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First published online 13 May 2003
doi: 10.1242/jcs.00497

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Centrin deficiency in Chlamydomonas causes defects in basal body replication, segregation and maturation

Department of Botany, University of Cologne, Gyrhofstr. 15, D-50931 Cologne, Germany

View larger version (66K): [in a new window]   Fig. 8. Centrin and bb development. (A) Isolated cytoskeletons of N41 stained with anti-centrin (a-c; pCen2) and anti-acetylated tubulin (a'-c'). a''-c'', merged images. Centrin was absent from probasal bodies (arrowheads in a'' and b''), which in some cells occurred in aberrant numbers (b). Arrowhead in c'', additional, nonflagellate bbs containing centrin emphasizing that bbs acquire centrin during their development. (B) Analysis of N41 isolated cytoskeletons with GT335 (a-d) and either monoclonal (BAS6.8; a',b') or polyclonal anti-centrin (pCen2; c',d'). Bar, 5 µm. Note the presence of bbs without or with only faint centrin staining. (C) Centrin content in flagellate and nonflagellate bbs of A7 or A10. Top, bbs stained with anti-centrin; adjustments of contrast and brightness were identical for all images. The rectangle shows the size of the box used for intensity measurements. Arrowheads, flagella; arrow, bald bb. Bottom, fluorescence intensities obtained by anti-centrin staining in arbitrary units. Note the reduced amount of centrin in bbs of A10, especially bald ones. Standard deviations and the number of measurements are indicated.

View larger version (55K): [in a new window]   Fig. 1. Expression of a sense-antisense hybrid construct induced centrin deficiency. (A) Design of the sense-antisense hybrid construct of centrin used to induce RNAi in C. reinhardtii. The positions and restrictions sites of the primers are indicated and +1 indicates ATG of the coding region. (B) Western blot of control (lane C) and the RNAi strains A7, A10 and N41. Membrane strips were stained with amido black or developed with anti-centrin (pCen1). (C) Anti-centrin (mAB6.8) staining of methanol-permeabilized control (a) and the centrin-RNAi strains. In the latter, centrin was concentrated at the bbs, and NBBCs were absent. In A10 (c) and especially N41 (d), numerous cells contained no centrin signal. (e) Overlay image of an isolated nucleus of A7 stained with DAPI (black) and centrin (white). Bar, 10 µm. (D) The average number of flagella () and centrin dots () was plotted against the amount of residual centrin as determined by western blotting. The data show that bb and flagellar development depend on centrin.

View larger version (83K): [in a new window]   Fig. 5. Over-expression of centrin-GFP disrupts the NBBCs. (A) Isolated cytoskeletons of control (a), Cen-GFP3 (b) and Cen-GFP8 (c) cells stained with anti-centrin (a'-c'), corresponding GFP images (phase contrast for the control) are shown in a-c. Over-expression of centrin-GFP disturbed the NBBCs (c), whereas moderate expression allowed incorporation into the proximal parts of the NBBCs and into the distal connecting fiber (arrow in b). Bar, 1 µm. (B) Dividing cells of Cen-GFP8 stained with anti-tubulin combined with either anti-centrin (a-c) or GT335 (d,e). (a-c) Small arrowheads, bb pairs; arrows, centrin-GFP clumps; large arrowheads, centrin/centrin-GFP located at the spindle poles. (d,e) Arrowheads, bb pairs detected by GT335 staining. Bar, 5 µm. (C) Phase contrast (a) and corresponding GFP image (a') of Cen-GFP8 cytoskeletons, which often disintegrated into basal apparatus (arrowhead) and cell nucleus (arrows). (D) Histogram showing the frequency of intact (solid bars) and defect (open bars) cytoskeletons. Data were averaged from three independent experiments. Cytoskeletons of the centrin-RNAi strain A7 and the centrin-GFP-overexpressing strain GFP8 were less stable.

View larger version (52K): [in a new window]   Fig. 2. Stability of centrin-RNAi in strain N41. (A) The average flagellar number per cell is plotted against the time in days after transformation. Data were recorded from cultures grown continuously in liquid medium (1, ), or inoculated from stock cultures of the same strain maintained in solid medium after 152 (2, ) and 204 days (3, ). The points of time for western blotting (WB), indirect immunofluorescence (IF) and embedding for EM are indicated. Arrows on abscissa mark the start of different cultures in liquid medium. (B) Western blot corresponding to the experiment shown in A comparing the amount of centrin in control cells (lane C) and the three cultures of N41. (C) Indirect immunofluorescence corresponding to A and B using anti-centrin on control cells and on three cultures of N41. Note the decrease in the intensity of centrin-RNAi with time as indicated for example by the presence of centrin on the nucleus and residual NBBCs in N41-1. Accordingly, increased amounts of residual centrin were detected in the corresponding western blot and the average number of flagella per cell was close to wild-type. By contrast, the centrin-RNAi effect was still strong in N41-3, a culture retrieved from slowly growing stocks about two weeks prior to analysis.

