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. 2017 Nov 21;21(8):2118–2133. doi: 10.1016/j.celrep.2017.10.084

Figure 4.

Figure 4

Actin is required for S135 phosphorylation-dependent capture of Syt4 DCVs at presynaptic sites

(A) GFP-tagged WT, S135A and S135E Syt4 transfected cells immunostained with MAP2.

(B and C) (B) Quantitation of WT, S135A, and S135E GFP puncta in axons (MAP2-negative) and dendrites (C; MAP-2-positive).

(D and E) Cropped axonal regions of hippocampal neurons co-transfected with syp-GFP and mCherry-tagged WT, S135A or S135E Syt4 in control (D) and latrunculin-treated (E) conditions.

(F) Quantitation of percentage of mCherry-tagged WT, S135A or S135E Syt4 colocalizing with syp-GFP, in control and latrunculin-treated conditions (n=37, 33 and 33 cells in control conditions, and n=31, 25 and 28 cells in latrunculin-treated conditions for WT, S135A and S135E, respectively, from 3 cultures).

(G and H) (G) Quantitation of velocity and mobile vesicle percentage (H) of WT, S135A and S135E Syt4 vesicles in control and and latrunculin-treated conditions, normalized to WT control (n=5109, 5609 and 2317 vesicles for velocity and n=22, 21 and 15 videos for mobile percentage for WT, S135A and S135E vesicles, respectively, from 4 cultures).

(I and J) (I) Quantitation of velocity and mobile vesicle percentage (J) of WT, S135A and S135E Syt4 vesicles in control and jasplakinolide-treated conditions, normalized to WT control (n=5625, 5977 and 8556 vesicles for velocity, and n=13, 13 and 15 videos for mobile percentage for WT, S135A and S135E vesicles, respectively, from 3 cultures). Scale bars = 5 μm. Significance determined by Student’s t-test with Bonferroni correction; error indicates s.e.m.; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001).