Figure 4: CTB retrograde carrier flux is composed of small aligned vesicular compartments.

(a) Dual-colour SIM on axon channels of microfluidic chambers labelled with CTB under either low K⁺ (left) or high K⁺ (right) conditions, β-tubulin III was used to label the axon fibers. Scale bar, 5 μm. Boxes are magnified in the lower panels; original wide-field images are compared with the SIM images of the same region (Scale bar, 1 μm), and individual CTB carriers are further magnified in yellow boxes (Scale bar, 100 nm). The resolution is ∼100 nm. Quantification of total (b) and small CTB carriers (diameter<150 nm) (c) from SIM images (Mander’s coefficient, mean±s.e.m., n=27 for low K⁺, n=31 for high K⁺, ***P<0.001, data from four independent preparations, Student’s t-test). (d) Electron microscopy of axons from neurons pulse-chased with CTB-HRP as described in a. Small vesicular carriers—red arrows, multivesicular body—blue arrow. Scale bar, 200 nm. (e) Quantification of the number of carriers per μm neurite length per axon channel. (mean±s.e.m., n=7 (low K⁺) or 16 (high K⁺) channels from two independent neuron preparations. **P<0.01) (f) Size distribution of CTB carriers in low K⁺ and high K⁺ stimulated cells (n=3 independent neuron preparations). (g) Confocal microscopy of axons from neurons pulse-chased with CTB as described in (a). Endogenous TrkB immunostaining is shown in green, CTB vesicles that co-localized with TrkB are indicated with arrowheads. Scale bar, 5 μm. (h) Quantification of CTB-positive (CTB⁺) and TrkB-positive (TrkB⁺) carriers after low K⁺ or high K⁺ treatments (n=10 and 11 for high K⁺ and low K⁺, *P<0.05, **P<0.01, data from two independent preparations; NS, not significant, Student’s t-test).