HMM-Bayes analysis of the internalized VAMP2 in presynapses and axonal segments. (A) Frequency of purely diffusive motion or a combination of diffusive and transport motion in presynapses (Ps) and axons (Ax). D indicates a diffusive state, and DV indicates a transport state. (B) An example SV trajectory displaying D-DV-DV motion inferred by HMM-Bayes analysis. The timeline shows the order and duration of the motion states in the trajectory. (C and D) Examples of three-state D-DV-DV and D-D-D models in two presynapses, inferred from a set of VAMP2–pHluorin–bound Atto647N nanobody trajectories. Circles represent the different states, and their area is proportional to the percentage occupation of VAMP2–pHluorin–bound Atto647N nanobodies in the respective state. P_ij, where i = 1, 2, or 3 and j = 1, 2, or 3, are the transition probabilities, with i = j indicating the probability of staying in the same state and i ≠ j indicating the probability of switching to another state. The models in C and D also indicate the apparent diffusion coefficients (D) and velocity magnitudes (V) for the different states. The apparent diffusion coefficient distributions (E), the velocity magnitude distributions (F), and the probability of remaining in the same state in the next time step from all mobility states (G), including both diffusive and transport states, in axons and presynapses. In E–G, the horizontal line indicates the mean. n = 31 presynapses (12,000 trajectories) and 29 axonal segments (16,700 trajectories) from seven and six hippocampal neurons, respectively, in individual cultures.