Journal: The Journal of Cell Biology

Article Title: Clathrin promotes centrosome integrity in early mitosis through stabilization of centrosomal ch-TOG

doi: 10.1083/jcb.201205116

Figure Lengend Snippet: Clathrin localizes at centrosomes of interphase and metaphase cells. (A) HeLa cells were methanol fixed and processed for IF to detect CHC (X22 mAb) or CLC (rabbit polyclonal) and centrosomal markers PCNT2 (goat polyclonal) and γ-tubulin (mAb) at indicated cell cycle phases. Colocalization of green and red is shown as yellow. Bars, 10 µm. (B) HeLa-SNAP-uLCa clone 3.3 cells were methanol fixed and processed for IF to detect LCa with mAb X16 (CLC), PCM1 (rabbit polyclonal), and PCNT2 (goat polyclonal). Merged colors showing colocalization between labeled proteins are shown as yellow (red and green) and white (red, green, and yellow or cyan and red). Bars: (top) 2 µm; (bottom) 10 µm. (C) HeLa-SNAP-uLCa clone 3.3 cells were methanol fixed and processed for IF to detect TfR (mAb) or EEA1 (mAb), CLC with polyclonal rabbit antibody, and PCNT2 (goat polyclonal). Merged colors showing colocalization between CLC and PCNT2 are shown as pink. Bar, 10 µm. (A–C, insets) Boxes in each frame are magnified threefold. Bars, 2 µm. Images shown are 3D maximum projections.

Article Snippet: Commercially available antibodies were used to detect β-actin (mAb; Sigma-Aldrich), γ-tubulin (mAb; Santa Cruz Biotechnology, Inc.), α-tubulin (mAb from Sigma-Aldrich and rabbit polyclonal from Santa Cruz Biotechnology, Inc.), PCNT2 (goat polyclonal; Santa Cruz Biotechnology, Inc.), centrin-2 (mAb; Abcam), SNAP-tag (rabbit polyclonals from both GenScript and New England Biolabs, Inc.), TACC3 (mAb; Santa Cruz Biotechnology, Inc.), ch-TOG (rabbit polyclonal from AbCam and rabbit polyclonal from BioLegend), PCM1 (rabbit polyclonal; Cell Signaling Technology), glyceraldehyde 3-phosphate dehydrogenase (GAPDH; mAb; EMD Millipore), TfR (mAb; BD), and EEA1 (mAb; BD).

Techniques: Labeling

Journal: The Journal of Cell Biology

Article Title: Clathrin promotes centrosome integrity in early mitosis through stabilization of centrosomal ch-TOG

doi: 10.1083/jcb.201205116

Figure Lengend Snippet: Covalent cross-linking of SNAP-uLCa disrupts CHC17-dependent pathways of transferrin uptake and recycling. (A) HeLa-SNAP-uLCa clone 3.3 cells were treated with SNAP-tag homodimerizer (BG-GLA-BG, 10 µM) or mock treated (0 µM) with solvent (DMSO) equivalent to the amount used for the BG-GLA-BG treatment. Cell lysates were immunoblotted for dimerized SNAP-uLCa (114** kD), monomeric SNAP-uLCa (57* kD), and endogenous uLCa (32 kD) using antibodies to the SNAP-tag or LCa (mAb X16), as indicated (left). β-actin was detected as a loading control. Protein mass based on migration relative to marker proteins is shown on the right. (B) Clone 3.3 cells were treated (2 h) with 5 µM BG-GLA-BG or equivalent DMSO (mock). Internalization of prebound (4°C) Alexa Fluor 488–transferrin (Tf) was visualized after 10 min or 1 h at 37°C after IF labeling with antibodies to the SNAP-tag or CHC17. Red-blue colocalization in merge is pink. Three-color colocalization in merge is white. Bars, 10 µm. (C) Clone 3.3 cells internalized prebound Alexa Fluor 488–transferrin for 1 h and were labeled by IF with antibodies to the SNAP-tag and EEA1. Red-green colocalization in merge is yellow. Three-color colocalization in merge is white. Bars, 10 µm. (B and C, insets) Boxes are magnified threefold. Bars, 1 µm. Images are 2D. (D) Clone 3.3 cells were treated with BG-GLA-BG or DMSO (mock), as in B, labeled with NHS-S-S-biotin, and then kept on ice (0* and 0 min) or incubated at 37°C for 5, 10, or 15 min. After reduction (stripping) of all samples except 0*, cells were lysed and analyzed by immunoblotting for total TfR and GAPDH (lysate), or biotinylated proteins were SA bound and immunoblotted to detect internalized TfR or control GAPDH. Protein mass based on migration relative to marker proteins is shown on the right. (E) TfR uptake (signal at internalization time point minus signal at time 0) was quantified by densitometry and plotted relative to total biotinylated TfR (0* minus signal at time 0) for four independent experiments, performed as in D for clone 3.3 cells treated with BG-GLA-BG (shaded squares) or DMSO (mock; open circles). Internalized TfR is shown in arbitrary units. Data represent the mean ± SEM of three separate experiments.

