Figure 5.
Regulation of PKC-3/PAR-6 Cluster Association by PAR-3/CDC-42 Balance Tunes Responsiveness to Cortical Flows
(A) Representative cortical images of PAR-6::GFP at late-establishment and maintenance-phase embryos for indicated conditions, shown along with a zoom of inset region (white boxes).
(B) Cluster index measurements of full dataset reveal a gradient of cluster association across conditions. Note that clustering decreases when embryos enter maintenance phase, except for CRT90/par-3(RNAi) embryos, which show minimal clustering even in establishment phase.
(C) ASI measurements of midsection images taken at late-establishment phase for a similar set of embryos as in (A) and (B), but expressing GFP::PKC-3, show a similar trend.
(D) Profiles of membrane signal for zygotes in (C) showing average (solid line) and full range of data (shaded) reveal shift of the PKC-3 domain boundary (arrows) toward the anterior in cdc-42(RNAi) embryos and toward the posterior in CRT90-treated zygotes, resulting in significantly (p < 0.01) smaller and larger domain sizes, respectively.
(E) Combining clustering data from pseudo-TIRF imaging in (A) and (B) and (F) and (G) with ASI measurements of a complete GFP::PKC-3 dataset for late-establishment phase across all conditions allows us to plot cluster index versus ASI for the mean of each condition, revealing a strong correlation (linear regression: R2 = 0.85, p < 0.01).
(F–I) Representative cortical images (F, full zygote and inset zoom), cluster index (G), representative midsection images (H), and ASI (I) for late-establishment phase zygotes expressing GFP::PKC-3 in combination with either CDC-42(WT) or CDC-42 (Q61L). Yellow arrowheads in (H) highlight PKC-3 domain boundaries.
(J) Profiles of membrane signal for zygotes in (I) showing average (solid line) and full range of data (shaded) highlight the posterior shift (black arrow) of the PKC-3 boundary in Q61L-expressing zygotes (p < 0.05). Profiles for wild-type (solid black line) and CRT90-treated (dashed line) from (D) shown for comparison.
(K) Quantification of the difference in boundary position between PAR-3 and PKC-3 in dual-labeled fixed zygotes for indicated conditions. Mean ± 95% CI (N) is shown. Positive values indicate reduced PKC-3 segregation relative to PAR-3. Representative images of PAR-3 and PKC-3 in zygotes overexpressing CDC-42(WT) of CDC-42 (Q61L). Yellow arrowheads indicate the posterior boundary of the PAR-3 or PKC-3 domains.
(L and M) Comparison of cortical flow velocities (L) and PKC-3 asymmetry (M, ASI) for DMSO, CRT90, or mlc-4(RNAi) embryos taken at late-establishment phase.
(N) A plot of PKC-3 ASI versus cortical flow rates for individual zygotes treated with DMSO, CRT90, or mlc-4(RNAi). Data points for individual embryos are shown with a 90% confidence boundary (shaded region).
∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. ns, not significant. Scale bars, 10 μm. See also Figure S6.