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Cell cycle-dependent control of polarised development by a cyclin-dependent kinase-like protein in the Fucus zygote

¹ Station Biologique, UMR 1931 CNRS and Laboratoires Goëmar, 29680 Roscoff, France
² Marine Biological Association, The Laboratory, Citadel Hill, Plymouth PL1 2PB, UK

View larger version (50K): [in a new window]   Fig. 1. (A-O) Time course of the first cell cycle and early development in Fucus spiralis zygotes. Zygotes were stained with mithramycin A at various times after fertilisation (AF): 1 hour (A); 3 hours, just before pronuclei fusion (B); 4 hours, after fusion (C); 14 hours, decondensed nuclei before germination (D,J); 16-17 hours, nuclear envelope breakdown and chromatin condensation (E,F); 18-20 hours, prophase (G), metaphase in a germinated cell (H,K), anaphase (I), telophase (L); 24 hours, cytokinesis (M). At 26 hours AF, embryo exhibiting two cells in metaphase (N). Note that the metaphase plate is transverse to the polar axis. (O) At 30 hours AF, subsequent parallel and transverse divisions in the rhizoid and thallus cells. Arrowheads in L-N indicate the germination site. Scale bar: 30 µm in A,B,N,O; 25 µm in K,M; 20 µm in C,E,J,L; 10 µm in H,I; 5 µm in D,F,G. (P) Temporal relationships between polarisation and G2 phase. Two representative experiments are shown (100 cells for each time point). The proportion of cells in late S or G2 phase (as determined by their ability to divide in the presence of aphidicolin), as well as the percentage of photopolarised cells (germination in response to a light vector) or those with a fixed axis (irreversible anchoring of polarity), are reported for each time point of development. (Q) Temporal relationships between cell cycle events and stages in zygote polarisation. The beginning of S phase occurs at 5-6 hours AF, as determined by measuring the expression of histone H3, a gene expressed at the onset of S phase. A peak of H1 kinase activity, which is representative of CDK activity, is detected at the time of mitosis (Corellou et al., 2000b). The purine derivative olomoucine inhibits cell cycle progression at the G1/S transition (preventing DNA replication and expression of histone H3) and at the G2/M transition, and through mitosis, by specifically inhibiting CDK-like proteins. Aphidicolin, by contrast, inhibits DNA replication but not the expression of histone H3. Whereas photopolarisation occurs before G2 phase, axis fixation begins in late S phase and ends in G2 phase in the majority of zygotes (as deduced from an analysis of 10 independent populations).

View larger version (92K): [in a new window]   Fig. 2. Effects of CDK inhibitors on early morphogenesis of Fucus zygotes. Treatments from 2 hours AF with either 100 µM olomoucine (A,E), 30 µM roscovitine (B,F) or 5 µM amino-purvalanol (C,G) prevent nuclear division, as revealed by staining DNA with mithramycin A (A-C), as well as the polar localisation of cell wall sulphated compounds, as detected by staining isolated walls with TBO (E-G). Arrowheads in A-C point to the decondensed nuclei. (D) Control embryo treated from 2 to 24 hours AF with 400 µM isoolomoucine. (H) Cell wall of 12-hour-old control zygotes exhibiting sulphated compounds localised at the future rhizoid pole. (I,J) Zygotes treated from 2 hours AF with either 20 µM aphidicolin or with lower doses of olomoucine (35 µM) germinated and elongated a rhizoid as observed at 36 hours AF. Note the presence of dispersed chromosomes after treatment with 35 µM olomoucine (arrow in J). (K) Two-week-old embryo recovering from a 28 hour-incubation with 100 µM olomoucine from 2 hours AF. Note that the multicellular embryo is is devoid of any rhizoids. By contrast, an apical hair (ah) is present. Scale bar: 25 µm in A-C; 30 µm in E-H; 40 µm in D,J; 50 µm in I; 80 µm in K.

View larger version (21K): [in a new window]   Fig. 3. Time-dependent effect of olomoucine and amino-purvalanol on germination. Olomoucine (100 µM) or amino-purvalanol (5 µM) were added at various times AF (as indicated on the x-axis) and the proportion of germinated zygotes was scored 36 hours later (red and white triangles, repectively). To determine the percentage of cells in G2 phase at the time of drug addition (red squares), aphidicolin (20 µM) was added at various times AF (x-axis) and the proportion of divided cells was scored 36 hours later. Photopolarisation (black circles) and axis fixation (white circles) are reported for each time point. This graph is representative of three independent experiments.

View larger version (45K): [in a new window]   Fig. 4. Effect of microinjection of anti-PSTAIRE antibody on germination. Injection of the anti-PSTAIRE antibody at 1 hour AF inhibited germination and cell division in 18 out of 20 zygotes, as determined 36 hours (black in F) to 48 hours later (zygote in A). By contrast, injections at 8 hours AF allowed 19 out of 25 zygotes to germinate in response to unidirectional light and to divide as determined at 36 hours (white in F) or 48 hours AF (zygote in B). Twenty out of 25 zygotes, injected at 1 hour AF with the same concentration of control ascite fluid germinated and divided, as determined at 30 hours (C), 36 hours (white in F) and 48 hours AF (D,E) though the division planes were sometimes misaligned (D) compared with controls injected with injection buffer only (E). Arrowheads point to the rhizoids. Scale bar: 40 µm in A,C,D,E; 50 µm in B.

View larger version (25K): [in a new window]   Fig. 5. Affinity purification of p34 on amino-purvanol-sepharose beads. The effect of amino-purvalanol was tested in vitro on the histone H1 kinase activity of extracts from cells arrested at the G1/S transition by a treatment with 100 µM olomoucine from 3 to 36 hours AF (black in A), or in mitosis by a treatment with 0.33 µM nocodazole from 3 to 36 hours AF (white in A). Methyl-amino-purvalanol (30 µM) was used as a control. (B) Although two PSTAIRE CDK-like proteins, p32 and p34, were bound to p9CKShs1 (p9CKShs1) in extracts from 48-hour-old embryos, only one (p34) was retained on amino-purvalanol-sepharose beads (APV), as detected with the anti-PSTAIRE antibody. Note that p34 could not be completely depleted from cells extracts using amino-purvalanol beads, as the supernatant still contained significant amounts of p34 that could be subsequently fixed on p9CKShs1 beads (Sup APV/p9CKShs1). Throughout the first cell cycle, from 2 to 10 hours AF, only the p34 PSTAIRE CDK-like protein was retained on amino-purvalanol beads but not on methyl-amino-purvalanol beads, as detected with the anti-PSTAIRE antibody. The data shown are representative of the results of three independent experiments.

View larger version (22K): [in a new window]   Fig. 6. Possible pathways for the control of early development by p34 in Fucus zygotes. (A) P34 positively controls both the entry in S phase and photopolarisation. Inhibition of p34 activity (in black) prevents both events. Alternatively (B), p34 controls the G1/S transition, which itself is required to activate polarisation events. (C) A third possibility is that cell cycle and polarisation belong to two independent pathways, but a checkpoint prevents polarisation when the cell cycle is arrested before S phase.