Figure 2.

Membrane Localization of PAR-6/PKC-3 Is Decoupled from PAR-3 when PKC-3 Is Inactive

(A–F) Representative midsection confocal images of live and fixed zygotes at establishment and maintenance phase comparing control (DMSO, wild-type), pkc-3(RNAi), and PKC-3-inhibited (CRT90, pkc-3(ts)) conditions. PAR-6 (A and B) and PKC-3 (C and D) show loss of asymmetric membrane staining in PKC-3-inhibited zygotes at both establishment and maintenance phase (posterior localization indicated by white arrowheads). In pkc-3(RNAi), PAR-6 is absent from the membrane at all times. PAR-3 (E and F) still polarizes in PKC-3-inhibited zygotes, but becomes weaker and less asymmetric during maintenance phase. Note that (B), (D), and (F) show the same wild-type and TS zygotes with the PAR-3 boundary position in TS indicated (red arrowheads) to allow comparison: PAR-6 and PKC-3 are clearly visible at the posterior membrane (white arrowheads), while PAR-3 is undetectable, as in wild-type. Bright foci in (D) are non-specific centrosome staining.

(G and H) Normalized ASI measurements for late establishment phase datasets represented in (A) to (F). ASI is normalized to control (wild-type [WT] or DMSO) for each protein.

(I and J) Anterior to posterior membrane distributions of PAR-3 (red) and PKC-3 (black) in wild-type (I) and pkc-3(ts) (J) embryos. Arrows highlight the posterior extension of PKC-3 relative to PAR-3. Mean ± SD is shown.

(K) Close-up view of the boundary region showing PAR-3 (top) and PKC-3 (bottom) for one representative zygote for wild-type (WT) and pkc-3(ts) backgrounds as indicated. Dashed rectangular selection denotes regions where PKC-3 is present in absence of PAR-3.

(L) PKC-3 to PAR-3 ASI ratio for wild-type (WT) and pkc-3(ts).

(M) Dual labeling of PAR-2 and PKC-3 in live, CRT90-treated zygotes (top) and fixed, pkc-3(ts) embryos (bottom) reveal overlap of aPAR and pPAR proteins.

^∗∗p < 0.01, ^∗∗∗p < 0.001, ^∗∗∗∗p < 0.0001. ns, not significant. Scale bars, 10 μm. See also Figures S3 and S4; Movie S2.