Figure 1. Ca 2 -binding Syt1 is required... | Rockefeller University Press

Figure 1.

Ca2+-binding Syt1 is required for migrasome formation. (A) Confocal image of migrasomes of L929 cells treated with 0, 2.5, 5, and 10 μM BAPTA and cell migrate on chamber overnight for 12 h. L929 are labeled with wheat germ agglutinin (WGA) (green). Scale bar, 10 μm. (B) The number of migrasomes per 100 μm RF length per cell from images like those shown in A was quantified. Data shown 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. (C) Confocal image of migrasomes from NRK and NRK Syt1-GFP. Cells are labeled with WGA (magenta). Scale bar, 10 µm or 2 µm (inset). (D) Quantification of the number of migrasomes per 100 μm RF length per cell in NRK and NRK Syt1-GFP cells shown in C. 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. (E) Quantification of the Max Feret’s diameter of migrasomes per cell in NRK and NRK Syt1-GFP cells shown in C. 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. (F) Confocal image of migrasomes from NRK Syt1-GFP treated with/without 1 mM Ca2+ for 12 h. Scale bar, 10 µm. (G) Quantification of the number of migrasomes per 100 μm RF length per cell in NRK Syt1-GFP cells shown in F. Data represent the mean ± SEM. P values were calculated using a two-tailed, unpaired t test. n = 50 for NRK Syt1-GFP; n = 50 for NRK Syt1-GFP treated with Ca2+. The experiments were performed three times. (H) Confocal image of migrasomes from NRK, NRK Syt1-GFP, and NRK Syt1-GFP treated with 5 μM BAPTA for 12 h. NRK are labeled with WGA (magenta). Scale bar, 10 μm. (I) The number of migrasomes per 100 μm RF length per cell from H was quantified. Data shown 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. (J) Schematic representation of Syt1 showing transmembrane domain, C2A domain, C2B domain, the phospholipid binding region, the calcium-binding sites, and the sites on the tips of membrane-binding loops. (K) Confocal image of migrasomes from NRK, NRK Syt1-mCherry, NRK Syt1(△C2A)-mCherry, NRK Syt1(△C2B)-mCherry, and NRK Syt1(△C2AB)-mCherry. The △C2A represents the truncation of C2A, the △C2B represents the truncation of C2B. The △C2AB represents the truncation of the phospholipid binding region. Cells are labeled with WGA (green). Scale bar, 10 µm. (L) Quantification of the number of migrasomes per 100 μm RF length per cell in NRK, NRK Syt1-mCherry, NRK Syt1(△C2A)-mCherry, NRK Syt1(△C2B)-mCherry, and NRK Syt1(△C2AB)-mCherry cells shown in K. Data represent the mean ± SEM. P values were calculated using a two-tailed, unpaired t test. n = 51 cells per group. The experiments were performed three times. (M) Confocal image of migrasomes from NRK, NRK Syt1-GFP, NRK Syt1(C2A*)-GFP, NRK Syt1(C2B*)-GFP, and NRK Syt1(C2A*B*)-GFP. The C2A* represents the calcium—binding site mutant (D178A, D230A, and D232A) on C2A, the C2B* represents the calcium—binding site mutant (D309A, D363A, and D365A) on C2B, the C2A*B* represents the calcium—binding site mutant (D178A, D230A, D232A, D309A, D363A, and D365A) on C2AB. Cells are labeled with WGA (magenta). Scale bar, 10 µm. (N) Quantification of the number of migrasomes per 100 μm RF length per cell in NRK, NRK Syt1-GFP, NRK Syt1(C2A*)-GFP, NRK Syt1(C2B*)-GFP, and NRK Syt1(C2A*B*)-GFP cells shown in M. 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. (O) Confocal image of migrasomes from NRK Syt1-GFP and NRK Syt1(4A)-GFP. The Syt1(4A) represent the mutant on the tips of the Syt1 membrane-binding loops (M173A, F234A, V304A, and I367A). Scale bar, 10 µm. (P) Quantification of the number of migrasomes per 100 μm RF length per cell in NRK Syt1-GFP and NRK Syt1 (4A)-GFP shown in O. Data represent the mean ± SEM. P values were calculated using a two-tailed, unpaired t test. n = 51 cells per group. The experiments were performed three times.

