Microtubule stabilization drives 3D centrosome migration to initiate primary ciliogenesis

Abstract

Primary cilia are sensory organelles located at the cell surface. Their assembly is primed by centrosome migration to the apical surface, yet surprisingly little is known about this initiating step. To gain insight into the mechanisms driving centrosome migration, we exploited the reproducibility of cell architecture on adhesive micropatterns to investigate the cytoskeletal remodeling supporting it. Microtubule network densification and bundling, with the transient formation of an array of cold-stable microtubules, and actin cytoskeleton asymmetrical contraction participate in concert to drive apical centrosome migration. The distal appendage protein Cep164 appears to be a key actor involved in the cytoskeleton remodeling and centrosome migration, whereas intraflagellar transport 88's role seems to be restricted to axoneme elongation. Together, our data elucidate the hitherto unexplored mechanism of centrosome migration and show that it is driven by the increase and clustering of mechanical forces to push the centrosome toward the cell apical pole.

Figure 1.

Adhesive micropatterns facilitate the study of centrosome migration during primary ciliogenesis. (A) Primary ciliogenesis is a multistep process that is proposed to begin with centrosome maturation and the formation of a ciliary vesicle at the distal end of the mother centriole, after which the centrosome migrates to the apical surface and attaches to the cortex. Full extension of the axoneme occurs once the mother centriole is anchored to the cortex. (B) Side view of micropatterned RPE1 cells expressing EGFP-centrin1 (white), cultured in the presence or absence of serum for 24 h, and stained with phalloidin to visualize F-actin, acetylated tubulin antibody to label the cilium, and DAPI to stain the DNA. (C) Measurement of centrosome z position as a percentage of nuclear height. Migration started within 2 h of serum starvation and appeared completed 6 h later. Measurement of the proportion of ciliated cells showed a delayed process compared with centrosome migration (one experiment, n = 60 cells per condition). Error bars represent standard deviation. (D) Side view of a representative time-lapse imaging of serum-starved RPE1 EGFP-centrin1 cells on micropatterns. Centrosome migration was engaged 1 h after starvation and completed 2 h later. (E) Representation of time-lapse centrosome movement in serum-starved RPE1 EGFP-centrin1 (data of three independent experiments, n = 53 cells). (i) The graph represents all the raw data. (ii) The centrosome trajectories are synchronized, i.e., they start when the centrosome leaves the basal pole. (iii) The plotting of the centrosome trajectories is limited to the maximal z position, i.e., they come to an end when centrosomes reach the apical pole. (F) Frequency distribution of centrosome migration velocity from basal pole to apical pole. Bars, 5 µm.

Figure 2.

Implication of known ciliogenesis effectors in centrosome migration. (A) RPE1 EGFP-centrin1 cells were treated with siRNAs targeting known primary ciliogenesis effectors for 24 h to investigate their potential role in centrosome migration during primary cilium formation. The proportion of cells displaying a centrosome located >3 µm above the basal pole was determined and normalized to that of the nontargeting control siRNA for each condition. See Table S1 for the siRNA sequences and Fig. S1 (A and B) for the effect on the rate of ciliated cells and the length of primary cilia. (B) Side views of serum-starved RPE1 EGFP-centrin1 cells stained with DAPI to label the DNA and an antibody to α-tubulin to stain microtubules. Bars: (x and y) 10 µm; (z) 2.5 µm. (C) Staining of RPE1 cells with DAPI and with antibodies to acetylated tubulin and γ-tubulin. Images show maximal projection of z stacks. Bars: (top) 10 µm; (bottom) 1 µm. (D) Representation of synchronized time-lapse centrosome movement in serum-starved RPE1 EGFP-centrin1 treated with nontargeting control siRNA (one experiment, n = 32 cells) or siRNA against siCep164 (one experiment, n = 25 cells; left and middle). The graph represents the maximal centrosome z position for cells treated with nontargeting control siRNA and with siRNA against siCep164 (right). ****, P < 0.0001. Error bars represent standard deviation. Horizontal bars show mean values.

