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. Author manuscript; available in PMC: 2022 May 11.
Published in final edited form as: Cell Rep. 2022 Apr 12;39(2):110652. doi: 10.1016/j.celrep.2022.110652

Figure 1. Engineered PAR-3 trimers are sufficient for aPAR segregation and normal development.

Figure 1.

(A) Illustration of our strategy to engineer PAR-3 to specific sizes. (i) During polarity establishment, PAR-3 clusters exist as a heterogeneous population of various sizes. (ii) PAR-3* monomers in which the oligomerization domain is disrupted are shown. (iii) Transgenic nAb::EOD constructs contain the EOD (trimer is shown as an example), a fluorescent label, and a GFP-binding nanobody. (iv) PAR-3 of controlled oligomer size was generated by combining YFP::PAR-3*(RRKEEE) and EOD constructs.

(B) YFP fluorescence intensity cortical YFP::PAR-3* in EOD-expressing embryos dissected from mlc-4 RNAi-treated worms. mlc-4 RNAi was used for these measurements to prevent crowding of PAR-3 clusters into the anterior domain, which makes accurately measuring intensity more difficult. Data show only clusters that are stabilized on the cortex for more than 10 frames (0.5 s), because these are the particle tracks we used in the quantitative analysis below. We obtained equivalent results with or without the track-length filter (Figure S1F). Red bars indicate means.

(C) Upper panels: live images of cortical YFP::PAR-3* in the indicated EOD strains. Scale bar: 10 μm. Lower panels: quantification of fluorescence intensity in anterior and posterior is shown. n = 3 embryos of each strain.

(D) Percentage of embryos from each EOD strain that displays effective PAR-3 polarization. n = 10 embryos of each strain.

(E) Cortical flow speed measured from DIC videos of live embryos dissected from wild-type, YFP::PAR-3*(RRKEEE), and YFP::PAR-3*; nAB::BFP::EOD strains. n = 3 embryos from each strain. Horizontal bars indicate means.

(F) Embryo lethality of each EOD strain. “Monomer” is a control construct comprising the nanobody and fluorescent tag but no EOD. Horizontal bars indicate means.

(G) Adult sterility of each EOD strain. Monomer is a control construct comprising the nanobody and fluorescent tag but no EOD. Horizontal bars indicate means.