Ca ²⁺ -binding Syt1 is required for migrasome formation. (A) Confocal image of migrasomes of L929 cells treated with 0, 2.5, 5, and 10 μM BAPTA and cell migrate on chamber overnight for 12 h. L929 are labeled with wheat germ agglutinin (WGA) (green). Scale bar, 10 μm. (B) The number of migrasomes per 100 μm RF length per cell from images like those shown in A was quantified. Data shown 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. (C) Confocal image of migrasomes from NRK and NRK Syt1-GFP. Cells are labeled with WGA (magenta). Scale bar, 10 µm or 2 µm (inset). (D) Quantification of the number of migrasomes per 100 μm RF length per cell in NRK and NRK Syt1-GFP cells shown in C. 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. (E) Quantification of the Max Feret’s diameter of migrasomes per cell in NRK and NRK Syt1-GFP cells shown in C. 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. (F) Confocal image of migrasomes from NRK Syt1-GFP treated with/without 1 mM Ca²⁺ for 12 h. Scale bar, 10 µm. (G) Quantification of the number of migrasomes per 100 μm RF length per cell in NRK Syt1-GFP cells shown in F. Data represent the mean ± SEM. P values were calculated using a two-tailed, unpaired t test. n = 50 for NRK Syt1-GFP; n = 50 for NRK Syt1-GFP treated with Ca²⁺. The experiments were performed three times. (H) Confocal image of migrasomes from NRK, NRK Syt1-GFP, and NRK Syt1-GFP treated with 5 μM BAPTA for 12 h. NRK are labeled with WGA (magenta). Scale bar, 10 μm. (I) The number of migrasomes per 100 μm RF length per cell from H was quantified. Data shown 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. (J) Schematic representation of Syt1 showing transmembrane domain, C2A domain, C2B domain, the phospholipid binding region, the calcium-binding sites, and the sites on the tips of membrane-binding loops. (K) Confocal image of migrasomes from NRK, NRK Syt1-mCherry, NRK Syt1(△C2A)-mCherry, NRK Syt1(△C2B)-mCherry, and NRK Syt1(△C2AB)-mCherry. The △C2A represents the truncation of C2A, the △C2B represents the truncation of C2B. The △C2AB represents the truncation of the phospholipid binding region. Cells are labeled with WGA (green). Scale bar, 10 µm. (L) Quantification of the number of migrasomes per 100 μm RF length per cell in NRK, NRK Syt1-mCherry, NRK Syt1(△C2A)-mCherry, NRK Syt1(△C2B)-mCherry, and NRK Syt1(△C2AB)-mCherry cells shown in K. Data represent the mean ± SEM. P values were calculated using a two-tailed, unpaired t test. n = 51 cells per group. The experiments were performed three times. (M) Confocal image of migrasomes from NRK, NRK Syt1-GFP, NRK Syt1(C2A*)-GFP, NRK Syt1(C2B*)-GFP, and NRK Syt1(C2A*B*)-GFP. The C2A* represents the calcium—binding site mutant (D178A, D230A, and D232A) on C2A, the C2B* represents the calcium—binding site mutant (D309A, D363A, and D365A) on C2B, the C2A*B* represents the calcium—binding site mutant (D178A, D230A, D232A, D309A, D363A, and D365A) on C2AB. Cells are labeled with WGA (magenta). Scale bar, 10 µm. (N) Quantification of the number of migrasomes per 100 μm RF length per cell in NRK, NRK Syt1-GFP, NRK Syt1(C2A*)-GFP, NRK Syt1(C2B*)-GFP, and NRK Syt1(C2A*B*)-GFP cells shown in M. 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. (O) Confocal image of migrasomes from NRK Syt1-GFP and NRK Syt1(4A)-GFP. The Syt1(4A) represent the mutant on the tips of the Syt1 membrane-binding loops (M173A, F234A, V304A, and I367A). Scale bar, 10 µm. (P) Quantification of the number of migrasomes per 100 μm RF length per cell in NRK Syt1-GFP and NRK Syt1 (4A)-GFP shown in O. Data represent the mean ± SEM. P values were calculated using a two-tailed, unpaired t test. n = 51 cells per group. The experiments were performed three times.