Acute inactivation of clathrin during S phase causes centrosome fragmentation in early mitosis. (A) To inactivate clathrin at specific times during the cell cycle, cells expressing the SNAP-uLCa were enriched in S phase by T/T and treated (2 h) with BG-GLA-BG immediately (T0) upon thymidine washout (0–2 h after T/T) or at 7 h (T7; 7–9 h after T/T). The timing of treatment relative to cell cycle phase is shown, as empirically determined for the transfected HeLa clones in this study. Corresponding cellular events are illustrated with the nucleus (N), microtubule networks (MT), Golgi (G), centrosomes (C), and focal adhesion structures (FA) indicated. (B) Clone 3.3 cells were enriched to S phase and then treated (2 h) with 5 µM BG-GLA-BG or mock treated, starting at T0 or T7 after T/T. Lysates prepared 2, 4, 6, 8, and 12 h after T/T for the T0 treatment and at 12 h after T/T (12*) for the T7 treatment were immunoblotted with antibodies to the SNAP-tag or β-actin (loading control). Migration positions of the SNAP-uLCa dimer (**), monomer (*), and molecular mass marker proteins are shown on the right. (C) Clone 3.3 cells were enriched to S phase by T/T block, and, at T0, cells were treated with BG-GLA-BG (10 µM for 2 h), MLN8054 (500 nM for 4 h), or DMSO (mock; 4 h). 4 h after T0, cells were methanol fixed and processed for IF to detect CLC (X16), PCM1, and PCNT2. Red-blue localization is pink in merge, and three-color localization is white. Bar, 10 µm. (D) Clone 3.3 cells were enriched to S phase by T/T block, and then, at T0, cells were treated with BG-GLA-BG (10 µM for 2 h), MLN8054 (500 nM for 8 h), or DMSO (mock; 8 h). As cells entered mitosis (8 h after T0), they were fixed with PFA and processed for IF to detect the SNAP-tag, γ-tubulin, and α-tubulin. Red-blue localization is pink in merge, and green-blue localization is cyan. Bar, 10 µm. (C and D, insets) Boxed regions are magnified threefold. Bars, 2 µm. Images are 3D maximum projections. (E) Clone 3.3 cells were enriched to S phase by T/T block, and, at T0, cells were treated with BG-GLA-BG (10 µM for 2 h), MLN8054 (500 nM for 4 or 8 h), or DMSO (mock; 4 or 8 h, as in C and D). Treated cells were processed for IF to detect γ-tubulin and α-tubulin and were scored for centrosome fragmentation (γ-tubulin fragments shown by arrowheads in D). Data represent the mean ± SEM of two separate experiments (***, P < 0.001). 70 cells were scored per experiment. (F) From the experiment in D, 3D maximum projections of images of prometaphase and metaphase cells were analyzed for the distribution of γ-tubulin at the spindle pole–associated centrosome using the Radial Profile Extended plug-in in ImageJ. The normalized, integrated fluorescence intensity (y axis) is compared with the distance (pixels) of detected γ-tubulin at and surrounding the centrosome for cells treated with BG-GLA-BG, MLN8054, or DMSO (mock). The inset graph shows a magnified portion of the main graph and highlights the γ-tubulin fragments detected further out from the spindle pole–associated centrosome. Data represent the mean ± SEM of two to five prometaphase or metaphase cells (***, P < 0.001).
