Figure 4.

Bipolar spindle formation and anaphase onset are delayed in cells lacking intact centrioles. (A) CEP152-KO and STIL-KO cells contain disorganized mitotic spindles. Cells were stained with antibodies against CDK5RAP2 and α-tubulin. DNA is shown in blue. Asterisks mark cells with disorganized spindles. (B) Unfocused MTs contact kinetochores in STIL-KO cells. Cells were stained with antibodies against the kinetochore protein, CENP-O, and α-tubulin. DNA is shown in blue. A single focal plane is shown along with higher magnification of framed area. (C) Graphs show the percentage of mitotic cells with disorganized spindles in CEP152-KO and STIL-KO (n = 3, >165 cells per genotype per experiment; means ± SD). (D) Still frames from time-lapse experiments show GFP-tubulin–expressing WT, CEP152-KO, and STIL-KO cells (Videos 1, 2, and 3). NEBD marks the first frame after NEBD. Times are given in hours, minutes, and seconds. (E) Graph shows distribution of time intervals from NEBD to anaphase onset. Medians and interquartile ranges are indicated, from Kruskal–Wallis and Dunn’s test. (F) MT regrowth in mitotic cells after depolymerization. Note the absence of MTs at 0 min and enrichment of MTs near chromatin in STIL-KO cells after 3 min of regrowth. Cells were stained with antibodies against CDK5RAP2 and α-tubulin. Bars, 5 µm.