RBM14 prevents assembly of centriolar protein complexes and maintains mitotic spindle integrity. (2015)
Figure 4

HsSAS-6-independent formation of centriolar protein complexes occurs in the cytoplasm in RBM14-depleted cells

A U2OS cells treated with control siRNA, RBM14 siRNA alone or RBM14 + HsSAS-6 siRNAs were stained with antibodies against centrin-2 (green) and HsSAS-6 (magenta). DNA is shown in blue. Insets show approximately twofold magnified views of fluorescent foci around the centrosome. Scale bar, 5 μm. Histograms represent frequency of mitotic cells with excess centrin foci in the indicated conditions. Values are mean percentages ± SEM from three independent experiments (n = 30 for each condition). n.s., not significant (one-tailed t-test).

B Live imaging of cycling RBM14-depleted HeLa cells expressing GFP-centrin1 (green) and RFP-H2B (magenta). Insets show approximately 1.5-fold magnified images of fluorescent foci. Time is denoted in hh:min, and scale bar is 10 μm. Time zero corresponds to the onset of excess centrin foci formation. Quantification of the number and total signal intensity of centrin foci in HeLa cells treated with control or RBM14 siRNA over time. Means ± SEM are shown (n = 5).

C Ectopic formation of centriolar intermediates likely occurs from late G1 to S phase. Time-lapse recording across the cell cycle of HeLa cells expressing GFP-centrin (green) and RFP-H2B (magenta) and treated with RBM14 siRNA. Time is denoted in hh:min, and scale bar is 10 μm. Time zero corresponds to the metaphase onset.

D Ectopic formation of centriolar protein complexes occurs during S phase arrest induced by HU treatment but not G1 phase arrest induced by lovastatin treatment. Insets show threefold magnified images of fluorescent foci. Scale bar, 5 μm. Histograms represent frequency of mitotic cells with excess centrin foci in the indicated conditions. Values are mean percentages ± SEM from three independent experiments (n = 30 for each condition). **P < 0.01, n.s., not significant (one-tailed t-test).

E Amorphous electron-dense structures containing microtubules are formed in RBM14-depleted cells (see also Supplementary Fig S5I). Electron micrographs from U2OS cells depleted of RBM14. Squares indicate the ectopic electron-dense structures, whereas circles indicate the pre-existing centrioles. Scale bar, 500 nm and 200 nm (magnified views). Average diameter of pre-existing centrioles and the amorphous electron-dense structures are 225 ± 8 nm (n = 10) and 130 + 11 nm (n = 16), respectively. Insets show magnified views of the squares. Note that microtubules (black arrowheads) are observed within the electron-dense structures (white arrowheads).

F Histograms represent frequency of the cells with ectopic foci of GFP-CPAP in the indicated conditions. U2OS cells expressing GFP-CPAP were fixed and stained 24 h after RNAi treatment and induction of GFP-CPAP. Values are mean percentages ± SEM from three independent experiments (n = 30 for each condition). **P < 0.01 (one-tailed t-test).

G U2OS cells expressing GFP-CPAP (green) and treated with RBM14 siRNA were analyzed using CLEM. Separate z-plane images of the cell with GFP-CPAP foci and the threefold magnified images are aligned from bottom to top along z-axis (left LM panels). White broken lines represent cell shapes, and blue broken lines represent nuclear shapes. Corresponding regions of EM images are shown on the right. White squares (a–d) represent the region around GFP-CPAP foci. Note that other than pre-existing centrioles (a, 1–4), ectopic small CPAP foci are recognizable as amorphous electron-dense structures (b–d). One of pre-existing centrioles (1) is elongated because of exogenous GFP-CPAP expression. Scale bar, 10 μm (LM), 2 μm (EM, low magnified image), 500 nm [magnified images of (a) and (d)), 200 nm (magnified images of (b) and (c)].