Figure 4.
Rescue of monomeric PAR-3 segregation by ectopic membrane targeting and/or clustering. (A) Schematic of rescue constructs fused to mNG::PAR-3(∆69–82) shown along with example images of embryos taken at the cortex or at midplane at establishment and maintenance phases. Arrowheads indicate regions with posterior localization (RitC). Note midplane images are shown with identical intensity scaling to highlight relative levels, while cortex images have been individually scaled to improve visibility of the clusters at the cortex. Scale bars = 10 µm. (B) Quantification of membrane to cytoplasm (M:C) ratio at the anterior and posterior pole at maintenance phase highlights reduced ability of PAR-3(RitC) to remain excluded from the posterior pole compared to clustered variants (Tukey’s multiple comparison test). (C) Posterior clearance is unaffected by par-1(RNAi). M:C ratios at the posterior pole were calculated from embryos subject to ctl or par-1(RNAi). Mean values indicated. Differences between ctl and par-1 were not significant, unpaired t test, Holm-Šídák correction. (D) Fit values for advection velocity parallel (υx, red) and perpendicular (υy, blue) relative to the flow axis are similar for all rescued variants. Lines connect paired values from single embryos. (E) Coupling coefficients are similar for all variants. Coupling coefficients calculated for all molecules in individual embryos are shown with means. Tukey’s multiple comparison test—variants are not significantly different from WT or between themselves. (F and G) All PAR-3 variants exhibit similar increases in diffusivity relative to PAR-3(WT). HaloTag labeling was used to sparsely label PAR-3 variants and effective diffusivity calculated from mean perpendicular step size for τ = 0.2 s. Mean diffusivity per embryo (F) and for all molecules (G) shown with mean indicated (RitC: 11,692 particles, 15 embryos, 2mer: 5,519 particles, 12 embryos, 4mer: 2,376 particles, 8 embryos). WT and PAR-3(∆69–82) data reproduced from Fig. 3 F for comparison. Mean diffusivity per embryo values for variants are significantly different from wild type, but not from each other. Tukey’s multiple comparison test. (H) Effect of clustering on membrane-tethered PAR-3. GFP fusions to either PAR-3(WT) or PAR-3(∆69–82) were tethered to the membrane via a membrane-associated nanobody (PH::GBP) and their ability to segregate in response to flows tested. Note that PAR-3(WT) segregates normally and is fully excluded from the posterior pole despite constitutive targeting to the plasma membrane and is absent from the posterior P1 daughter cell. By contrast, PAR-3(∆69–82) is less efficiently cleared and partially reinvades the posterior once flows cease such that it is also present on the plasma membrane of the P1 daughter cell. Arrowheads highlight posterior GFP signal. Scale bar = 10 µm.

Rescue of monomeric PAR-3 segregation by ectopic membrane targeting and/or clustering. (A) Schematic of rescue constructs fused to mNG::PAR-3(∆69–82) shown along with example images of embryos taken at the cortex or at midplane at establishment and maintenance phases. Arrowheads indicate regions with posterior localization (RitC). Note midplane images are shown with identical intensity scaling to highlight relative levels, while cortex images have been individually scaled to improve visibility of the clusters at the cortex. Scale bars = 10 µm. (B) Quantification of membrane to cytoplasm (M:C) ratio at the anterior and posterior pole at maintenance phase highlights reduced ability of PAR-3(RitC) to remain excluded from the posterior pole compared to clustered variants (Tukey’s multiple comparison test). (C) Posterior clearance is unaffected by par-1(RNAi). M:C ratios at the posterior pole were calculated from embryos subject to ctl or par-1(RNAi). Mean values indicated. Differences between ctl and par-1 were not significant, unpaired t test, Holm-Šídák correction. (D) Fit values for advection velocity parallel (υx, red) and perpendicular (υy, blue) relative to the flow axis are similar for all rescued variants. Lines connect paired values from single embryos. (E) Coupling coefficients are similar for all variants. Coupling coefficients calculated for all molecules in individual embryos are shown with means. Tukey’s multiple comparison test—variants are not significantly different from WT or between themselves. (F and G) All PAR-3 variants exhibit similar increases in diffusivity relative to PAR-3(WT). HaloTag labeling was used to sparsely label PAR-3 variants and effective diffusivity calculated from mean perpendicular step size for τ = 0.2 s. Mean diffusivity per embryo (F) and for all molecules (G) shown with mean indicated (RitC: 11,692 particles, 15 embryos, 2mer: 5,519 particles, 12 embryos, 4mer: 2,376 particles, 8 embryos). WT and PAR-3(∆69–82) data reproduced from Fig. 3 F for comparison. Mean diffusivity per embryo values for variants are significantly different from wild type, but not from each other. Tukey’s multiple comparison test. (H) Effect of clustering on membrane-tethered PAR-3. GFP fusions to either PAR-3(WT) or PAR-3(∆69–82) were tethered to the membrane via a membrane-associated nanobody (PH::GBP) and their ability to segregate in response to flows tested. Note that PAR-3(WT) segregates normally and is fully excluded from the posterior pole despite constitutive targeting to the plasma membrane and is absent from the posterior P1 daughter cell. By contrast, PAR-3(∆69–82) is less efficiently cleared and partially reinvades the posterior once flows cease such that it is also present on the plasma membrane of the P1 daughter cell. Arrowheads highlight posterior GFP signal. Scale bar = 10 µm.

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