QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online June 8, 2005
doi: 10.1242/10.1242/jcs.02353

This Article Summary Full Text Full Text (PDF) Supplementary Material Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Corellou, F. Articles by Brownlee, C. Search for Related Content PubMed PubMed Citation Articles by Corellou, F. Articles by Brownlee, C. Social Bookmarking                         What's this?

Spatial re-organisation of cortical microtubules in vivo during polarisation and asymmetric division of Fucus zygotes

Florence Corellou², Susana M. B. Coelho^2,*, François-Yves Bouget² and Colin Brownlee^1,

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

View larger version (79K): [in a new window]   Fig. 1. Specificity of incorporation of fluorescent tubulin in different MT arrays of unpolarised zygotes. Zygotes are positioned with the light coming from the top of the page and were microinjected on the left side. (A) Oospheres 4 hours after injection (1 optical section). (B) Cortical area of a 5 hours zygote (projection of the 2 upper optical sections): MTs are homogeneously distributed and randomly oriented. (C) One 15 µm deep section shows the cytoplasmic MT array in a 6-hour-old zygote. (D) Projection of section from 30 to 38 µm deep nuclear MT array in a 6-hour-old zygote. (E-L). Effect of nocodazole on MT arrays. (E-H) 7-hour-old zygote before and (I-L) after 2 hours incubation with nocodazole. (E,I) Cortical MTs showing only a few MTs and fluorescent foci (free non incorporated tubulin) remaining after nocodazole treatment. (F) Cytoplasmic region (projection from 7.5 to 27 µm deep inside the zygote) showing a dense cortical MT array at the periphery of the zygote. After incubation with nocodazole (J) very few and short MTs remain. (G,K) Nuclear region (projection from 28.5 to 33 µm deep inside the zygote). (G) The tubulin has not been well incorporated in the nuclear area, which is only faintly labelled. (H,L) Nuclear area from another zygote before (H) and after (L) nocodazole incubation. The bright patches on the left of zygotes in A, E, I, correspond to unincorporated tubulin on the zygote surface near the injection site. Bar, 15 µm.

View larger version (103K): [in a new window]   Fig. 2. Time course of cortical MT array reorganisation in polarised zygotes. (A-H) Polarisation, germination and mitosis entry in the same zygote. Light direction is from the left. (A-E) All sections are projected to reflect the shape of the zygote. (A) 17 hours (B) 19 hours, (C) 21 hours and (D) 23 hours AF showing progressive localisation of cortical MTs towards the rhizoid pole (projection of all sections). (E) 26 hours AF (projection of all sections). The rhizoid has germinated and long cortical MTs are absent in the zygotes with a prophase spindle but bright tubulin structures remain at the rhizoid collar. (F-H) Different areas of the same 26-hour-old zygote showing that tubulin structures at the collar start at the cortex and go deep inside the cytoplasm: (F) Cortical area through the upper 10 µm of the same zygote. Bright tubulin structures are localised as a collar around the rhizoid subapex. (G) Cytoplasmic area (11-22 µm) and (H) nuclear area (32-37 µm) with the prophase spindle. (I-N) MT reorganisation during rhizoid elongation (projection of all sections). (I) 17 hours AF with cortical MTs localised to the rhizoid hemisphere. (J,K,L) 19, 22 and 24 hours AF showing rhizoid emergence and the parallel arrangement of MTs to the rhizoid axis. (M,N) 25 and 27 hours AF: MTs depolymerise from the rhizoid tip to bright cortical star-shaped structures located at the collar. (O-Q) Projection of all sections during rhizoid germination and growth. (O) 16 hours AF showing convergence of MTs towards the site of rhizoid emergence. (P) 20 hours AF: MT bundles form a crescent at the periphery of the growing rhizoid. (Q) 27 hours AF showing disappearance of MTs from the rhizoid apex and MT bundles parallel to the growth axis extending from the rhizoid collar to a region below the apex. (R-U) Cortical network disappearance during mitosis and reformation after mitosis (projection of all sections). In this zygote rhizoid germination occurred downwards out of the horizontal scanning plane but on the right side of the zygote. (R) 16-hour-old zygote. Cortical MTs are more localised to the rhizoid hemisphere and the nuclear envelope is labelled. (S) 20 hours AF. Cortical MTs are more focused to the rhizoid and all cortical MTs are shortening. (T) Aster nucleation begins 26 hours AF showing mitosis entry. Both asters are detected in the cell centre. Only cortical star-shaped structures remain at the cortex (arrows). (U) 38-hour-old zygote showing reformation of the cortical network, particularly at the rhizoid pole. Bar, 15 µm.

