RT-PCR

To determine the genetic landscape of fkb-6(tm2614), we performed RT-PCR analysis. This was done using the Superscript III OneStep RT PCR kit (12574-026; Thermo Fisher Scientific) and primers 5′-CTTGAAGCGCTTCTTGTCACGC-3′ and 5′-CCAACCACTGGAACGACCGTG-3′. Our analysis revealed a deletion of the last 119 amino acids of the 431-aa protein. There was also a simultaneous insertion of 52 amino acids. This deletion removes the third TPR domain and part of the second TPR domain. Our analysis also showed that transcript levels are not different from a wild-type background. RT-PCR of other fkb-6 mutations, created through CRISPR-Cas9, was performed. Using the technique described earlier, we found that fkb-6(iow4) is a 230-bp deletion that contains a 166-bp deletion of the promoter and 64-bp of the first exon; this deleted region encodes for the first 79 amino acids of the FKB-6 protein, which includes the first PP domain. Another small deletion mutation leading to sterility, fkb-6(iow3), was isolated and had a frameshift around the PAM site of the sgRNA (5′-GTTGAAACTCATCAAGAAGGAGG-3′). Both of these mutants were examined by DAPI and SYP-1 staining and were shown to exhibit a phenotype indistinguishable from that of the fkb-6(tm2614) allele.

Stage L4 worms were moved to individual NGM plates and allowed to lay eggs for 24 h. These single adult worms were then moved to new NGM plates and allowed to lay eggs again. Adult worms were moved a total of three times. These experiments were performed with three different worms for N2 and fkb-6(tm2614) strains.

Adult hermaphrodites were dissected 20–24 h after L4 to release gonads. Antibodies used for immunostaining as in Colaiácovo et al. (2003) are as follows: SYP-1 (1:500), RAD-51 (1:10,000; ModEncod), SYP-2 (1:100; gift from A. Villeneuve, Stanford University, Stanford, CA), SYP-4 (1:100), HTP-3 (1:500; gift from A. Dernburg, University of California, Berkeley, Berkeley, CA), HTP-1/2 (1:500; gift from A. Dernburg, McGill University, Montreal, Canada), HIM-3 (1:500; gift from M. Zetka, University of Iowa, Iowa City, IA), LAB-1 (1:300; gift from M. Zetka), agglutinin (1:10, FL-1071; Vector Laboratories), phalloidin (1:10, A12380; Molecular Probes), KT3 (1:10, PGL-3; Developmental Studies Hybridoma Bank), SUN-1 (1:500; Novus Biologicals), and HIM-8 (1:1,000; Sdix). Secondary antibodies used were α-goat Alexa Fluor 549, α-rabbit Alexa Fluor 488, α-guinea pig Alexa Fluor 488, and α-mouse Alexa Fluor 488. HIM-8 (1:500; gift from A. Dernburg) staining was done as in Nabeshima et al. (2004). Secondary antibody was α-guinea pig Alexa Fluor 488. V5 staining was performed with the V5 antibody (1:200; Novus Biologicals) and was performed as in Reid et al. (2014), with a few minor changes. Methanol fix after dissection was limited to 1 min, and acetone fix was removed. Formaldehyde fix was lowered to 2 h from 12 h. Secondary antibody was α-rabbit Cy3 (gift from J. Weiner). Staining for phosphorylated histone 3 (PH3) was done as in Hsu et al. (2000). PH3 antibody (1:100; EMD Millipore) staining was performed with adult hermaphrodites dissected 20–24 h after L4. The secondary antibody used was α-rabbit Alexa Fluor 555. Microtubule staining was done as in Dennis et al. (2012). 12G10 (1:10; Developmental Studies Hybridoma Bank) antibody staining was done with adult hermaphrodites dissected 20–24 h after L4. Secondary antibody used was α-mouse Alexa Fluor 488. All gonads were stained with DAPI (1:2,000 dilution of a 5-mg/ml DAPI stock in PBS-Tween) for 10 min.