Journal: The Journal of Cell Biology

Article Title: Centriolar cap proteins CP110 and CPAP control slow elongation of microtubule plus ends

doi: 10.1083/jcb.202406061

Figure Lengend Snippet: Subcellular localization of GFP-tagged CP110 and its fragments, CEP97, and CEP97^CP110 chimera. (A) U2OS transiently transfected with the indicated GFP-tagged constructs were fixed and stained with antibodies against CEP192 (magenta), GFP (green), and tyrosinated tubulin (gray). White box highlights region with centrioles, which are enlarged in zoom. (B) U-ExM images of centrioles from U2OS cells overexpressing the indicated constructs and stained for acetylated tubulin (blue), CP110 (magenta), and GFP (green). CP110 full-length and CEP97^CP110 both localize to the distal cap of the mother centriole (white arrowhead) and distal cap of the daughter centriole.

Article Snippet: HEK293T cells (ATCC) and the Flp-In T-REx U2OS host cells ( ) were cultured in DMEM and Ham’s F10 (1:1) supplemented with 10% FCS and 5 U/ml penicillin and 50 μg/ml streptomycin.

Techniques: Transfection, Construct, Staining

Journal: The Journal of Cell Biology

Article Title: Centriolar cap proteins CP110 and CPAP control slow elongation of microtubule plus ends

doi: 10.1083/jcb.202406061

Figure Lengend Snippet: Characterization of the effects of disrupting CPAP–CP110 interaction on centriole length regulation at interphase. (A) Scheme showing the generation of the inducible transgenic cell lines expressing either GFP-tagged WT full-length CPAP (CPAP-FL WT ) or full-length CPAP with L149A/K150A mutation (CPAP-FL MUT ). U2OS cells (Control) were used to integrate with the Tet repressor, a single FRT site, and the lacZ-Zeocin fusion gene by lentivirus to generate the Flp-In T-REx U2OS host cell line (Host). pcDNA5/FRT/TO vectors for doxycycline-inducible expression of GFP-CPAP-FL WT or GFP-CPAP-FL MUT were co-transfected together with Flp recombinase-encoding pOG44 vector into the Flp-In T-REx U2OS host cell line to induce their integration into the FRT site of the host cell genome in a Flp recombinase-dependent manner. The expression of GFP-CPAP-FL WT or GFP-CPAP-FL MUT was controlled by the inducible hybrid human cytomegalovirus (CMV)/Tet operator 2 (TetO2) promoter. The endogenous CPAP gene was knocked out using a CRISPR/Cas9–based approach. (B) Mean ± SD of the normalized CPAP levels based on western blots shown in ( n = 3 trials). Cell lines used for quantification are shown in magenta, where cell line pairs 1 and 2 (p1 and p2, respectively) are highlighted. Nonsignificant (ns), P > 0.05 calculated using an unpaired two-tailed Mann–Whitney U test. (C and E) Immunofluorescence images acquired using Airyscan 2 confocal microscope of centrioles at G1/S (C) and G2/M (E) and stained for acetylated tubulin (blue), CP110 (green), and GFP-CPAP (magenta). (D) Median ± IQR of mother centriole length at G1/S measured from proximal end of centriole (determined by acetylated tubulin) to distal end (determined by the geometric center of CP110 signal) (scheme in panel F). n , number of analyzed centrioles: control cell line, n = 113; host, n = 105; CPAP-FL WT#3 , n = 132; CPAP-FL WT#4 , n = 131; CPAP-FL MUT#1, n = 84; CPAP-FL MUT#5, n = 170; CPAP-FL MUT#4, n = 81; nonsignificant (ns); and ****P < 0.001 calculated using Kruskal–Wallis ANOVA test. (F) Median ± IQR of centriole length at G2/M measured as in D. n , number of analyzed mother centrioles (MC) and daughter centrioles (DC): control cells, n = 80 MC, 75 DC; host, n = 72 MC, 59 DC; CPAP-FL WT#3 , n = 67 MC, 69 DC; CPAP-FL WT#4 , n = 64 MC, 57 DC; CPAP-FL MUT#1 , n = 71 MC, 80 DC; CPAP-FL MUT#5 , n = 78 MC, 79 DC; CPAP-FL MUT#4 , n = 79 MC, 77 DC; nonsignificant (ns); and ****P < 0.001 calculated using Kruskal–Wallis ANOVA test.

Article Snippet: HEK293T cells (ATCC) and the Flp-In T-REx U2OS host cells ( ) were cultured in DMEM and Ham’s F10 (1:1) supplemented with 10% FCS and 5 U/ml penicillin and 50 μg/ml streptomycin.

Techniques: Transgenic Assay, Expressing, Mutagenesis, Control, Transfection, Plasmid Preparation, CRISPR, Western Blot, Two Tailed Test, MANN-WHITNEY, Immunofluorescence, Microscopy, Staining

Journal: The Journal of Cell Biology

Article Title: Centriolar cap proteins CP110 and CPAP control slow elongation of microtubule plus ends

doi: 10.1083/jcb.202406061

Figure Lengend Snippet: Key resources table

Article Snippet: HEK293T cells (ATCC) and the Flp-In T-REx U2OS host cells ( ) were cultured in DMEM and Ham’s F10 (1:1) supplemented with 10% FCS and 5 U/ml penicillin and 50 μg/ml streptomycin.

Techniques: Recombinant, Protease Inhibitor, Plasmid Preparation, Expressing, Software, Imaging