Figure 3.
Advective velocity of PAR-3 is independent of clustering. (A) Schematic of full-length PAR-3 and PAR-3(∆69–82) and a crystal structure of the PAR-3 CR1 domain dimer (rat PAR3-NTD, PDB accession no. 4I6P) are shown. Individual CR1 molecules are shown in gray and blue. Amino acids 69–82 (orange) are located at the oligomerization interface and color-coded in the schematics. (B) PAR-3(∆69–82) does not accumulate on the anterior plasma membrane. Embryos expressing mNG fusions to either PAR-3(WT) or PAR-3(∆69–82) are shown in a cortical plane by HILO microscopy (Pre-Symmetry-Breaking, Establishment) or at midplane (Maintenance). Note general lack of signal at the plasma membrane, consistent with the requirement of oligomerization for stable membrane association. Scale bar, 10 µm. Time mm::ss relative to symmetry breaking. (C–E) PAR-3(∆69–82) leads to loss of P0 division asymmetry, embryonic lethality, and sterility. Data for Halo::PAR-3 fusions shown (see Fig. S1 for mNG::PAR-3). (C) Daughter cell asymmetry (AreaAB/AreaAB + P1). (D) Percentage of embryos reaching L4 stage. (E) Surviving adults exhibiting sterility. Individual embryos (C) or replicates (D and E) shown with mean indicated. (F) PAR-3(∆69–82) exhibits increased diffusivity relative to PAR-3(WT). HaloTag labeling was used to sparsely label PAR-3(∆69–82) and effective diffusivity calculated from mean perpendicular step size for τ = 0.2 s (WT: 12,810 particles, 13 embryos, ∆69–82: 3,616 particles, 14 embryos). (i and ii) Distribution of diffusivities for all molecules, mean indicated (i) and mean diffusivity per embryo, means indicated (ii) (WT data reproduced from Fig. 2 G for comparison). (G) PAR-3(∆69–82) displacements are biased along the flow axis. Plots of per-embryo mean displacements parallel and perpendicular to the flow axis for τ = 0.5 s. (H) PAR-3(∆69–82) couples to cortical flow. Coupling coefficients for PAR-3(∆69–82) shown for all molecules or molecules binned by diffusivity shown compared to PAR-3(WT) and NMY-2. Datapoints for PAR-3(WT) and NMY-2 represent single embryos. Due to reduced numbers of molecules per embryo for PAR-3(∆69–82) embryos, each data point in PAR-3(∆69–82) bins reflects molecules combined from multiple embryos to obtain sufficient numbers for fitting. Due to small sample size, significance assessed by Mann–Whitney U test.

Advective velocity of PAR-3 is independent of clustering. (A) Schematic of full-length PAR-3 and PAR-3(∆69–82) and a crystal structure of the PAR-3 CR1 domain dimer (rat PAR3-NTD, PDB accession no. 4I6P) are shown. Individual CR1 molecules are shown in gray and blue. Amino acids 69–82 (orange) are located at the oligomerization interface and color-coded in the schematics. (B) PAR-3(∆69–82) does not accumulate on the anterior plasma membrane. Embryos expressing mNG fusions to either PAR-3(WT) or PAR-3(∆69–82) are shown in a cortical plane by HILO microscopy (Pre-Symmetry-Breaking, Establishment) or at midplane (Maintenance). Note general lack of signal at the plasma membrane, consistent with the requirement of oligomerization for stable membrane association. Scale bar, 10 µm. Time mm::ss relative to symmetry breaking. (C–E) PAR-3(∆69–82) leads to loss of P0 division asymmetry, embryonic lethality, and sterility. Data for Halo::PAR-3 fusions shown (see Fig. S1 for mNG::PAR-3). (C) Daughter cell asymmetry (AreaAB/AreaAB + P1). (D) Percentage of embryos reaching L4 stage. (E) Surviving adults exhibiting sterility. Individual embryos (C) or replicates (D and E) shown with mean indicated. (F) PAR-3(∆69–82) exhibits increased diffusivity relative to PAR-3(WT). HaloTag labeling was used to sparsely label PAR-3(∆69–82) and effective diffusivity calculated from mean perpendicular step size for τ = 0.2 s (WT: 12,810 particles, 13 embryos, ∆69–82: 3,616 particles, 14 embryos). (i and ii) Distribution of diffusivities for all molecules, mean indicated (i) and mean diffusivity per embryo, means indicated (ii) (WT data reproduced from Fig. 2 G for comparison). (G) PAR-3(∆69–82) displacements are biased along the flow axis. Plots of per-embryo mean displacements parallel and perpendicular to the flow axis for τ = 0.5 s. (H) PAR-3(∆69–82) couples to cortical flow. Coupling coefficients for PAR-3(∆69–82) shown for all molecules or molecules binned by diffusivity shown compared to PAR-3(WT) and NMY-2. Datapoints for PAR-3(WT) and NMY-2 represent single embryos. Due to reduced numbers of molecules per embryo for PAR-3(∆69–82) embryos, each data point in PAR-3(∆69–82) bins reflects molecules combined from multiple embryos to obtain sufficient numbers for fitting. Due to small sample size, significance assessed by Mann–Whitney U test.

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