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. 2015;14(6):902-19.
doi: 10.1080/15384101.2014.1000197.

Centrosomal nucleolin is required for microtubule network organization

Xavier Gaume  1 , Anne-Marie Tassin, Iva Ugrinova, Fabien Mongelard, Karine Monier, Philippe Bouvet
Affiliations

Centrosomal nucleolin is required for microtubule network organization

Xavier Gaume et al. Cell Cycle. 2015.

Abstract

Nucleolin is a pleiotropic protein involved in a variety of cellular processes. Although multipolar spindle formation has been observed after nucleolin depletion, the roles of nucleolin in centrosome regulation and functions have not been addressed. Here we report using immunofluorescence and biochemically purified centrosomes that nucleolin co-localized only with one of the centrioles during interphase which was further identified as the mature centriole. Upon nucleolin depletion, cells exhibited an amplification of immature centriole markers surrounded by irregular pericentrin staining; these structures were exempt from maturation markers and unable to nucleate microtubules. Furthermore, the microtubule network was disorganized in these cells, exhibiting frequent non-centrosomal microtubules. At the mature centriole a reduced kinetics in the centrosomal microtubule nucleation phase was observed in live silenced cells, as well as a perturbation of microtubule anchoring. Immunoprecipitation experiments showed that nucleolin belongs to protein complexes containing 2 key centrosomal proteins, γ-tubulin and ninein, involved in microtubule nucleation and anchoring steps. Altogether, our study uncovered a new role for nucleolin in restricting microtubule nucleation and anchoring at centrosomes in interphase cells.

Keywords: centrosome; interphase; mature centriole; microtubules; nucleolin.

