A genomewide screen for suppressors of par-2 uncovers potential regulators of PAR protein-dependent cell polarity in Caenorhabditis elegans

Abstract

The PAR proteins play an essential role in establishing and maintaining cell polarity. While their function is conserved across species, little is known about their regulators and effectors. Here we report the identification of 13 potential components of the C. elegans PAR polarity pathway, identified in an RNAi-based, systematic screen to find suppressors of par-2(it5ts) lethality. Most of these genes are conserved in other species. Phenotypic analysis of double-mutant animals revealed that some of the suppressors can suppress lethality associated with the strong loss-of-function allele par-2(lw32), indicating that they might impinge on the PAR pathway independently of the PAR-2 protein. One of these is the gene nos-3, which encodes a homolog of Drosophila Nanos. We find that nos-3 suppresses most of the phenotypes associated with loss of par-2 function, including early cell division defects and maternal-effect sterility. Strikingly, while PAR-1 activity was essential in nos-3; par-2 double mutants, its asymmetric localization at the posterior cortex was not restored, suggesting that the function of PAR-1 is independent of its cortical localization. Taken together, our results identify conserved components that regulate PAR protein function and also suggest a role for NOS-3 in PAR protein-dependent cell polarity.

Figure 1.—

nos-3(q650) suppresses the early embryonic phenotypes of par-2 mutants. (A) While nos-3(q650) embryos have a wild-type pattern of early development (n = 22), such a pattern is rarely observed in par-2(it5ts) (n = 24) or in par-2(lw32) (n = 19) mutant embryos. This phenotype is suppressed in nos-3(q650); par-2(it5ts) (n = 22) and nos-3(q650); par-2(lw32) (n = 31) double-mutant embryos. (B–I) Images from time-lapse movies of embryos undergoing first (B, D, F, and H) or second (C, E, G, and I) division. Phenotypes were scored in wild-type (B and C), nos-3(q650) (D and E), par-2(it5ts) (F and G), or nos-3(q650); par-2(it5ts) (H and I) embryos at 25°. In B–I, anterior is to the left and arrowheads indicate centrosome positions.

Figure 2.—

PAR-3 and PAR-6, but not PAR-2 and PAR-1, localization is restored in nos-3(q650); par-2(it5ts) mutants. (A–D) In wild-type embryos, PAR-3 (A) and PAR-6 (B) localize at the anterior cortex while PAR-2 (C) and PAR-1 (D) localize at the posterior cortex. (E–H) In nos-3 mutants, PAR protein localization is as in wild type: 100% of embryos showed anterior PAR-3 (E, n = 32), anterior PAR-6 (F, n = 24), posterior PAR-2 (G, n = 20), and posterior PAR-1 (H, n = 27). (I–L) In par-2(it5ts) mutants, the localization of PAR-3 (I) and PAR-6 (J) is expanded toward the posterior pole (in 90% of embryos, n = 19 for PAR-3 and n = 29 for PAR-6) whereas PAR-2 (K) and PAR-1 (L) are absent from the cortex (in 100% of embryos, n = 15 each). (M–P) In nos-3(q650); par-2(it5ts) mutants, wild-type localization of PAR-3 (M) and PAR-6 (N) is restored [in 68% of embryos for PAR-3 (n = 22) and in 74% of embryos for PAR-6 (n = 19)] whereas PAR-2 (O) and PAR-1 (P) are absent from the cortex [in 100% of embryos for PAR-2 (n = 20) and in 91% of embryos for PAR-1 (n = 22)]. All animals were shifted to 25° at 24 hr before dissection and staining. In A–P, anterior is to the left.

Figure 3.—

Fertility and P-granule localization are restored in nos-3(q650); par-2(it5ts) mutants. (A) Proportion of sterile animals determined for the indicated genotype at 15° and 25°. Maternal-effect sterility was scored in all cases except in par-2 mutant animals at 25° because of embryonic lethality. However, escapers were previously shown to have a fully penetrant sterile phenotype (Watts et al. 1996). (B–E) P-granule localization in wild-type (B), nos-3(q650) (C), par-2(it5ts) (D), and nos-3(q650); par-2(it5ts) (E) four-cell-stage embryos. In every wild-type and nos-3 embryo observed, P granules were restricted to the posterior-most cell (n = 10) whereas they were localized in several cells in all embryos mutant for par-2(it5ts) (n = 10). P granules were restricted to the posterior-most cell in 66% of nos-3(q650); par-2(it5ts) embryos (n = 18). All animals were shifted to 25° at 24 hr before dissection and staining. In B–E, anterior is to the left.