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Asymmetric cell division in fucoid algae: a role for cortical adhesions in alignment of the mitotic apparatus

University of Utah, Department of Biology, 257 South 1400 East, Salt Lake City, UT 84112-0840, USA

View larger version (37K): [in a new window]   Fig. 1. Centrosomal alignment (A,B) and distances between the centrosomes (C) in zygotes progressing into and through mitosis. (A) Fifteen populations were developmentally staged by assaying the percent of zygotes at or past metaphase; the alignment angles were measured on zygotes stained with anti--tubulin antibodies and the angle between the cell plate and the growth axis was measured on living embryos stained with FM® 4-64. Each symbol designates a different population and data points are the mean angles±s.e.m. in each population. (B) Distribution of alignment angles in one population of zygotes (A, grey circles). Each bar represents the mean from three timepoints±s.d. (C) Distances between centrosomes; bars indicate the mean of three populations±s.d.

View larger version (77K): [in a new window]   Fig. 2. Microtubule arrays during centrosomal alignment. (A-D) A projection of confocal sections representing a section 10-20 µm thick in the midplane of zygotes. The zygotes are positioned with rhizoids towards the bottom of the page. (A,B) Prior to spindle formation, microtubules extended from the centrosomes to the cell cortex, and were concentrated in the rhizoid. (C) Metaphase spindles had short astral microtubules. (D) At telophase, microtubules again reached the cell cortices at both rhizoid and thallus poles. (E-G) Projections of confocal images from the outermost 1.5 µm of the thallus cortex in premetaphase (E), metaphase (F) and telophase (G). Arrows indicate cortical microtubules parallel to, and just beneath, the plasma membrane and arrowheads indicate microtubules that terminate at right angles to the surface. Scale bar in A also applies to B-D; scale bar in E also applies to F and G.

View larger version (33K): [in a new window]   Fig. 3. Effects of low concentrations of oryzalin (0.1 or 0.3 µM) or paclitaxel (1 µM). Treatments were initiated 16-17 hours AF. (A) Centrosomal alignment angles were measured on zygotes prior to treatment and during mitosis. (B) Distances between centrosomes at telophase. Data points in A and bars in B are the means±s.d. of two or three experiments. (C-F) Microtubule arrays in 0.3 µM oryzalin (C), 0.1 uM oryzalin (D), or 1 uM paclitaxel (E,F).

View larger version (26K): [in a new window]   Fig. 4. Centrosomal alignment (A) and separation (B) in latrunculin B. Either 30 or 100 nM latrunculin B was applied to zygotes 16-17 hours AF and samples of latrunculin-treated and controls were taken 4 hours later. Data points and bars are the means±s.d. of two experiments.

View larger version (25K): [in a new window]   Fig. 5. Plasmolysis disrupts premetaphase alignment. Treated zygotes were transferred to ASW containing either 0.6 or 0.7 M sucrose at 15 hours AF. (A) Alignment angles. Data points are the means±s.d. of three experiments. (B) The distance between the centrosomes at telophase; bars are the means±s.d. of two experiments.

View larger version (25K): [in a new window]   Fig. 6. Centrosomal alignment and separation in Brefeldin A. (A) Centrosomal alignment angles were measured before transfer to Brefeldin A (at 15.5 hours AF) and again during mitosis (21.5 hours AF) on either untreated zygotes or on zygotes incubated in 5 µg/ml Brefeldin A. Data points are the means±s.d. of two experiments. (B) The distances between centrosomes at telophase were measured on zygotes cultured in 5 µg/ml Brefeldin A from 9 hours AF and bars indicate the means±s.d of three experiments.

View larger version (25K): [in a new window]   Fig. 7. Effects of externally applied proteases. Treated zygotes were incubated in 0.1% (w/v) thermolysin and 0.1% (w/v) proteinase K beginning 15-17 hours AF. (A) Centrosomal alignment angles were measured on zygotes prior to treatment and during mitosis. (B) Distances between centrosomes at telophase. Data points in A and bars in B are the means±s.d of three experiments. Premetaphase (C) and telophase (D) microtubule arrays in protease-treated zygotes. Scale bar in C also applies to D.

View larger version (28K): [in a new window]   Fig. 8. Inhibition of cell wall deposition. Zygotes were transferred to ASW containing isoxaben and/or low sulfate ASW at 0-1 hours AF. (A) Centrosomal alignments in 10 µM isoxaben; (B) low sulfate ASW±10 µM isoxaben. Data points are the means±s.d. of three experiments.

View larger version (42K): [in a new window]   Fig. 9. Centrosomal alignment in olomoucine. (A) Alignment angles were measured on four populations of either untreated zygotes progressing through mitosis or zygotes incubated in 35 µM olomoucine beginning 5 hours AF. In two of the populations, zygotes were released from olomoucine 24 hours AF and alignments of the centrosomal axes were measured on premetaphase and metaphase zygotes. Data points are means±s.d. (B) An elongated nucleus in an untreated zygote 16 hours AF. (C,D) Nuclei in olomoucine-treated zygotes 25.5 hours AF are also elongated. (E) An oval nucleus in a zygote cultured in olomoucine from 5 to 15.5 hours AF and then olomoucine and 30 nM latrunculin B from 15.5 hours AF until 39.5 hours AF. Scale bar in B also applies to C-E.