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Figures

Figure 1 (See previous page).
Figure 1 (See previous page).
Nucleolin decorates one of the 2 centrioles throughout interphase in U2OS-centrin-1-GFP cells. (A) Co-visualization of nucleolin, centrin-1-GFP (C1G) and nuclear counterstain (DAPI) shown as individual projections and as a 2- and 3-color merged projections. Nucleolin was detected with a monoclonal antibody and revealed with an Alexa555 secondary antibody [red] and centrin-1-GFP detection was enhanced with a GFP booster [green], while nuclei were counterstained with DAPI [cyan]. On the top left image, arrows show nucleoli and the centrosome area. Enlarged images of the centrosome area (boxed on the 3 color image) are shown as insets. Full size images and insets on the first row correspond to the projection of 5 consecutive optical sections centered on the focal plane, that have been previously submitted to a 3D constrained iterative deconvolution process. Insets on second row correspond to the individual optical sections of the centrosome area, separated by 0.2 μm steps, used to obtain the projection on the first row. Scale bars represent 5 μm on full size images and 1 μm on enlarged insets. (B) Co-visualization of nucleolin, γ-tubulin, centrin-1-GFP (C1G) and nuclear counterstain (DAPI) shown as individual projection and as a 3-color merged projection with nuclear outline. Nucleolin, centrin-1-GFP and DAPI were detected similarly as in A, while γ-tubulin was detected with a primary antibody directly coupled to TRITC [in white on the unmerged image and in blue on merged images]. Enlarged projections of the centrosome area (boxed on the 3 color image) are shown as 2-color insets. Scale bars represent 5 μm on full size images and 1 μm on enlarged insets. (C) Quantification of the number of centrioles (centrin-1-GFP label) positive for nucleolin per cell, for A and B experiments. Cells exhibiting 0/2, 1/2 and 2/2 centrioles labeled are reported as percentages of the number n of cells analyzed. ND stands for “not determinable” and mainly corresponds to cases where the centrosomal signal is superimposed to the strong nucleolar signal obtained with nucleolin. (D and E) Fluorescent intensity profiles, along a line (displayed on the 0.4 μm section of the z-stack gallery in A for D and on the 3-color inset in B for E) of an individual section of the centrosome area, obtained for nucleolin [NCL, red], centrin-1-GFP [C1G, green] and for E, γ-tubulin [blue]. The x axes represent the distance along the line in μm and the y axes correspond to fluorescence intensity in arbitrary units. (F and G) Co-visualization of nucleolin and centrin-1-GFP (C1G) shown as individual projections and as a 2-color merged projection in G1 and S cell cycle phases (F) and in G2 phase (G). Asynchronously growing cells were selected under the microscope thanks to the 4 centrin dots specific of G2 cells (see 2 arrows pointing to the 2 separated centrosomes on left image). Asynchronously growing cells were incubated with the nucleotide analog EdU for 30 min before fixation, detected with Alexa647 [white] and visualized as an individual projection and on the 4-color merged projection in F. Nucleolin, centrin-1-GFP and DAPI were detected similarly as in A. Enlarged projections of the centrosome area (boxed on the 2 color merged image) are presented in the insets. Scale bars represent 5 μm on full size images and 1 μm on enlarged insets. (H) Quantification of the number of centrioles (centrin-1-GFP label) positive for nucleolin per cell, for F and G experiments, in G1 (left), S (middle) or G2 (right) cell cycle phases. G1 and S cells exhibiting 0/2, 1/2 and 2/2 centrioles labeled are reported as percentages of the number n of cells analyzed. G2 cells exhibiting 0/4, 1/4 and more than 4 (>1/4) centrioles labeled are reported as percentages of the number n of cells analyzed. As in C, ND stands for “not determinable."
Figure 2 (See previous page).
Figure 2 (See previous page).
Nucleolin is stably bound to centrosomes independently of microtubules. (A–C) Nucleolin is present in pure centrosome preparations. (A and B) Fluorescent Western blots of cell lysates and biochemically purified centrosomes (pur. centr.) probed with a nucleolin monoclonal antibody (A), an anti γ-tubulin polyclonal antibody (B, upper), and an anti B23 monoclonal antibody (B, lower). Monoclonal antibodies were detected with a secondary antibody coupled to Alexa680, while the polyclonal antibody was detected with a secondary antibody coupled to IRdye800. C/ Co-visualization of nucleolin and γ-tubulin on purified centrosomes, derived from same experiment as in A and B. Purified centrosomes were spun down on coverslips, fixed in cold methanol and submitted to immunofluorescence using a nucleolin monoclonal antibody (detected with a secondary antibody coupled to Alexa488) [red] and an anti γ-tubulin antibody directly coupled to TRITC [green]. The corresponding DIC (Differential Interference Contrast) image is shown in gray. Scale bar