Journal: Molecular Biology of the Cell
Article Title: Centrosome-intrinsic mechanisms modulate centrosome integrity during fever
Figure Lengend Snippet: The centrosome is a substrate organelle for molecular chaperone Hsp70. (See also Supplemental Figures S1–S3.) (A) Left, images of maximum projections from cntrl and HS cells as indicated show that upon HS, Hsp70 accumulates at the centrosome and in the nucleus (inset, arrow points at the centrosome); bar, 10 μm. Semiquantitative analysis of integrated intensity (×10 5 arbitrary units) of centrosomal Hsp70 before and after HS (15–20 centrosomes/sample, mean ± SEM). (B) Superresolution images (OMX) demonstrate loss of γ-tubulin (red) from HS cells and recruitment of Hsp70 (green) as an outer layer in stressed cells; bar, 0.2 μm. (C) Immunoprecipitation (IP) of Hsp70 pulls down PCNT. Immunoglobulin G (IgG), control. (D) Centrosome-targeted Hsp70 (cHsp70) protects γ-tubulin from HS, whereas membrane-targeted Hsp70 (mHsp70) does not. Data are shown as semiquantitative profiles of confocal microscopy images. Right, percentage of cells with centrosomal γ-tubulin after HS in stably expressing centrosome-(cHsp70) or membrane-(mHsp70) targeted Hsp70-expressing cells (four experiments, 500–600 cells/sample, mean ± SD, one-way analysis of variance [ANOVA] combined with Tukey’s multiple comparison test). (E) cHsp70, but not the chaperone-negative mutant cHsp70D10S, protects γ-tubulin signal at the centrosome; semiquantitative analysis of integrated γ-tubulin signal intensity (×10 5 arbitrary units, mean ± SEM). (F) cHsp70 protects centrosomal Centrin2 from HS, whereas mHsp70 does not. Data are shown as semiquantitative profiles of confocal microscopy images. Right, percentage of cells with centrosome-localized Centrin2 after HS in stably expressing cHsp70- or mHsp70-targeted Hsp70 cells (three experiments, 500–600 cells/sample, mean ± SD, one-way ANOVA combined with Tukey’s multiple comparison test).
Article Snippet: The following commercial antibodies were used: ninein (ab4447; Abcam [Cambridge, MA]), pericentrin (ab4448; Abcam), cenexin/ODF2 (ab43840; Abcam), centrobin (ab70448; Abcam), Hsp70 (SPA-810; Stressgen [Plymouth Meeting, PA]), PCM1 (5213; Cell Signaling [Danvers, MA]), glyceraldehyde-3-phosphate dehydrogenase (sc-32233; Santa Cruz Biotechnology [Dallas, TX]), rootletin (sc-67824; Santa Cruz Biotechnology), SAS-6 (sc-81431; Santa Cruz Biotechnology), Cep83 (HPA038161; Atlas [Stockholm, Sweden]), Septin7 (sc-20620; Santa Cruz Biotechnology), MLKP (sc-867; Santa Cruz Biotechnology), ubiquitin (550944; BD Transduction [San Jose, CA]), p150 (P50520-050; BD Biosciences), RacGAP (ab2270; Abcam), acetylated tubulin (T 6793; Sigma-Aldrich [St. Lous, MO]), EB1 (610534; BD Transduction), CREST (15-234-0001; Antibodies Incorporated [Davis, CA]), CenpE (ab5093; Abcam), ATP5A (ab14748; Abcam), mitochondrial Hsp70 (MA3-028; Affinity Bioreagents [Golden, CO]), GFP (DSHB-GFP-12A6; DSHB Biology [Iowa City, IA]), and GFP (sc-8334; Santa Cruz Biotechnology).
Techniques: Immunoprecipitation, Confocal Microscopy, Stable Transfection, Expressing, Mutagenesis