Induction of Pk1 puncta. (A) Pk1 puncta and plaque dynamics at surface of Pk1-venus– and mb-RFP–labeled PCM cells. Pk1 puncta/plaques can fuse (arrows). (B) Dissociated PCM cells labeled with Pk1-venus and mb-RFP on BSA, FN, and plastic. Arrows indicate Pk1 puncta outside the contact in cell pairs. Scale bars, 10 μm. Time in minutes. Scale bars, 10 μm. (C) Number of puncta per cell in single cells, pairs, and small clusters (≤15 cells). On BSA: single cells, n = 49; cell pairs and clusters, n = 28. On FN: single cells, n = 18; cell pairs and clusters, n = 32. On plastic: single cells, n = 12; cell pairs and clusters, n = 12. n, number of cells. **, P ≤ 0.01; ****, P ≤ 0.0001 in a two-tailed Student’s t test. Data distribution was assumed to be normal but was not formally tested. (D) Single Pk1-venus–labeled PCM cells on BSA in 1× MBS, 2× MBS (H), 0.25× MBS (L), or 0.25 M sorbitol in 1× MBS. Scale bars, 10 μm. (E) Fraction of PCM cells showing Pk1 puncta formation. Count of puncta induced cells was pooled from multiple experiment replicates to calculate fractions. Control, n = 40; H MBS, n = 89; L MBS, n = 23; sorbitol, n = 71. n, number of cells. ****, P ≤ 0.0001 in a two-tailed Student’s t test. Data distribution was assumed to be normal but was not formally tested. (F) Fractions of induced Pk1 puncta that are <1, 1–5, or >5 μm long in single PCM cells. Puncta count was pooled from multiple experiment replicates to calculate the fractions. H MBS, n = 36; sorbitol, n = 48. (G) Number of cell surface puncta per cell. H MBS, n = 36; sorbitol, n = 48. Two-tailed Student’s t test. Data distribution was assumed to be normal but was not formally tested.