represents 1 μm. (D–I) Nucleolin remains associated with centrosomes after nocodazole- (F and G) or cold- (I) induced microtubule depolymerization in U2OS-centrin-1-GFP cells. Co-visualization of nucleolin and centrin-1-GFP (D, F, H and I) or α-tubulin and centrin-1-GFP (E and G). Asynchronously growing cells were treated with nocodazole (F and G) or incubated at 4°C (I) for 3 hours before fixation. Control untreated cells are presented in (D, E and H). Centrin-1-GFP detection was enhanced with a GFP booster [green], nucleolin (D, F, H and I) and α-tubulin (E and G) were detected with a monoclonal antibody (detected with a secondary antibody coupled to Alexa555) [red], while in (D, F, H and I) nuclei were counterstained with DAPI [cyan]. In (D, F, H and I), enlarged images of the centrosome area (displayed on the 3 color merged images) are presented in the insets. In D and F, the DAPI images were not submitted to a 3D constrained iterative deconvolution process. Scale bars represent 5 μm on full size images and 1 μm on enlarged insets.
Figure 3 (See previous page).
Figure 3 (See previous page).
Non-random distribution of nucleolin toward the mature centriole. (A–C) Co-visualization of nucleolin and centrin-1-GFP (C1G) together with ninein (A), cenexin (B) or CEP164 (C) in U2OS-centrin-1-GFP cells. Enlarged images of the centrosome area (displayed on the 3 color merged images) are presented in the insets as 2 color merged images to facilitate colocalization visualization. (D, E) Co-visualization of nucleolin (NCL) and centrin-1-GFP (C1G) together with ninein (NIN, D) and with CEP164 (C164, E) in U2OS-centrin-1-GFP cells, exhibiting 4 centrin dots, characteristic of G2 cells. The arrow on the left image shows the centrosome area. Enlarged images of the centrosome area (displayed on the 3 color merged images) are presented in the upper insets for a single channel and in the lower insets as 2 color merged images to facilitate colocalization visualization. In A-E, nucleolin was detected with a monoclonal antibody (detected with a secondary antibody coupled to Alexa647) [red]; centrin-1-GFP detection was enhanced with a GFP booster [green], while ninein, cenexine and CEP164 were detected with polyclonal antibodies (detected with a secondary antibody coupled to Alexa555) [in white on the unmerged image and in blue on merged images]. Outline of the nuclei, counterstained with DAPI (not shown) is highlighted on the merged images. Scale bars represent 5 μm on full size images and 1 μm on enlarged insets. (F) Quantification of the number of centrioles (centrin-1-GFP label) positive for ninein [dark bars] and nucleolin [gray bars] per cell in G1/S cell cycle phase, from experiment in A. Cells exhibiting 0/2, 1/2 and 2/2 centrioles labeled for each marker are reported as percentages of the number n of cells studied. ND stands for not determinable and mainly corresponds to cases where the centrosomal signal of nucleolin is superimposed to the strong nucleolar signal. (G) Quantification of co-distribution between both centrosomal markers, recorded in F. The presence of ninein on nucleolin positive centrioles is shown on the left, while the presence of nucleolin on ninein positive centrioles is shown on the right, expressed as percentages of the number n of cells studied. Similarly as in F, ND stands for “Not Determinable." (H) Quantification of the number of centrioles (centrin-1-GFP label) positive for ninein [dark bars] and nucleolin [gray bars] per cell in G2 cell cycle phase, from experiment in D and E. Cells exhibiting 0/4, 1/4 and >1/4 centrioles labeled for each marker are reported as percentages of the number n of cells studied. As in F, ND stands for “Not Determinable." (I) Quantification of co-distribution between both centrosomal markers, recorded in H. The presence of ninein on nucleolin positive centrioles is shown on the left, while the presence of nucleolin on ninein positive centrioles is shown on the right, expressed as percentages of the number n of cells studied. As in F, ND stands for “Not Determinable."
Figure 4 (See previous page).
Figure 4 (See previous page).
Nucleolin depletion leads to amplification of immature centriole markers. (A–E) Presence of centrosome amplification in nucleolin-silenced cells during interphase. Co-visualization of γ-tubulin, centrin-1-GFP (C1G) and nuclear counterstain (DAPI) shown as individual projections and as a 2- and 3-color merged projections in untransfected control (A, B and C) or nucleolin siRNA transfected (D, E) U2OS-centrin-1-GFP cells. Cells were harvested 4 days after transfection. Centrin-1-GFP detection was enhanced with a GFP booster [green] and γ-tubulin was detected with a primary antibody directly coupled to TRITC [red]. Nuclei were counterstained with DAPI [cyan]. Asynchronously growing cells were selected under the microscope with reference to the number of centrin dots (2 dots for G1/S cells and 4 dots for G2 cells). Early G2 and late G2 control cells are shown in B and C respectively. Enlarged projections of the