Figure 3.

Microtubule network remodeling during centrosome migration. (A and B) Microtubule network organization was studied by time-lapse imaging of RPE1 cells expressing EGFP-centrin1 and MAP4-RFP. Two examples are shown: one where the centrosome (indicated by white arrowheads) migrated to the apical pole (A) and another where it did not (B). Orthogonal and top views are shown. Microtubule network symmetry breaking and densification are shown with yellow arrows. (C) Measurement of centrosome z position (left) and α-tubulin fluorescence intensity in a 5-µm box surrounding the centrosome (right) in thymidine-synchronized serum-starved RPE1 cells expressing EGFP-centrin1 for various delays after serum removal (n = 75 cells per condition). Horizontal bars show mean values. Error bars represent standard deviation. (D) The graph shows the microtubule network density at the centrosome against centrosome z position at various time points after serum starvation in thymidine-synchronized RPE1 cells expressing EGFP-centrin1. In all conditions, the two parameters were correlated. (E) RPE1 cells expressing EGFP-centrin1 (centrosome indicated by white arrowheads) were fixed 120 min after serum withdrawal and stained for α-tubulin and DAPI. The side views facilitated the visualization of microtubule bundle orientation and quantification of cell proportion exhibiting either a microtubule bundle toward the basal pole (images and bar graph with yellow outlines), a microtubule bundle toward the apical pole (images and bar graph with green outlines), or no detected microtubule bundle (images and bar graph with red outlines; results of four independent experiments, n = 116 cells). (F) Staining of serum-starved RPE1 cells expressing EGFP-centrin1 with an antibody to EB1. The graph shows EB1 fluorescence intensity measurements in a 5-µm box surrounding the centrosome (one experiment, n = 60 cells per condition). Bars: (x and y) 10 µm; (z) 5 µm. a.u., arbitrary units. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Figure 4.

Microtubule stabilization after serum starvation promotes centrosome migration. (A) Identification of cold-resistant microtubules. Serum-starved RPE1 cells were subjected to cold shock (on ice for 12 min) and fixed and stained with an antibody to α-tubulin (lookup table [LUT] fire [ImageJ, National Institutes of Health]). Images show five examples of serum-starved and five examples of serum-fed cells (LUT fire). The graph shows measurements of α-tubulin fluorescence intensity after cold shock for various delays after serum removal (results of three independent experiments, n = 125 cells per condition). (B) 3D numerical simulations of microtubule growth from the centrosome at the basal pole. They showed that longer microtubules, assembled by reducing the catastrophe rate, induced a symmetry break in the network architecture that was capable of pushing the centrosome to the apical surface. The graph shows the centrosome z position according to the catastrophe rate. (C and D) Identification of cold-resistant microtubules in serum-fed cells treated with either control siRNA or siRNA against the tubulin sequestering protein stathmin 1. The same conditions as in A. Images show five examples of serum-fed cells treated with control siRNA and five examples of serum-fed cells treated with stathmin 1 siRNA (C). (D) Graph shows measurements of α-tubulin fluorescence intensity after cold shock (results of two independent experiments, control siRNA, n = 125 cells; stathmin 1 siRNA, two siRNA sequences, n = 125 cells each). Horizontal bars show mean values. (E) Stathmin 1 was depleted by siRNA from RPE1 cells cultured in the presence of serum to promote microtubule growth and observe its effect on centrosome position. Cells were fixed and stained for α-tubulin, γ-tubulin, and DNA. Centrosomes are indicated by white arrowheads, and the microtubule bundle is shown with a yellow arrow (left side view images). The graph shows cell percentage displaying basal centrosome (located from 0 to 2 µm above the glass substrate), intermediate centrosome (between 2 and 3 µm above the glass substrate), and apical centrosome (located >3 µm above the glass substrate; results of three independent experiments, control siRNA, n = 100 cells; stathmin 1 siRNA, two siRNA sequences, n = 150 cells each). (F) Time-lapse imaging for 80 min of serum-fed RPE1 cells expressing EGFP-centrin1 treated with control siRNA or siRNA against stathmin 1, then fixation/immunostaining for α-tubulin. The left graph shows the percentage of cells exhibiting a moving centrosome toward the apical pole in each condition (siCTR vs. siStathmin 1). The right graph shows microtubule bundle orientation frequency for each condition (one experiment, control siRNA, n = 63 cells; siRNA against stathmin 1, n = 45 cells). Bars: (x and y) 10 µm; (z) 2.5 µm. a.u., arbitrary units. ****, P < 0.0001.

