The recruitment of Syt1 induces an unstable swelling of migrasomes prior to the recruitment of TSPAN4. (A) Time-lapse imaging of NRK cells stably expressing Syt1-mCherry and TSPAN4-GFP. Confocal microscopy images were captured every 2 min 54 s. Scale bar, 2 μm. (B) Statistical analysis of normalized fluorescence intensity of Syt1 and TSPAN4 at migrasome formation sites during migrasome formation shown in A. Data are presented as mean ± SEM; n = 30. (C) Time-lapse imaging of NRK Syt1-GFP migrasome formation. Green signal, Syt1-GFP. The time-lapse imaging from top to bottom represent three independent migrasomes. Scale bar, 2 µm. (D) Time-lapse imaging of migrasomes from NRK Syt1-mCherry, NRK TSPAN4-GFP, and NRK Syt1-mCherry TSPAN4-GFP. Scale bar, 2 µm. (E) Statistical analysis of the shrinking back migrasomes to all migrasomes ratio shown in D. Data are presented as mean ± SEM; P values were calculated using a two-tailed, unpaired t test. n = 50. The experiments were performed three times. (F) Quantification of the life time of migrasome shown in D. Data represent the mean ± SEM. P values were calculated using a two-tailed, unpaired t test. n = 50 cells per group. The experiments were performed three times. (G) Time-lapse imaging of migrasomes from NRK Syt1-GFP treated with/without 1 mM Ca2+ for 12 h. Scale bar, 2 µm. (H) Quantification of the life time of migrasome shown in G. Data represent the mean ± SEM. P values were calculated using a two-tailed, unpaired t test. n = 100 cells per group. The experiments were performed three times.
