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. 2016 Dec 16;7:13660. doi: 10.1038/ncomms13660

Figure 3. Hidden Markov modelling reveals the existence of distinct Sx1A-mEos2 diffusive states.

Figure 3

(a) About 1,500 trajectories from one NMJ chain were colour-coded with the most likely diffusive state inferred by vbSPT analysis. Blue, green and red indicate immobile, slow mobile and fast mobile diffusive states, respectively. Scale bar, 3 μm. (b–f) A high-magnification view of colour-coded trajectories from the boxed region in a. Trajectories either continued to remain in the (b) immobile, (c) slow mobile or (d) fast mobile state, or (e) stochastically switched between different diffusive states. Scale bar, 1 μm. (f) An example trajectory undergoing stochastic switching between the three diffusive states. The timeline shows the inferred state occupation at different time points. Scale bar, 0.5 μm. (g) A three-state model and the parameters inferred by the vbSPT analysis of trajectories from NMJ chains at 30 °C without dTRPA1 expression. Circles represent the different states and are colour-coded as in a–f. The areas of the circle represent the average state occupation (%) of Sx1A-mEos2 in their respective states. Coloured arrows indicate the average transition probabilities between different diffusive states per time frame. DI, DS and DF are the average apparent diffusion coefficients of immobile, slow mobile and fast mobile states, respectively. Only changes in the state occupation (see white arrows within the circle) on dTRPA1 expression at 30 °C are shown. Grey circle areas correspond to state occupation of Sx1A-mEos2 in different states at 30 °C with dTRPA1 expression. (h–j) Apparent diffusion coefficients and state occupations inferred by analysing trajectories from NMJ chains with or without expression of dTRPA1 at (h,i) 30 and (j,k) 25 °C. Statistical tests were performed using Mann–Whitney U-test (NS, not significant, *P<0.05 and **P<0.01). n=12 and 11 NMJ chains at 25 and 30 °C without dTRPA1 expression, respectively, and 14 and 15 NMJ chains at 25 and 30 °C with dTRPA1 expression, respectively. Mean±s.e.m. are plotted.