Figure 2.
Figure 2. An analysis of centriole and cilia behavior in WT and CP110Δ primary spermatocytes. (A–G) 3D-SIM images of centrioles in primary spermatocytes stained for endogenous CP110 (A and B) or overexpressed CP110-GFP (C–G, green) and either Asl (A–F) or acetylated tubulin (G, red). (A and B) CP110 is detectable at the distal tips of the short centrioles in very young WT spermatocytes (A, arrows) but is no longer detectable once the centrioles start to elongate in G2 (B). (C–G) Both overexpressed CP110S-GFP (C and D) and CP110L-GFP (E–G) are detected at the distal ends of young centrioles (C and E, arrows), and on fibers that extend from the distal tip of the elongating centrioles (D and F, arrows). CP110-GFP is also sometimes detectable at the proximal end of mother centrioles (E and F, arrowheads). The CP110-GFP fiber appears to be located within the centriolar MTs stained with acetylated tubulin antibodies (G). (H–K) Panels show EM images of centrioles (H and I) and cilia (J and K) in WT (H and J) and CP110Δ (I and K) primary spermatocytes. The centriole appears slightly elongated and the axoneme slightly shortened in the CP110Δ cilium (K). (L) Quantification of centriole and axoneme length by EM in 7 WT (blue) and 5 CP110Δ (red) cilia confirmed this trend, although the difference in centriole length was not statistically significant (perhaps due to the small numbers analyzed). (M) A statistically more robust quantification of centriole length in WT (blue) and CP110Δ (red) mature primary spermatocytes (measured by immunofluorescence with the centriole markers GTU88* and Ana1) revealed no significant difference in centriole length. Total number of centrioles analyzed; total number of spermatocyte cysts analyzed are: 1504;52, 1029;39, 1624;52, and 1090;43, respectively (order as shown in graph). In this and all subsequent bar charts error bars denote SEM and significance testing was conducted using the Mann-Whitney test. P > 0.05 (ns); *, P = 0.01–0.05; **, P = 0.001–0.01; ***, P = 0.0001–0.001; and ****, P < 0.0001. Note that an average centriole length for each spermatocyte cyst was calculated and the number of spermatocyte cysts analyzed was then used as the sample number for statistical analysis. Bars: (A–G) 1 µm; (H–K) 100 nm.

An analysis of centriole and cilia behavior in WT and CP110Δ primary spermatocytes. (A–G) 3D-SIM images of centrioles in primary spermatocytes stained for endogenous CP110 (A and B) or overexpressed CP110-GFP (C–G, green) and either Asl (A–F) or acetylated tubulin (G, red). (A and B) CP110 is detectable at the distal tips of the short centrioles in very young WT spermatocytes (A, arrows) but is no longer detectable once the centrioles start to elongate in G2 (B). (C–G) Both overexpressed CP110S-GFP (C and D) and CP110L-GFP (E–G) are detected at the distal ends of young centrioles (C and E, arrows), and on fibers that extend from the distal tip of the elongating centrioles (D and F, arrows). CP110-GFP is also sometimes detectable at the proximal end of mother centrioles (E and F, arrowheads). The CP110-GFP fiber appears to be located within the centriolar MTs stained with acetylated tubulin antibodies (G). (H–K) Panels show EM images of centrioles (H and I) and cilia (J and K) in WT (H and J) and CP110Δ (I and K) primary spermatocytes. The centriole appears slightly elongated and the axoneme slightly shortened in the CP110Δ cilium (K). (L) Quantification of centriole and axoneme length by EM in 7 WT (blue) and 5 CP110Δ (red) cilia confirmed this trend, although the difference in centriole length was not statistically significant (perhaps due to the small numbers analyzed). (M) A statistically more robust quantification of centriole length in WT (blue) and CP110Δ (red) mature primary spermatocytes (measured by immunofluorescence with the centriole markers GTU88* and Ana1) revealed no significant difference in centriole length. Total number of centrioles analyzed; total number of spermatocyte cysts analyzed are: 1504;52, 1029;39, 1624;52, and 1090;43, respectively (order as shown in graph). In this and all subsequent bar charts error bars denote SEM and significance testing was conducted using the Mann-Whitney test. P > 0.05 (ns); *, P = 0.01–0.05; **, P = 0.001–0.01; ***, P = 0.0001–0.001; and ****, P < 0.0001. Note that an average centriole length for each spermatocyte cyst was calculated and the number of spermatocyte cysts analyzed was then used as the sample number for statistical analysis. Bars: (A–G) 1 µm; (H–K) 100 nm.

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