 10 µm. Note that, interestingly, the fluorescence of MiDASes alternated on each side in 20% of the cells examined (10/44; see supplementary material Fig. S1).

View larger version (118K): [in this window] [in a new window]   Fig. 2. Distribution of MiDASes, nuclei and centrosomes in multinucleated HS1 cells. Shown is a series of vertical optical sections of a representative multinucleated HS1 cell during cell division. To visualize F-actin and nuclei (A-G), HS1 cells expressing GFP-histone1 (green), which represents the position of nuclei, were fixed and stained with tetramethyl rhodamine-phalloidin (red). (H-N) The distribution of F-actin (red) and GFP–-tubulin (green). The optical sections are 1.5 µm in thickness and separated from each other by 0.6 µm (A-F,H-M). Panels G and N show 3D-reconstructed images from panels A-F and H-M, respectively. The positions of MiDASes (arrows) correspond to those of nuclei and centrosomes. Bars, 10 µm.

View larger version (102K): [in this window] [in a new window]   Fig. 3. Centrosomes and astral microtubules determine the position of MiDASes. (A) MiDASes and centrosomes were observed simultaneously in live HS1 cells expressing mCherry-actin and GFP–-tubulin. (B) Sequential images in the box of panel A. The centroid of the MiDAS (closed circle) relocated, following the movement of the centrosome (open circles). (C) Trajectories of a centrosome (green line) and a MiDAS (red line) observed for 99.9 seconds. Arrowheads indicate the original positions and black arrows indicate the final positions of the centrosome and the MiDAS, respectively. (D) TIRF microscopy of a typical dividing multinucleated cell possessing four MiDASes, showing the distal ends of microtubules (green) in the MiDAS regions (arrows). These images were captured by an exposure of 100 mseconds. (E) Sum of traces of microtubules (green) and outlines of MiDASes (red) acquired for 15 seconds, indicating that the distal ends of microtubules reaching to the ventral cell cortex are limited mainly to the MiDAS regions. Bars, 2 µm.

View larger version (135K): [in this window] [in a new window]   Fig. 4. Astral microtubules are required for the formation and maintenance of MiDASes. HS1 cells expressing GFP–-tubulin (A) or GFP-actin (B) were exposed to thiabendazole, an inhibitor of microtubules, under confocal microscopy. Shortly after the addition of thiabendazole, astral microtubules shortened (A), and MiDASes (arrowheads in B) gradually regressed in size and finally disappeared (0-65 seconds in panel B). When thiabendazole was removed by washing with buffer 65 seconds after addition of thiabendazole, MiDASes gradually reappeared underneath the centrosomes (105-160 seconds). The arrows indicate the restored MiDASes (160 seconds). Bars, 5 µm.

View larger version (72K): [in this window] [in a new window]   Fig. 5. Loss of MiDASes results in the failure of cytokinesis. Dividing HS1 cells expressing GFP-actin were observed by confocal microscopy. When one of the MiDASes (arrows) disappeared by accident, the other half of the cell resorbed this half, and eventually the cell failed to complete cytokinesis (A). The arrows indicate the position of the MiDAS in the resorbed half of the cell. (B) Time-course of area of left (filled squares) and right (filled triangles) halves, which are divided by a line in panel A. Note that the area of the resorbing half (open arrowhead) rapidly decreased after the disappearance of the MiDAS (closed arrowhead). Also, when the disappearance of MiDASes was artificially induced by treatment with thiabendazole, all of the cells (n=26 cells) failed to divide (C). Bars, 10 µm.

View larger version (143K): [in this window] [in a new window]   Fig. 6. Dividing cells attach to the substratum at MiDAS regions. A representative dividing multinucleated HS1 cell simultaneously observed by fluorescence microscopy and interference reflection microscopy (IRM). Left panels show fluorescence images of GFP-actin, and right panels show IRM images. (A) IRM images represent a darker tone (arrowheads) at MiDAS regions compared with that present at other ventral membrane regions. Therefore, the cell membrane at MiDAS regions is closer to the substratum than the other ventral cell membrane regions. (B,C) Cell bodies blown away by a jet of buffer from a pipette under confocal microscopy. Left panels show a half of dividing cells before blowing, and right panels show the same areas after blowing away the cell bodies, as revealed by fluorescence microscopy for GFP actin and phase-contrast microscopy. The figures are representative results from 20 cells. Note that only MiDAS regions remained attached to the substratum, indicating that dividing cells strongly attach to the substratum mainly at the MiDAS regions. Bar, 5 µm.

View larger version (93K): [in this window] [in a new window]   Fig. 7. Actin rapidly turns over in MiDASes. The fluorescence of a part of a MiDAS region (within black squares) was bleached in HS1 cells expressing GFP-actin (A). The fluorescence of the bleached region recovered shortly after photobleaching (B). The half-time of recovery was 2.15±0.89 seconds (mean ± s.d., n=17). (C,D) A high-magnification view of the fluorescence recovery experiments. (D) Images inside the square in panel C. After photobleaching, fluorescence appeared as individual small fluorescent dots (arrowheads) and finally formed an aggregate. Bar, 2 µm.

View larger version (132K): [in this window] [in a new window]   Fig. 8. MiDASes comprise dots of actin. HS1 cells expressing GFP-actin were observed by TIRF microscopy. In the early stage of mitosis, a nascent MiDAS (arrowhead) was formed by aggregation of many small actin dots (A). These small actin dots did not change their positions; they appeared and then disappeared at the same position (panel A, arrows). By contrast, during the final stage of cytokinesis, these actin dots in MiDASes gradually disappeared, and the size of the MiDAS decreased gradually (B). Bar, 2 µm.

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