Figure 5.

Contractility increase breaks actin cytoskeleton symmetry and promotes microtubule stabilization. (A) Reorganization of the actin and microtubule cytoskeletons upon serum starvation. RPE1 cells were fixed 4 h after serum withdrawal, stained with phalloidin to visualize F-actin, immunostained with antibodies against α-tubulin, and compared with serum-fed cells. Images show four examples of serum-starved and serum-fed cells. (B) Nucleus positioning upon serum starvation. RPE1 cells were fixed 4 h after serum starvation, stained with phalloidin and DAPI and compared with serum-fed cells. Images show four examples of serum-starved and serum-fed cells. White dotted lines indicate the cell symmetry axis. The top graph shows measurements of nucleus height, and the bottom graph shows measurements of nucleus position in the x–y plane in fed and starved cells at various time points after serum starvation (results of two experiments, n = 75 cells). Error bars represent standard deviation. (C) Immunostaining against phosphomyosin showed an intense staining along the noncircular actin bundle (green) in serum-starved cells. White arrowheads point at centrosomes detected with anticentrin1 antibodies (white). RPE1 cells were fixed 4 h after serum withdrawal. (D) Averaging of phosphomyosin fluorescence intensity levels (LUT fire), obtained by stacking and averaging 50 images per condition, showed that the myosin phosphorylation increased after serum starvation. (E) Identification of cold-resistant microtubules in serum-fed and serum-starved cells in the presence or absence of the myosin II ATPase inhibitor blebbistatin. RPE1 cells were fixed 2 h after serum withdrawal. Images show five examples of serum-starved cells, five examples of serum-fed cells, and five examples of serum-starved cells treated with blebbistatin for 2 h (LUT fire). The graph shows measurements of α-tubulin fluorescence intensity after cold shock (results of two independent experiments, n = 110 cells for each condition). Bars: (x and y) 10 µm; (z) 5 µm. **, P < 0.01; ****, P < 0.0001. Horizontal bars show mean values. a.u., arbitrary units.

Figure 6.

The ciliogenesis effector Cep164 affects microtubule stabilization and actomyosin contractility upon serum starvation. (A) Identification of cold-resistant microtubules in serum-starved and serum-fed cells treated with siRNA against Cep164. Images show four representative examples of Cep164-depleted serum-starved and Cep164-depleted serum-fed cells (experiments obtained with two distinct Cep164 siRNA sequences). Cells fixed after a brief cold shock of 12 min after 3 h of serum starvation. The graph shows measurements of α-tubulin fluorescence intensity after cold shock (LUT fire; results of two independent experiments, n = 110 cells per condition). (B) Same as in A with siRNA against IFT88 (results of two independent experiments, n = 110 cells per condition). (C) Averaging of phosphomyosin fluorescence intensity levels (LUT fire), obtained by stacking and averaging 50 images per condition. Cells were fixed 4 h after serum starvation. Averaged images showed that myosin phosphorylation increased in control and IFT88-depleted cells, but not in Cep164-depleted cells. Bars, 10 µm. *, P < 0.05; ****, P < 0.0001. a.u., arbitrary units.

Figure 7.

Proposed sequence of events driving centrosome migration to the cell apical pole. These schemes show a side and top view of cytoskeleton rearrangements after serum withdrawal. Microtubule network densification and actin network contraction break the symmetry of both networks, which results in the production of pushing forces moving the centrosome to the dorsal surface. Upon contact, the centrosome promotes local surface maturation into an apical pole and centriole anchoring and elongation to form the primary cilium.