Loss of FZR-1 results in elevated SAS-5 levels.

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Loss of FZR-1 results in elevated SAS-5 levels. Loss of FZR-1 results in elevated SAS-5 levels. (A) Images of centrosomes stained for SAS-5 (green) at the first anaphase. Bar, 5 μm. (B) Quantification of centrosome-associated SAS-5 levels at the first anaphase. SAS-5 levels are normalized to the average fluorescence intensity in wild-type centrosomes. fzr-1(bs31) embryos exhibit increased levels of centrosomal SAS-5 (1.41 ± 0.42 fold, n = 18; P < 0.001) relative to wild-type controls (1.00 ± 0.28 fold, n = 38). In zyg-1(it25) fzr-1(bs31) double mutants, centrosomal SAS-5 levels are restored to near wild-type levels (0.95 ± 0.44-fold, n = 20; P = 0.003), compared to zyg-1(it25) embryos that show decreased levels of centrosomal SAS-5 (0.64 ± 0.28-fold, n = 16). n is the number of centrosomes. Each dot represents a centrosome. Box ranges from the first through third quartile of the data. Thick bar indicates the median. Solid gray line extends 1.5 times the interquartile range or to the minimum and maximum data point. ** P < 0.01, *** P < 0.001 (two-tailed t-test). (C) Quantitative western blot analyses show that (left panel) fzr-1(bs31) mutant embryos possess increased levels of both SAS-5 isoforms, SAS-5A (1.56 ± 0.16-fold) and SAS-5C (1.48 ± 0.19-fold), compared to wild-type (N2) embryos. However, there were no significant differences in levels of either SAS-6 (1.09 ± 0.08-fold) or TBG-1 (1.08 ± 0.07-fold) between fzr-1(bs31) mutant and wild-type embryos. Four biological samples and eight technical replicates were used. Average values are presented and error bars are SD. Right panel: Representative western blot using embryonic lysates from fzr-1(bs31) mutants and N2 animals. Tubulin was used as a loading control. (D) IP using anti-GFP suggests that FZR-1 physically interacts with SAS-5. Both SAS-5 isoforms (SAS-5A, SAS-5C) coprecipitate with GFP::FZR-1 or FZR-1::GFP. Wild-type (N2) control embryos were used as a negative control of IP; ∼1% of total embryonic lysates was loaded in the input lanes. > indicates a nonspecific detection by the SAS-5 antibody. Jeffrey C. Medley et al. G3 2017;7:3937-3946 ©2017 by Genetics Society of America