Article Snippet: Commercially available antibodies were used to detect β-actin (mAb; Sigma-Aldrich), γ-tubulin (mAb; Santa Cruz Biotechnology, Inc.), α-tubulin (mAb from Sigma-Aldrich and rabbit polyclonal from Santa Cruz Biotechnology, Inc.), PCNT2 (goat polyclonal; Santa Cruz Biotechnology, Inc.), centrin-2 (mAb; Abcam), SNAP-tag (rabbit polyclonals from both GenScript and New England Biolabs, Inc.), TACC3 (mAb; Santa Cruz Biotechnology, Inc.), ch-TOG (rabbit polyclonal from AbCam and rabbit polyclonal from BioLegend), PCM1 (rabbit polyclonal; Cell Signaling Technology), glyceraldehyde 3-phosphate dehydrogenase (GAPDH; mAb; EMD Millipore), TfR (mAb; BD), and EEA1 (mAb; BD).

Techniques: Solvent, Control, Migration, Marker, Labeling, Incubation, Stripping Membranes, Western Blot

Journal: The Journal of Cell Biology

Article Title: Clathrin promotes centrosome integrity in early mitosis through stabilization of centrosomal ch-TOG

doi: 10.1083/jcb.201205116

Figure Lengend Snippet: 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 (T 0 ) upon thymidine washout (0–2 h after T/T) or at 7 h (T 7 ; 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 T 0 or T 7 after T/T. Lysates prepared 2, 4, 6, 8, and 12 h after T/T for the T 0 treatment and at 12 h after T/T (12*) for the T 7 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 T 0 , 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 T 0 , 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 T 0 , 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 T 0 ), 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 T 0 , 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).

Article Snippet: Commercially available antibodies were used to detect β-actin (mAb; Sigma-Aldrich), γ-tubulin (mAb; Santa Cruz Biotechnology, Inc.), α-tubulin (mAb from Sigma-Aldrich and rabbit polyclonal from Santa Cruz Biotechnology, Inc.), PCNT2 (goat polyclonal; Santa Cruz Biotechnology, Inc.), centrin-2 (mAb; Abcam), SNAP-tag (rabbit polyclonals from both GenScript and New England Biolabs, Inc.), TACC3 (mAb; Santa Cruz Biotechnology, Inc.), ch-TOG (rabbit polyclonal from AbCam and rabbit polyclonal from BioLegend), PCM1 (rabbit polyclonal; Cell Signaling Technology), glyceraldehyde 3-phosphate dehydrogenase (GAPDH; mAb; EMD Millipore), TfR (mAb; BD), and EEA1 (mAb; BD).

Techniques: Expressing, Transfection, Clone Assay, Control, Migration, Marker, Blocking Assay, Fluorescence