PAR-3 and CDC-42 Have Opposing Regulatory Roles in an In Vivo PKC-3 Activity Assay
(A) C1B targeting strategy for inducing PKC-3 membrane loading by PMA. PKC-3 kinase activity is monitored by following loss of PAR-2 from the membrane.
(B) Zygotes expressing GFP::C1B alone (GFP::C1B-Ø) or GFP::C1B-PKC-3 along with mCherry::PAR-2 were subject to the indicated treatment. Note that uniform membrane targeting of C1B-PKC-3 leads to reduction of PAR-2 domain size, whereas omitting PMA or expressing C1B alone has no effect. Right: cartoon representation of results.
(C) Quantification of PAR-2 domain size ratio for embryos shown in (B).
(D) PAR-2 retention in GFP::C1B-PKC-3 expressing zygotes treated with PMA and CRT90 confirms that induced PAR-2 loss is dependent on PKC-3 kinase activity.
(E) Zygotes expressing mCherry::PAR-2 with GFP::C1B-Ø or GFP::C1B-PKC-3 subject to par-6, cdc-42, or par-3(RNAi) before and 5 min after PMA addition.
(F) Quantification of PAR-2 cortex retention comparing GFP::C1B-PKC-3 and GFP::C1B-Ø zygotes after treatment with PMA as in (E).
Representative midsection confocal images are shown in (B), (D), and (E) before and 5 min after PMA/DMSO addition. ∗∗∗p < 0.001. ns, not significant. Scale bars, 10 μm. See also Figure S7; Movies S6 and S7.