Dynamic Pk1 puncta and plaques at separating cell–cell contacts. (A) PCM expressing Pk1-venus and mb-RFP. Punctate Pk1 (arrows) at cell–cell contacts. BCR is to the top. Scale bar, 10 μm. (B) Fractions of Pk1 plaques (>1 μm) at lateral cell contacts, cell tails, and front ends. Count of plaques was pooled from multiple experiment replicates to calculate fractions. Error bars indicate SD. n = 225. (C and D) Dynamics of Pk1 puncta at cell contacts in PCM expressing Pk1-venus and mb-RFP. Time in minutes. Scale bars, 10 μm. (E and F) Dynamics of separation at puncta-stained (E; Video 6) and puncta-less (F) cell contacts in PCM expressing Pk1-venus and mb-RFP. Time in minutes. Scale bars, 10 μm. (G) Separation angle θ_s and membrane curvature angle θ_c at cell contact between front and rear ends of migrating PCM cells. (H and I) Membrane curvature angle θ_c of each cell (puncta-stained contacts, n = 15; puncta-less contacts, n = 11) and contact angle θ_s during tail retraction (puncta-stained contacts, n = 18; puncta-less contacts, n = 18). ***, P ≤ 0.001; ****, P ≤ 0.0001 in a two-tailed Student’s t test. Data distribution was assumed to be normal but was not formally tested. (J) Rates of contact shortening during cell separation. Each data point represents the average changes of contact lengths from each contact over the period of separation (WT, n = 8; puncta-stained, n = 6; puncta-less, n = 5). ****, P ≤ 0.0001 in a two-tailed Student’s t test. Data distribution was assumed to be normal but was not formally tested. (K) Rates of puncta movement (n = 161), growth (n = 166), and shrinkage (n = 178) at contacts, calculated by changes in puncta lengths. n, number of puncta.