Figure 4.

Effects of PCM exaggeration on the number of daughter centrioles. (A) Overexpression of pericentrin in S-phase arrested CHO cells results in the formation of a large cloud of pericentriolar material that contains numerous centrin-GFP aggregates cloud. overexpressing pericentrin. (A) Lower-magnification view of the centrosome in a cell expressing pericentrin (visualized with an anti-HA antibody). Inset in the Υ-tubulin frame depicts the size of the Υ-tubulin cloud of normal centrosomes in CHO cells shown at the same magnification. (B) Higher-magnification view of the region boxed in “(A) Merged”. (C) Some but not all centrin aggregates in pericentrin-overexpressing cells are centrioles. Centrin-GFP distribution in two cells that were analyzed by serial-section EM (see Fig. S7 for EM data). In the cell fixed 25 hr after transfection (left frame) centrin-GFP distribution is generally diffused with two brighter and several dimmer discrete spots. The brighter spots (M1 and M2) correspond to full-length mother centrioles while five of the dimmer spots (D1-D5) correspond to short randomly-oriented daughter centrioles (see Fig. S7A). In the cell fixed 45 hrs after transfection (right frame) discrete centrin spots are more prominent. Two brightest spots (M1 and M2) correspond to mother centrioles and twelve of the dimmer spots (D1-D12) correspond to daughter centrioles (see Fig. S7B). (D) 3-D organization of centrosomes with multiple daughter centrioles. Notice that centrin spots corresponding to the distal ends of centrioles may reside within the Υ-tubulin cloud or protrude outside. In contrast, the proximal ends of daughter centrioles (marked by SAS-6) consistently reside within the PCM although they are not always in the proximity of the mother centriole . See Videos 9-11 for surface-rendered models of the centrosomes presented here.