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Influence of the centrosome in cytokinesis of brown algae: polyspermic zygotes of Scytosiphon lomentaria (Scytosiphonales, Phaeophyceae)

Muroran Marine Station, Field Science Center for Northern Biosphere, Hokkaido University Muroran 051-0003, Japan

View larger version (44K): [in a new window]   Fig. 1. Immunofluorescence images of spindle formation in polyspermic zygotes of S. lomentaria. (A,C,E,G) DIC images; (B,D,F,H) merged images of immunolocalization of ß-tubulin (green), centrin (red) and DAPI staining (blue). (A,B) Just before mitosis. Two pairs of centrioles derived from male gametes locate at one side of the nucleus and duplicate there. Therefore, four anti-centrin-positive spots can be observed (arrow in B). (C,D) Migration of four centrosomes to the future mitotic poles. Arrowheads in D show anti-centrin-positive spots. (E,F) Tri-polar spindle is formed and one of the three poles has two centrosomes. Arrowheads in F show four anti-centrin positive spots. (G,H) Tri-polar spindle is formed. One of the four centrosomes is positioned away from the spindle. Arrowheads in H show four anti-centrin-positive spots.

View larger version (135K): [in a new window]   Fig. 2. Ultrastructural images of a multipolar spindle in a polyspermic zygote of S. lomentaria. Three sections are shown from the consecutive serial sections of the same cell. The cell is at the same point in mitosis as shown in Fig. 1H. Arrows show centrioles. (A) One pole of the tripolar spindle. Spindle MTs from around the centriole (arrow), elongate towards the chromosomes and become arranged at equators of the multipolar spindle. The nuclear envelope is almost intact, except at the spindle poles. (B) Another spindle pole (arrow). Golgi bodies (G) exit at nearby centrioles. (C) One centrosome is located away from the spindle. The Golgi body is close to the centrosome. The bottom arrow shows the same mitotic pole as in B.

View larger version (50K): [in a new window]   Fig. 3. Immunofluorescence images of cytokinesis in polyspermic zygotes of S. lomentaria. (A,C,E,G) DIC images; (B,D,F,H) merged images of immunolocalization of ß-tubulin (green), centrin (red) and DAPI staining (blue). (A,B) Anaphase of a multipolar spindle. Four daughter nuclei are formed. Arrowheads in B show four anti-centrin-positive spots. (C,D) Four daughter cells are produced. Each of the four daughter cells contains one nucleus and one centrosome. Arrows in C show cytokinetic planes and arrowheads in D show anti-centrin-positive spots. (E,F) Three daughter cells are produced. Each of the three daughter cells contains one nucleus. One of the three cells contains two centrosomes. These centrosomes are close to each other and function as one MTOC. Arrows in E show cytokinetic planes and arrowheads in F show anti-centrin-positive spots. (G,H) Four daughter cells are produced. Each of the four daughter cells contains one centrosome. The top cell has no nucleus. Arrows in G show cytokinetic planes and arrowheads in H show anti-centrin-positive spots.

View larger version (38K): [in a new window]   Fig. 4. Fluorescence images of chloroplasts and nuclei in polyspermic zygotes. Red shows autofluorescence of chlorophyll in chloroplasts and blue shows DNA staining with DAPI. (A) Before mitosis; there are three chloroplasts (C1-C3) and a pre-mitotic nucleus (N). (B) Metaphase. (C) Anaphase; three daughter chromosome masses are observed. Three chloroplasts do not show any change. (D) Four daughter cells are produced. Each of the cells has a nucleus and a chloroplast. (E) Three daughter nuclei are produced. One of the three chloroplasts (arrow) is constricted and there are two nuclei near it. There is no nucleus next to the top chloroplast (C1).

View larger version (135K): [in a new window]   Fig. 5. Ultrastuctural images of cytokinesis in a polyspermic zygote of S. lomentaria. These sections are shown from the consecutive serial sections of the same cell. (A) This polyspermic germinate has four nuclei after mitosis, and three nuclei (N) are observed in this section. Cell divisions (arrows) do not proceed simultaneously; cytokinesis proceeds further at the upper position than at the lower one. (B) Magnified image of the nascent membranous cell partition seen at the lower arrow in another section (A). Golgi-derived globular vesicles and flat tubular vesicles can be observed near the membranous cell partition. Note that one edge of the cell partition is connected to the plasma membrane. (C,D) The other cell division plane. Vesicle accumulation and fusion during cytokinesis progress add one chloroplast.

View larger version (43K): [in a new window]   Fig. 6. The patterns of spindle formation and subsequent cytokinesis in polyspermic zygotes of S. lomentaria after fertilization. (A-D) Fertilization and germination. (A) Two male gametes fuse to one female. (B) Just after fertilization, there are three chloroplasts and three pairs of centrioles. (C) Female centrioles disappear and two pairs of centrioles from male gametes remain. (D) Before mitosis, the two pairs of centrioles duplicate into four. (E-G) Spindle formation in polyspermic zygotes. (E) Tetra-polar spindle. (F) Tri-polar spindle. One of the three poles contains two pairs of centrioles. (G) Tri-polar spindle. One of the four pairs of centrioles locates away from spindle. (H-I) Cytokinesis in polyspermic zygotes. (H) Four daughter cells are produced. Each cell contains a nucleus, a centrosome and a chloroplast. (I) Three daughter cells are produced. Each cell contains a nucleus and a chloroplast. All cells have a centrosome, and one cell contains two centrosomes. At that time, two centrosomes adjoin and function as one MTOC. (J) Four daughter cells are produced. Each cell contains a centrosome and a chloroplast. Sometimes, a cell is produced without a nucleus. FC, centrioles derived from female gamete; FCh, chloroplast from female gamete; MC, centrioles derived from male gamete; MCh, chloroplast from male gamete.