Panel A shows a multiple sequence alignment of Ana1/Cep295 homologs, highlighting conserved residues involved in the Cep135-Ana1 interaction. The alignment includes sequences from various species, with residues color-coded by type. Panel B presents a line graph of Circular Dichroism (CD) analysis, showing the ellipticity (mdeg) on the y-axis and wavelength (nm) on the x-axis for WT Ana1 and two mutants (F283D and I286D), indicating that all proteins are largely helical. Panel C is a bar graph depicting the climbing ability of flies, with the percentage of flies climbing past the 5-cm mark within 2 seconds on the y-axis and different genotypes on the x-axis. Error bars indicate mean standard deviation, and the results are not significant (ns). Panel D is a bar graph showing the number of centriole pairs marked by Asl localization, with the number of centriole pairs on the y-axis and different genotypes on the x-axis. Error bars indicate mean standard deviation, and the results are not significant (ns). Panel E displays representative microscopy images of centriole elongation in primary spermatocytes at various stages from different genotypes, stained for EGFP-Ana1 (green) and Asl (red). The images show different stages of spermatocyte development, with a scale bar of 2 micrometers.
Cep135 binding–deficient Ana1 mutants fail to induce centriole overelongation. (A) MSA showing the conservation of residues involved in the Cep135-Ana1 interaction. Note that sequences of Ana1/Cep295 homologs are less well conserved through evolution, and thus, an MSA was performed using Drosophila sequences. (B) CD analysis showing that WT Ana1 and Ana1 mutant proteins are all largely helical in nature. (C) Climbing ability was assessed by measuring the percentage of flies per genotype that climbed past the 5-cm mark within 2 s. Each data point represents the percentage from 15 flies, with three independent biological replicates using distinct cohorts of flies per genotype (total of 45 flies per genotype). Error bars indicate the mean ± SD. ns, not significant (unpaired two-tailed Student’s t test). (D) Centriole pairs, marked by Asl localization, were quantified in 15–20 cells per testis from the indicated genotypes. Each data point represents the number of centriole pairs per testis, derived from five independent testes per genotype. Error bars indicate the mean ± SD. ns, not significant (unpaired two-tailed Student’s t test). (E) Representative images showing centriole elongation in primary spermatocytes at various stages from different genotypes, stained for EGFP-Ana1 (green) and Asl (red). Scale bar, 2 µm. WT, wild type; CD, circular dichroism; MSA, multiple sequence alignment.