View larger version (86K): [in a new window]   Fig. 3. Perinuclear MT re-organisation in zygotes having localised cortical MTs. Four representative zygotes are shown (A-C), (D-F), (G-I), (J-L). (A,D,G,J) Nuclear region (projection of 5-6 sections) and (B,E,H) cortical region (projection of all sections) of 10-hour-old zygotes. In all zygotes cortical MTs are located at the hemisphere where germination will occur. (C,F,I) Transmitted images of germinated zygotes at 28 hours AF. (A) Perinuclear MTs are homogeneously nucleated from the nuclear envelope and thicker MTs extend toward the cortex of the future rhizoid. Two non-separated centrosomes are detected (arrow). (D) Centrosomes (arrows), almost aligned with the growth axis start to nucleate MTs. (G) MT nucleation occurs from centrosomes that are aligned with the growth axis. (J) MTs radiating in opposite directions from both centrosomes (aligned with the growth axis) towards the cortex. (K) Nuclear area of the same zygote 25 hours AF. Only MTs connected to the rhizoid site remain and the nucleus is pulled toward that site in the germinated zygote. (L) Projection of all sections of the same zygote. The bright patches in H and L correspond to unincorprated tubulin aggregate on the zygote surface at the site of injection. Bar, 15 µm.

View larger version (130K): [in a new window]   Fig. 4. MT behaviour during mitosis entry. Upper panel (A-H): Mitosis entry and spindle rotation. (A-C) 22 hours AF. (A) Projection of all sections. Nuclear MTs are nucleated at centrosomes and are connected to the cortex of the rhizoid collar and to the rhizoid tip. (B) Projection of 6 upper cortical sections. Cortical MTs are nucleated at the rhizoid collar and are oriented parallel to the growth axis. (C) Nuclear area (projection of 6 sections). One of the centrosomes is connected via two long MT bundles to peripheral MTs. (D) 23 hours AF, the centrosomal axis has rotated slightly. The rhizoid has elongated and the rhizoid MT array is nucleated from a cortical site at the rhizoid collar. (E) 26 hours AF: Prophase spindle and cortical MT structures at the cortex of the collar (projection of all sections). All other cortical MTs have disappeared. Outline of the zygote is shown white. (F,G,H) 30 hours AF, respectively, all sections, seven upper cortical sections and nuclear area (projection of 12 sections). The metaphase spindle has rotated to become almost aligned with the growth axis. Cortical MT structures are present at the rhizoid collar and the rhizoid apex (arrows). (I-L) Projection of all sections during mitosis exit: (I) telophase spindle at 22 hours AF. Spindle asters are separated by a thick MT bundle. (J) At 23 hours AF nuclear envelopes are reformed and MTs appear at the rhizoid apex. In the inset, the focus on one deep section shows faint MT connections between the new rhizoid nucleus and the cortex. (K) At 24 hours AF MTs extend from both nuclei toward the future division plane at the centre of the embryo while cortical MTs reappear in the rhizoid. (M) Different sections of the zygote shown in (K) sampled every 6 µm. In the upper section cortical MTs are detected in both the thallus and the rhizoid. Numerous fine MTs connect both nuclei and interdigitate at the centre of the embryo. (L) 26 hours: cytokinesis is achieved. In the rectangle contrast has been enhanced to show MTs organised in the spindle interzone at the division plane. MTs extend horizontally to the edge of the embryo and perpendicularly other nuclear MTs reach the same plane. Bar, 15 µm.

View larger version (119K): [in a new window]   Fig. 5. Mitosis in 2 cell embryos. (A,B) Interphase in a 2 cell embryo. Cortical MT arrays are reformed in both the thallus and the rhizoid. (A) Projection of all sections, (B) single equatorial optical section. MTs connect the thallus nucleus to the upper cell cortex. MT arrays in the rhizoid are detected in the same plane. Inset shows the rhizoid nucleus with two centrosomes that separate around the nucleus. (C,D) Mitosis entry in a 2 cell embryo: (C) A few cortical MTs probably connected with nuclei are seen in both the thallus and the rhizoid cell. (D) One nuclear section in the thallus. A bundle of MTs connects one centrosome to the injection site. The other centrosome is connected through MT bundles to the cortex of the embryo (see C). (E) The rhizoid nucleus is rotating. Both centrosomes separated around the nuclei are connected to the rhizoid apex. (F) 3 hours later cortical MTs are reduced to star shaped structures and metaphase spindles are seen in both the rhizoid and the thallus cells (G). Note that the thallus spindle is perpendicular to the growth axis whereas the spindle in the rhizoid is not yet aligned with this axis (projection of all sections). Bar, 15 µm.

View larger version (40K): [in a new window]   Fig. 6. Sensitivity of MTs to nocodazole in polarised zygotes. (A) MT array in a 10-hour-old polarised zygote before and after a 2 hour incubation with 0.33 µM nocodazole. Cortical MTs remain localised to the rhizoid pole. Most MTs have disappeared after nocodazole treatment and only a few cortical MTs remain at the rhizoid pole. MTs connecting the nucleus to the rhizoid tip have persisted after the treatment and the nucleus is displaced towards the rhizoid. In the bright field image of the same zygotes 26 hours AF nocodazole has been washed out and zygotes grow normally. (B) Susceptibility of nocodazole-treated embryos to hypo-osmotic shock. 14- and 24-hour-old zygotes were pre-incubated for 2 hours in nocodazole and then submitted to hypo osmotic shock (40% SW). Results show % of zygotes that burst in response to the hypo-osmotic shock (means of three replicates). (C) Long-term effect of nocodazole on embryo morphology. Zygotes were incubated continuously with nocodazole. 48-hour-old embryos remain undivided and exhibit large and broader rhizoids. Bar, 15 µm.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter