The γ-tubulin complex–binding CM1 region of CDK5RAP2 is critical for acentriolar mitosis. (A) CDK5RAP2 schematic highlighting the N-terminal CM1 region; mutation of the conserved F75 residue to alanine blocks γ-tubulin binding (red asterisk). (B) Immunoblots of the indicated conditions. mClover was detected using an anti-GFP antibody; α-tubulin is a loading control; numbers below indicate relative total band intensities. (C) Mitotic outcomes observed by live imaging following centrinone treatment for the indicated conditions. (D) Mitotic duration in centrinone for the indicated conditions. Error bars are 95% CI. P value is from a t test. (E–G) Analysis of engineered CDK5RAP2 variants in the presence of endogenous CDK5RAP2. (E) Images of DMSO and centrinone-treated cells expressing WT or CM1 mutant CDK5RAP2; endogenous CDK5RAP2 is present in all conditions. (F) Immunoblots showing expression of the CM1 mutant, the C-terminal truncation, and the double mutant variants of CDK5RAP2. α-Tubulin is a loading control; numbers below indicate relative total band intensities. (G) Mitotic outcomes using live imaging following centrinone treatment for the indicated conditions. The key for the different outcomes is in C. ***, P < 0.001. IB, immunoblot; tub, tubulin.