centrosome area (boxed on the 3-color image) are shown as insets. Scale bars represent 5μm on full size images and 1 μm on enlarged insets. (F, G) Nucleolin silencing leads to an amplification of pericentrin. Co-visualization of centrin-1-GFP (C1G) and pericentrin in untransfected control (F) or in nucleolin-silenced (G) U2OS-centrin-1-GFP cells. Cells were harvested 4 days after transfection. Centrin-1-GFP detection was enhanced with a GFP booster [green] and pericentrin was detected with a monoclonal antibody (detected with a secondary antibody coupled to Alexa555) [red]. Nuclei were counterstained with DAPI [cyan]. Enlarged images of the centrosome area (displayed on the 3 color merged images) are presented in the insets. Scale bars represent 5 μm on full size images and 1 μm on enlarged insets. (H, I) Nucleolin silencing leads to a specific amplification of centriole immature mark. Co-visualization of centrin-1-GFP (C1G) with ninein and centrobin in untransfected control (H) or in nucleolin-silenced (I) U2OS-centrin-1-GFP cells. Cells were harvested 4 days after transfection. Centrin-1-GFP detection was enhanced with a GFP booster [green], ninein was detected with a polyclonal antibody (detected with a secondary antibody coupled to Alexa 555) [red] and centrobin was detected with a monoclonal antibody (detected with a secondary antibody coupled to Alexa 647) [in white on the unmerged images and in blue on the merge images]. Nuclei were counterstained with DAPI [cyan]. Enlarged images of the centrosome area (displayed on the 3 color merged images) are presented in the insets. Scale bars represent 5 μm on full size images and 1 μm on enlarged insets. (J, K) Quantification of the number of centrin-1-GFP dots (C1G, J), and of γ-tubulin positive centrin-1-GFP dots (γ-tubulin +, K) per cell in untransfected control (Cont., left) or nucleolin siRNA transfected (NCL siRNA, right), for A-E experiments. Cells exhibiting 2 dots [dark bars], 3 or 4 dots [gray bars] and more than 4 dots (>4) [white bars] are expressed as percentages of the number n of cells studied. The error bars correspond to standard deviations calculated for 3 independent experiments. *: Significant difference with a p value < 0.01 with control populations (left columns). n: non-significant difference with a p value < 0.01 with control populations (left columns). (L, M) Quantification of the number of ninein positive centrin-1-GFP dots (ninein +, L) and of centrobin positive centrin-1-GFP dots (centrobin +, M) per cell in untransfected control (Cont., left) or nucleolin siRNA transfected (NCL siRNA, right), for H and I experiments. Cells are distributed in different classes according to their number of dots (see individual legends), whose values are expressed as percentages of the number n of cells studied. (N, O) Quantification of γ-tubulin and ninein at the centrosome in absence of nucleolin. γ-tubulin (N) or ninein (O) fluorescence intensity were measured at the centrosome in untransfected control or nucleolin siRNA transfected U2OS-centrin-1-GFP cells. Fluorescence intensities of γ-tubulin or ninein were measured on the centrin-1-GFP dots positive for γ-tubulin or ninein respectively. These intensities were normalized to that of centrin-1-GFP. For each condition, the mean value is reported on the graph with standard deviation error bars.
Figure 5.
Figure 5.
Nucleolin belongs to protein complexes containing centrosomal proteins. (A) γ-tubulin coimmunoprecipitates with nucleolin. Immunoprecipitation (IP) assay was performed in HeLa cells with anti nucleolin polyclonal antibody (polyclonal antibody 5567). Following immunoprecipitation, proteins were separated on a SDS-PAGE gel and probed with an anti nucleolin polyclonal antibody (polyclonal antibody 134, first row), detected with a secondary antibody coupled to IRdye800, and with anti γ-tubulin (second row) and B23 (third row) monoclonal antibodies, detected with a secondary antibody coupled to Alexa680. (B) Nucleolin coimmunoprecipitates with ninein. Immunoprecipitation (IP) assay was performed in HeLa cells with anti ninein monoclonal antibody. Following immunoprecipitation, proteins were separated on a SDS-PAGE gel and probed with an anti nucleolin polyclonal antibody (polyclonal antibody 5567, lower row), detected with a secondary antibody coupled to IRdye800, and with anti ninein monoclonal antibody (upper row), detected with a secondary antibody coupled to Alexa680. IP without antibody (No Ab) and preimmune serum (IgG) were used as control. Input: input pre-immunoprecipitation fractions; IP: immunoprecipitated proteins; Supernatant: supernatant after immunoprecipitation.
Figure 6.
Figure 6.
