Supplementary Figure 2: Cortical tension by actomyosin contractility stimulates clustering of PAR-3. | Nature Cell Biology

Supplementary Figure 2: Cortical tension by actomyosin contractility stimulates clustering of PAR-3.

From: Cortical forces and CDC-42 control clustering of PAR proteins for Caenorhabditis elegans embryonic polarization

Supplementary Figure 2: Cortical tension by actomyosin contractility stimulates clustering of PAR-3.

(a) Representative images of live zygotes expressing PAR-3::GFP, PKC-3::GFP, and GFP::CDC-42 under control, par-6(RNAi), par-3(RNAi), and pkc-3(RNAi) conditions. All images are representative of at least 10 zygotes from 3 independent experiments. Scale bar, 5 μm. PAR-3::GFP formed cortical clusters in par-6(RNAi) and pkc-3(RNAi) zygotes, while PKC-3::GFP no longer appeared as clusters in par-3(RNAi) zygotes. Treatment with par-3(RNAi), pkc-3(RNAi), and par-6(RNAi) caused GFP::CDC-42 to appear throughout the cortex. (b) Time-lapse sequence of PAR-3::GFP cluster assembly in nmy-2(ne3409) zygotes treated with hypotonic buffer. The formation of 20 clusters was tracked from 2 zygotes, and the graphs show changes in intensity over time for 10 clusters. The times stated are with respect to the appearances of PAR-3::GFP cluster. PAR-3::GFP appeared as a small puncta at the cortex and progressively increased in intensity. (c) The histograms show the distribution of intensity of PAR-3::GFP and PKC-3::GFP clusters in nmy-2(ne3409) zygotes treated with or without hypotonic buffer and nmy-2(ne3409) zygotes depleted of CDC-42. Black and grey bars represent clusters with an R2 value from 2D Gaussian fitting over and below 0.6, respectively. Mean ± s.d. from n = 7 zygotes for control, n = 6 zygotes for PAR-3::GFP in nmy-2(ne3409) treated with hypotonic buffer, and n = 5 zygotes each for other conditions. (d) The histograms show the distribution of cortical cluster intensity of GFP-tagged PAR-3 in NIH3T3 cells treated with or without hypotonic buffer in the presence of Blebbistatin. Black and grey bars represent clusters with an R2 value from 2D Gaussian fitting over and below 0.6, respectively. Mean ± s.d. from n = 12 cells for control and n = 24 cells each for all other conditions. (e) Representative images of live NIH3T3 cells expressing GFP-tagged C. elegans PAR-3 with or without treatment with 1 μM Latrunculin-A, a drug that depolymerize filamentous actin. All images are representative of 5 cells at each condition from 3 independent experiments.

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