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First published online 22 March 2005
doi: 10.1242/jcs.02302

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Microtubule nucleation and anchoring at the centrosome are independent processes linked by ninein function

Institut Curie, Research Section/UMR144 du Centre National de la Recherche Scientifique, 26 rue d'Ulm, 75248 Paris Cedex 05, France

View larger version (100K): [in a new window]   Fig. 1. The C-terminal domain of ninein associates with both centrioles. (A) L929 cells were transiently transfected with GFP-tagged ninein N-terminal domain, coiled-coil region and C-terminal domain (green). 2 hours after transfection, cells were fixed and centrioles labelled with either GT335 antibody or -tubulin antibody (red), demonstrating that only the C-terminal domain was targeted to centrioles. Bars, 10 µm (4 x magnification in insets). (B) L929 cells expressing GFP/Cter-ninein for 2 hours were fixed and labelled with an antibody to C-Nap1 (a), which decorates the proximal ends of both centrioles (cartoon) or to ninein (b), which decorates essentially the distal end of the mother centriole (cartoon). The GFP signals did not colocalize with the C-Nap1 labelling but colocalized with ninein to the mother centriole, suggesting that GFP/Cter-ninein might be associated with the distal end of both centrioles (same magnification as insets in A).

View larger version (41K): [in a new window]   Fig. 3. The C-terminal domain of ninein displaces endogenous ninein from the centrosome. L929 cells transfected with the GFP/Cter-ninein construct for 6 hours (A) showing a decrease in endogenous ninein at centrioles (arrowhead) compared with control cells (arrow), as judged by staining with an anti-ninein antibody. (B) Distribution of ninein labelling relative intensity at centrosomes in non-expressing cells (white bars), cells 2 hours after transfection (grey bars) and 6 hours after transfection (black bars). Expression of Cter-ninein resulted in a reduction in the level of endogenous ninein at the centrosome, on average, decreases of 42% at 2 hours and of 62% at 6 hours were observed (P<0.001 in both cases). Bars, 10 µm (4 x magnification in insets).

View larger version (91K): [in a new window]   Fig. 2. The ninein N-terminus/C-terminus chimerical fusion is concentrated on the mother centriole. (A) L929 cells were transiently transfected with a GFP-tagged construct encoding a ninein version lacking the coiled-coil domain (Nter/Cter-Nin, green). 2 hours after transfection, cells were fixed and centrioles labelled with either GT335 or anti--tubulin antibody (red). Representative images show colocalization of centriole markers with the ninein fusion at one centriole. Bars, 10 µm (4 x magnification in insets). (B) L929 cells expressing GFP/Nter/Cter-ninein for 2 hours were fixed and labelled with an antibody to C-Nap1 (a), which decorates the proximal ends of both centrioles (cartoon), or to ninein (b), which essentially decorates the distal end of the mother centriole (cartoon). The GFP signals did not colocalize with C-Nap1 labelling but colocalized with ninein to the mother centriole, showing that GFP/Nter/Cter-ninein is associated with the distal end of the mother centriole or of both centrioles, depending on the cell (C) (same magnification as insets in A). (C) Quantification of Nter/Cter-ninein localization in living cells at 2 hours and 6 hours after transfection. Most cells presented localization on one spot representing the mother centriole or on two spots representing both centrioles.

View larger version (37K): [in a new window]   Fig. 4. Effect of the displacement of ninein from the centrosome on microtubule regrowth and anchorage. HCT116 cells transiently transfected with GFP/Cter-ninein for 5 hours and further treated with nocodazole for 1 hour were labelled using anti--tubulin antibodies to visualize microtubules upon regrowth. (A) One minute after nocodazole washout, expressing cells had no (a, arrowhead) or few (b, arrowhead) microtubules (compare with the control cells, arrow). When few microtubules were observed, as in b, they concentrated around the centrosome. (c) Quantification of microtubules and aster formation in expressing and non-expressing cells after 1-minute nocodazole washout, corresponding to the experiment illustrated in (a,b). (B) Ten minutes after nocodazole washout, microtubules polymerized in expressing cells but, in contrast to control cells (arrow), no asters were observed (a, arrowhead). (b) Quantification of microtubules and aster formation in expressing and non-expressing cells after 10 minutes nocodazole washout, corresponding to the experiment illustrated in a. ND (not determined) indicates cells in which microtubules were present but formation of an aster could not be discerned. Bars, 10 µm (2 x magnification in insets).

