Fig 1: The organization of mature centrioles and centrosomes. (A) Left: Electron micrograph of a mother centriole (MC, in cross-section) associated with a procentriole (PC, in longitudinal section) from an mIMCD3 (mouse inner medullary collecting duct) cell. Pericentriolar material (PCM) is visible around the MC. One microtubule (MT) triplet built of A, B, and C MTs is enlarged in the red box. Right: Electron micrograph of a mother centriole (MC) and associated procentriole (PC) from an mIMCD3 cell. Both centrioles are sectioned longitudinally. (B) Centrosomes in mitotic or interphase human cycling retinal pigmental epithelial (RPE-1) cells. Two mitotic centrosomes have expanded pericentriolar material (PCM), here labeled by anti-?-tubulin antibody, and are positioned on the opposite poles of the mitotic spindle. Each centrosome contains two centrioles, labeled with Centrin1-GFP. Chromosomes are aligning in the middle of the cell. In the interphase cell, two centrosomes, each harboring a duplicated mother centriole, are positioned near the nucleus and are associated with less abundant PCM. (C) Electron micrograph of a mature mother centriole with distal appendages (DA), subdistal appendages (SDA), and a cilium from a serum-starved RPE-1 cell. Ciliary axoneme (CA) is extending from the centriole distal end. DAs are adjacent to a ciliary pocket (CP), which continues into the ciliary membrane (CM) and surrounds the CA. Right: Cross sections through the region of centriole containing DAs (top) or SDAs (bottom) associated with MTs (red arrows). Electron microscopy was performed as described in Ref. [18]. (D) A montage of Stochastic Optical Reconstruction Microscopy (STORM) images of four centrosomal proteins immunolabeled as indicated and superimposed, to illustrate toroidal organization of centrosomal components with respect to centriole MTs (red). STORM and conventional microscopy were performed as described in Ref. [19] and using the antibodies as follows: ?-tubulin: Sigma; T5326, Cep63: Millipore; 06–1292, Pericentrin: Abcam; ab4448, and acetylated tubulin: Sigma; T7451. Scale bars: 400 nm in (A and C); 5000 nm in (B); 400 nm in (D).
Fig 2: . N-terminal segments of PCNT phase separate in a concentration-dependent manner in cells. (A) Alignments of 169 pericentrin orthologous proteins from vertebrates (167), fruit fly (1) and budding yeast (1), colored by the ClustalX coloring scheme in Jalview (Table S1). Conservation scores, locations of the predicted CCs, PACT motif, LCRs, putative dynein and ?-tubulin binding domains (BDs) of human PCNT, and the epitopes of the anti-PCNT antibody (Abcam, ab4448) are noted below the alignments. (B) Conservation scores within or outside of CC or LCRs of human PCNT. Data are median with the third quartile. (C) Representative time-lapse micrographs of GFP-PCNT (2-1960) condensates and GFP-PCNT (1954-3336) scaffolds 24 h post Dox induction in RPE-1 cells. Brackets denote an area with dynamic rearrangement of GFP-PCNT (2-1960) condensates; PCNT (1954-3336) scaffolds are non-dynamic (also see Movies 4, 5). (D) FRAP analyses of GFP-PCNT (2-1960) condensates and GFP-PCNT (1954-3336) scaffolds in RPE-1 cells. Dashed circles denote the bleached sites. Data are mean±95% c.i. n, number of condensates/scaffolds analyzed from more than three biological replicates. The percentage of recovery and half-life (t1/2) after photobleaching were calculated after fitting the data with non-linear regression. (E) Quantification of relative protein concentrations in live cells expressing various GFP-tagged PCNT segments after Dox induction (see Fig. S4 for details and representative images). PCNT (2-1960) and PCNT (854-1960) phase separated after reaching their respective critical concentrations (Csats), the concentrations of the light phase at which LLPS just occurred. Csats are mean±95% c.i. n, number of cells analyzed from three biological replicates. Statistical significance was determined by the Student's t-test (unpaired and two-tailed). ****P<0.0001. a.u., arbitrary unit. Scale bars: 5 µm.
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