Fig. 2: Activity-dependent bouton formation mechanistically resembles blebbing migration.

a Schematic of bleb life-cycle. Non-motile cells show: (1) initiation, (2) rapid expansion, (3) stabilization, and (4) slow retraction (a few min). Retraction is rarely observed in motile cells. b, c Time-lapse images of ghost boutons (GBs) induced by high-K⁺ stimulation showing bleb life phases. Neuronal membrane and F-actin were labeled with UAS-CD4-GFP (or CD8-GFP) and UAS-Lifeact-Ruby, both under the control of NSyb-Gal4. b Example of bouton formation and subsequent stabilization. c Example of sequential bouton formation without retraction. d, e Kymographs of (b) and (c). Kymographs for distinct ∆t values to highlight the dynamic nature of actin during bouton outgrowth. d Initially actin is low (∆t1) but subsequently assembles inside boutons and accumulates when bouton size stabilizes (∆t2 and ∆t3). e actin is absent in the forming bouton, but an F-actin punctum persists at the base. f Time-lapse images of bouton formation under unstimulated conditions also show bleb life phases and morphological characteristics. Example of bouton formation and stabilization. g Kymograph from f displaying actin dynamics found in boutons formed without stimulation, analogous to boutons induced by high-K⁺, N = 7 larvae/7NMJs. b–g White arrow indicates where bouton emerges, yellow arrow indicates ROI used for kymograph generation. h Time-lapse images showing the location where actin intensity was measured: squares of 1 µm² were positioned at the base (b) and edge (e) of the bouton in the beginning and at the end of expansion. i, j Bar plots (mean) with normalized values (min–max normalization) for F-actin intensity measured in new bouton edges and corresponding bases at the beginning or after expansion. i Actin levels at the edges were significantly lower than those of basal actin at both growth stages. j No significant actin flux occurred at the base or edge during bouton outgrowth. All data points are represented, and the lines connect the paired points. n = 14 larvae/53 boutons. Statistical significance determined using non-parametric Wilcox on matched-pairs signed rank test; ****p < 0.0001, ns is not significant. Scale bar, 2 µm for time-lapse images (b, c, f, h) and 1 µm for kymographs (d, e, g). Source data provided as a Source Data file.