Fig 1. CUL-4 E3 ligase complex mutants exhibit PC formation in meiotic prophase I.

A) A model of the CRL4 E3 ligase complex and its components examined in this study, based on what is known about its biochemical activity and composition from studies in other systems. The complex is presented in two states: active (neddylated/upper) and inactivated (deneddylated/lower), the active form is depicted before and after ubiquitination of the substrate. Gray subunits represent deletion mutations that do not result in a phenotype (RBX-1/2) or show minor phenotype (CAND-1) B) Full germline images of wild type (top) and cul-4(ok1891) mutants (bottom); blue (DAPI) and red (SYP-1). White lines are the border between the image and a black background that was added so a rectangular shape will be created. Scale bars are 20μm. C) Graphical comparison of germline length in wild type, csn-5(ok1064), and cul-4(ok1891) mutants (Mann-Whitney; p-values, * < 0.001). D-J) Graphical analyses of SYP-1 localization analysis (“No SYP-1” was defined as having no SYP-1 immunofluorescence present along chromosomes (DAPI). “SYP-1 PC” were nuclei with only PC formation present, no elongation of SYP-1 along DAPI. “SYP-1 PC and Some Linear” was the presence of PC(s) in the nucleus but also partial elongation of SYP-1, <50% of DAPI. “SYP-1 PC and Linear” was similar to “SYP-1 PC and Some Linear” with the exception that SYP-1 is elongated along >50% of DAPI. “SYP-1 Partial Linear” nuclei had elongated SYP-1 along up to 50% of DAPI. “SYP-1 Mostly Linear” nuclei had SYP-1 along up to 50% of DAPI but less than 100%. “SYP-1 Linear” nuclei had fully elongated SYP-1 along all DAPI. “Other” was defined as nuclei that had abnormal DAPI appearance.); statistical comparisons of each mutant were made against wild type (Fischer’s exact test; p-values, * < 0.001). Scale bars are 2μm. K) Analysis of percentage of germlines with PC formation within each individual genotype. The number of PCs per germline were grouped into three main categories: No PCs = germlines with no SC PC formation, Few PCs = germlines no more than 5 PCs or less than ½ of a zone with PCs, and Abundant PCs = germlines with more than 5 PCs or greater than ½ of a zone with PCs. Statistical comparisons were made to wild type for each mutant individually (Fisher’s exact test; p-values, * < 0.0001). L) Analysis of homologous and non-homologous pairing by FISH to the 5S locus coupled with SYP-1 immunostaining in pachytene nuclei. In wild type the majority of nuclei are paired and synapsed (green) while in cul-4(ok1891) mutants less nuclei are paired. Unpaired FISH foci frequently associated with SYP-1 on at least one locus (purple and red) indicating non-homologous synapsis occurs in cul-4(ok1891) mutants regardless if the nuclei contain or do not contain PC. cul-4(ok1891) nuclei with PCs also contain FISH singnals that do not associate with SC (black and gray).