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. 2026 Oct 28;12(44):eaei2661. doi: 10.1126/sciadv.aei2661

Fig. 7. Activity-induced presynaptic filopodia generate convective membrane flow.

Fig. 7.

(A) Hippocampal neuron coexpressing GPI-EGFP and SypmOr2 before and during electrical field stimulation (600 APs at 40 Hz). Black bars separate the filopodial (white arrow) and distal sides. Right: Difference image showing SypmOr2 accumulation toward the filopodium. Scale bar, 1 μm. (B) SypmOr2 fluorescence at filopodial and distal ROIs during stimulation (means ± SEM). P values, mixed-effects model with Sidak’s test. (C) Normalized EI for individual cells (green lines) and means ± SEM (filled symbols). Positive values denote accumulation toward filopodia. P values, one sample t test. (D) Pooled linear regression of flux over time. Cell traces, green; 95% confidence interval, shading. (E) Super-resolved uPAINT images of surface GPI-EGFP labeled with ATTO647N anti-GFP nanobody (magenta) over synaptophysin-RFP (gray), before and during stimulation. Black arrow, filopodium-forming bouton; green arrow, bouton without filopodia. Lines, intensity-profile axes. Scale bar, 1 μm. (F to H) GPI-EGFP–ATTO647N intensity profiles along the boutons oriented toward the filopodia before (F) and during (G) stimulation and their difference (H). Individual cells, gray; purple shaded band, means ± SEM. Intensity and length were normalized independently to 0 to 100%. P values, one-sample t test against zero. (I) uPAINT trajectories of GPI-EGFP–ATTO647N (rainbow) over synaptophysin-RFP–labeled boutons (gray). Yellow dashed circles, boutons; insets, boutons with (black arrows) or without (green arrows) filopodia. Scale bars, 1 μm. (J) Particle-velocity distributions at boutons without (left) and with (right) filopodia, fitted by two Gaussians (gray and yellow; combined fit, purple). Gaussian components (mean velocity in μm/s, weight): without filopodia (left), gray (0.55, 0.85) and yellow (1.27, 0.15); with filopodia (right), gray (0.48, 0.53), and yellow (0.98, 0.47). (K) Model-predicted release at measured flow speeds with or without filopodia. Release after 10 ms is asynchronous (orange dashed line). Asynchronous proportion: with filopodia, 0.15; without, 0.30.