Most of these mitochondria (99.4%) showed inter-frame velocities of < 0.1 μm/s and most of these APP-containing vesicles (99.0%) showed inter-frame velocities of < 0.25 μm/s. Therefore, mitochondria and APP-containing C59 wnt cell line vesicles were defined to be in pause when an inter-frame velocity was below 0.1 or 0.25 μm/s, respectively. From these considerations, we calculated the average velocities of mitochondria and APP-containing vesicles as averages of inter-frame velocities excluding the time points defined to be in pause. When reinitiation of moving occurred, the pause was defined as short pause (3 s ≤ pause duration < 30 min

for mitochondria; 1 s ≤ pause duration < 10 min for APP-containing vesicles; the majority of pauses were less than a few minutes).

In the other cases, when mitochondria were stationary through the imaging periods, mitochondria were defined to be in stationary state (long pause). Mitochondria and APP-containing vesicles that moved over 10 μm within an imaging period were used for the analysis of dynamic properties in mobile state. To examine a positional specificity of mitochondrial short pauses, random short-pause positions were made by a stochastic simulation. For the simulation, the total short-pause number and moving distance PD0332991 chemical structure of individual mitochondria, presynaptic distributions and sizes of moving mitochondria obtained experimentally were used. Distances between respective short-pause positions were set over 1 μm and calculations were repeated 500 000 times for

each mitochondrion. The expected Morin Hydrate means and SDs of the short-pause number near presynaptic sites were calculated. The short-pause position preference is expressed as (4) where Nexp and Nsim are the average numbers of short pauses near presynaptic sites obtained from experiments (Nexp) and simulation (Nsim); SDsim is the SD of the expected average number of short pauses near presynaptic sites. Higher values of short-pause position preference indicate that mitochondrial short pause occurred preferentially near presynaptic sites. The short-pause position preference was not high in the specific axonal region and was not dependent on the short-pause rates or sizes of moving mitochondria. Short-pause position preference was not significantly changed by alternation of the scale of spatial resolution for the simulation. APP-containing vesicles were used for a cargo control and stationary mitochondria localised away from synaptic sites were used for a positional control. In order to integrate the information about the properties of mobile mitochondria and probability of transition between stationary and mobile states, it is necessary to convert the parameters linked to individual mitochondria into parameters of events that take place per unit length of axons.