View larger version (97K): [in a new window]   Fig. 3. Centrin-RNAi cells contained additional, nonflagellate basal bodies. (A) Antibodies to centrin (a-c), polyglutamylated (e) and acetylated tubulin (f) were used to demonstrate the presence of nonflagellate bbs in A10 (a-c) and N41 (e,f). a'-c', corresponding anti--tubulin images. Arrowheads, bbs without attached flagella. GT335 also stained the cell nucleus because nucleosome assembly proteins are polyglutamylated in addition to bb tubulin (Regnard et al., 2000). Bar, 5 µm. (B) EM analysis of wild-type (a) or N41 bbs (b-e) in longitudinal section. (a) Wild-type bb with transition region (arrowhead) and dCF (arrow). (b-e) bbs of N41 showing extremely short (b) or no flagella (c-e). The transition region, in longitudinal section usually appearing as an H-like structure, was defective in N41 (arrowhead in b). Note that bbs in N41 are improperly docked to the plasma membrane (c,d) or reside within the cytoplasma (e), and sometimes contained electron-dense material in the lumen (arrowhead in c). Arrowhead in d, basal feet. Arrowhead in e, attached microtubules. Bar, 250 nm. (C) Wild-type (a) and N41 bbs in cross-section. The stellate structure (a) was absent in the centrin-RNAi strains (b), causing the central pair microtubules to penetrate the bb. Bar, 200 nm.

View larger version (97K): [in a new window]   Fig. 6. Aberrant numbers of bbs disturbed the microtubular cytoskeleton. (A) Indirect immunofluorescence of control (a-c), N41 (d) and A10 cells (e-g) using antibodies to -tubulin (a,e'-g'), acetylated (c,d) and polyglutamylated tubulin (b,g). (e,f) Corresponding anti-centrin staining. In control cells, microtubules are focused onto the flagellar apparatus (a), which contains two polyglutamylated bbs (b) and four acetylated microtubular bundles (arrows in c). Arrowheads, axonemes. (d) Methanol-permeabilized N41 cells showing bbs (cell 1 and 2) with an incomplete set of aceylated microtubules (2). Some cells showed only a few acetylated microtubules (cell 3), or no staining (cell 4). (e) Additional bbs were often surrounded by microtubules (arrowhead; compare also g and Fig. 3A,c). (f) Cells without dot-like centrin signals often showed dispersed microtubules. (g) Cell with three bbs (arrowheads) as recognized by GT335 and two microtubular asters (g'). Bars, 5 µm. (B, a) Strain D5 expressing GFP-SFA. (b-e) Strain D5 after induction of centrin-RNAi. Cells were stained for centrin (displayed in white color, not shown in a); GFP-SFA is shown in black. Note the defects in the usually cruciated SFA fibers in cells with bbs of low centrin content (arrows in b) and in cells with too many (c,d) or without (e) bbs. Bar, 5 µm.

View larger version (76K): [in a new window]   Fig. 4. Centrin deficiency caused mistakes during bb segregation. (A) Histograms showing the distribution of flagella (solid bars) and centrin dots (open bars) in control and A10 cells. Note the discrepancy between the numbers of centrin dots (presumably bbs) and that of flagella in the centrin-RNAi strain. (B) Analysis of mitotic and dividing cells from control (a-c), F11 (d-g), A10 (h-k) and N41 (l,m). Detergent-extracted cells (a-j) or methanol-permeabilzed cells (k-m) were stained with anti--tubulin (not in l,m) and anti-centrin (BAS6.8); cell nuclei were stained with DAPI. While centrin and the bbs were located at the spindle poles of wild-type cells (arrowheads in a), bbs were detached from the poles in centrin-deficient cells. Frequently, mistakes in bb segregation were observed in mitotic or dividing cells (arrowhead in g,i-m). Arrows, metaphase band microtubules; arrowhead in j, spindle without bbs; arrowheads in m, dividing cells; l, phase contrast. Bars, 5 µm (in j for a-j).

View larger version (63K): [in a new window]   Fig. 7. Centrin deficiency increased the number of multinucleated cells. (a) N41 cell with three pyrenoids (arrowheads). (b) Phase contrast of a detergent-permeabilized N41 cell stained with Syto25 (Molecular Probes, Leiden, NL) revealing three nuclei (c). Bars, 5 µm (a), 10 µm (b,c). (d) Histogram showing the frequency of multinucleated cells in control, A10 and N41.