View larger version (44K): [in a new window]   Fig. 5. Effect of the displacement of ninein on the localization of -TuRC components or -TuRC docking proteins. L929 (A,B) or Hela (C) cells transfected with GFP/Cter-ninein for 2 hours were fixed and labelled with different markers for -TuRC components (A) or for -TuRC docking proteins (B,C). (A) -Tubulin (a) and Spc98p (b) were displaced from the centrosome in expressing cells (arrowhead) compared with control cells (arrow). (B) Nlp (a) and pericentrin (b) were correctly localized to the centrosome in expressing cells (arrowhead) compared with control cells (arrow). (C) AKAP 450 was also present at the centrosome in expressing cells (arrowhead). Bars, 10 µm (the centrosome areas were magnified four times in insets).

View larger version (36K): [in a new window]   Fig. 6. Ninein interacts with -tubulin by its N-terminal domain. (A) GFP-ninein expressed for 2 hours in L929 cells recruited the microtubule-nucleation complex (arrowhead) as judged by -tubulin (a) or Spc98p (b) staining. The centrosome of a control cell is indicated by an arrow. Bars, 10 µm (the centrosome areas were magnified four times in insets). (B) Fusion constructs including the ninein N-terminal domain immunoprecipitated with myc/-tubulin. Extracts from L929 cells co-expressing myc/-tubulin and either one of the three different GFP-ninein constructs indicated were used for immunoprecipitation experiments with a polyclonal anti-GFP antibody. The weak band of -tubulin associated with immunoprecipitation of GFP/Cter-ninein is likely to be non-specific. (C) Two N-terminal domains (residues 1-246 and 1-496) of ninein were expressed as GST fusions and incubated with a lysate prepared from HeLa cells. -Tubulin was present in all of the ninein-GST pull-downs. A similar result was obtained for Spc98/GCP3.

View larger version (33K): [in a new window]   Fig. 7. The ninein N-terminus/C-terminus chimerical fusion displaces ninein but not -tubulin from the centrosome. (A) L929 cells expressing GFP Nter/Cter-ninein for 6 hours were fixed and immunolabelled with an anti--tubulin antibody (a), showing that, in expressing cells (arrowhead), -tubulin was not displaced from the centrosome compared with control cells (arrow). (b) Quantification of endogenous -tubulin labelling relative intensity at the centrosome in expressing (GFP/Nter/Cter-ninein 6 hours, black bars) and non-expressing (white bars) cells, corresponding to the experiment illustrated in a. (B) L929 cells expressing GFP/Nter/Cter-ninein for 6 hours were fixed and immunolabelled with an anti-ninein antibody (a), showing that, in expressing cells (arrowhead), ninein was displaced from the centrosome compared with control cells (arrow). (b) Quantification of endogenous ninein labelling relative intensity at the centrosome, in expressing (GFP/Nter/Cter-ninein 6 hours, black bars) and non-expressing (white bars) cells, corresponding to the experiment illustrated in a. In expressing cells, the relative intensity of ninein label decreased on average to 49% (P<0.001). Bars, 10 µm (4 x magnification in insets).

View larger version (34K): [in a new window]   Fig. 8. The ninein N-terminus/C-terminus chimerical fusion impairs microtubule anchoring but not microtubule nucleation. HCT116 cells transfected with GFP/Nter/Cter-ninein for 5 hours were further treated with nocodazole for 1 hour and analysed, using immunolabelling with anti--tubulin antibody to visualize microtubules upon regrowth. (A) One minute after nocodazole washout, expressing cells (a, arrowhead) formed asters comparable to those in control cells (arrow). (b) Quantification of microtubules and aster formation 1 minute after nocodazole washout, corresponding to the experiment illustrated in a. (B) Ten minutes after nocodazole washout, expressing cells (a, arrowhead) had no asters compared with control cells (arrow). (b) Quantification of microtubules and aster formation 10 minutes after nocodazole washout, corresponding to the experiment depicted in a. Bars, 10 µm.

View larger version (26K): [in a new window]   Fig. 9. Model for the role of ninein in microtubule organization by the centrosome. Microtubules are nucleated by the -TuRC, which is enriched at the centrosome. The newly nucleated microtubules could have different behaviours. (1) Microtubules are nucleated far from the subdistal appendages. They are transiently anchored by the -TuRC or released from the centrosome. (2) A proportion of the -TuRC is docked at the mother centriole by ninein. Microtubules nucleated by this -TuRC near or on the subdistal appendages would be quickly captured by the anchoring complex.