Microtubule network is disorganized in nucleolin depleted cells. (A) Co-visualization of α-tubulin, CEP164 and centrin-1-GFP (C1G) shown as individual projections and as 3-color merged projections in control or nucleolin siRNA transfected U2OS-centrin-1-GFP cells. Cells were harvested 4 days after transfection. Centrin-1-GFP detection was enhanced with a GFP booster [green], CEP164 was detected with a polyclonal antibody (detected with a secondary antibody coupled to Alexa647) [red] and α-tubulin was detected with a monoclonal antibody (detected with a secondary antibody coupled to Alexa555) [black]. Enlarged projections of the centrosome area (boxed on the 3-color image) are shown as insets. Scale bars represent 10 μm on full size images and 3 μm on enlarged insets.(B) Quantification of the number of microtubules emanating from the centrosome of untransfected control (cont.) and nucleolin siRNA transfected (NCL siRNA) U2OS-centrin-1GFP cells. Each black mark corresponds to a cell (cont. n = 50 and NCL siRNA n = 50) and the red lines correspond to the mean (cont. 27.72 +/− 2.74 and NCL siRNA 27.24 +/− 2.97). (C) Quantification of the changes in the interphase microtubule network organization following nucleolin depletion from experiment in A. The histogram shows the percentage of the number n of U2OS-centrin-1-GFP cells analyzed for untransfected control (cont., left), control siRNA transfected (Cont. siRNA, middle), or nucleolin siRNA transfected (NCL siRNA, right), harboring a microtubule network mainly organized from the centrosome [green bars, see example on first row of A], or disorganized [red bars, see examples on second and third row of A].
Figure 7 (See previous page).
Figure 7 (See previous page).
Alteration of microtubule regrowth in nucleolin-silenced cells after depolymerization. (A-B) Microtubule regrowth after microtubule induced depolymerization in untransfected control (A) and nucleolin siRNA transfected (B) U2OS-centrin-1-GFP cells. Co-visualization of α-tubulin (α-tub), centrin-1-GFP (C1G) and nuclear counterstain (DAPI) shown as 3-color merged projections (first row) and as individual sections for α-tubulin (inverted dynamics, second row) before depolymerization (A1, A5, B1 and B5), immediately after depolymerization (A2, A6, B2 and B6), after 2 minutes at 37°C (A3, A7, B3 and B7), and finally after 5 minutes at 37°C (A4, A8, B4, B8, and zooms A4, A8, B4, and B8). Centrin-1-GFP detection was enhanced with a GFP booster [green]; α-tubulin was detected with a monoclonal antibody (detected with a secondary antibody coupled to Alexa555) [red on the 3-color merged images and black on individual projections]. Enlarged projections of the centrosome area (boxed on the 3-color images) are shown as insets (A1-A3 and B1-B3) or as full sized pictures (A4, A8, B4, and B8 zooms). Arrows pointing to the centrosomal area appear red when no microtubule regrowth is observed (A6, B6 and B7) or green when microtubule regrowth is observed (A7). Arrowheads pointing to microtubules appear light green when microtubules are directed to the centrosomal area (A8 zoom) or orange when microtubules are not pointing to the centrosomal area (B8 zoom). Scale bars represent 10 μm on full size images, 5 μm on the zoomed images and the insets. (C–D) In C, quantification of the cells incubated for 2 minutes at 37°C (A7 and B7) exhibiting either microtubules regrowth (green bars on C, see also green arrows in A7) or no microtubule regrowth (red bars on C, see also red arrows in B7). In D, quantification of the cells incubated for 5 minutes at 37°C (A8 and B8) exhibiting either absence (green bars on D, see also cells with only green arrowheads on A8 zoom) or presence of cytoplasmic microtubules (orange bars on D, see also cell with orange arrowheads on B8 zoom). These classes are displayed for untransfected control cells (Cont., left), cells transfected with control siRNA (Cont. siRNA, middle) and nucleolin siRNA transfected cells (NCL siRNA, right), whose values are expressed as percentages of the total number n of cells studied. Error bars represent the standard deviation from 4 independent experiments. *: Significant difference with a p value < 0.01 with control populations (left columns). n: non-significant difference with a p value < 0.01 with control populations (left columns).
Figure 8.
Figure 8.
Analysis of microtubule regrowth after depolymerization using the microtubule plus-end tracking protein EB3-tagRFP. (A) Still frames from time-lapse experiments show EB3-tagRFP expressing untransfected control and nucleolin siRNA transfected U2OS-centrin-1-GFP cells, after microtubule depolymerization. Enlarged images of the centrosome area (displayed on the full size images) are shown under the full size images. For each time point (except for t = 0min), a projection of 10 temporal images prior to the image matter was performed. Scale bars represent 5 μm on full size images and 2 μm on enlarged images. Times are given in minutes (full videos are available in supplementary material as video S1 and S2). (B) Quantification of centrosomal fluorescence intensity at the indicated times in untransfected control and nucleolin siRNA transfected U2OS-centrin-1-GFP cells, after microtubule depolymerization from A. Mean values of 3 control [red line] and 5 nucleolin depleted (NCL siRNA) [blue line] cells were plotted.
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