Fig. 1: PDZD8 shows hotspots in close proximity to mitochondria by high-speed single molecule tracking.

a Diffraction-limited imaging of the ER (cyan) and the mitochondria (red) in the periphery of a representative COS7 cell with the simultaneously measured likelihood of finding a PDZD8 molecule in a 1-min window. The locations of the mitochondria in the probability map are indicated with dotted white lines. Boxes correspond to the mitochondria-associated hotspots (MitoHS, magenta) or non-mitochondria-associated hotspots (OtherHS, green) in b and c. Data are representative of 14 cells from two independent experiments. b Zooms of MitoHS in a showing individual PDZD8 trajectories engaging with the hotspots and the associated PDZD8 probability density. Dotted lines indicate hotspot boundaries as used for subsequent analysis. c Zooms of the OtherHS in a showing individual PDZD8 trajectories engaging with the hotspots and the associated PDZD8 probability density. Dotted lines indicate hotspot boundaries as used for subsequent analysis. The shape and size are consistent with endosomal contact sites, as described in Obara et al.²⁹. d Plots of the distance of individual PDZD8 molecules shown in different colors from the center of example hotspots over time. Plots are from MitoHS 2 (top panel) or OtherHS 3 (bottom panel) shown in a. Note the long stretches where single molecules remain engaged in the hotspots. e PDZD8 shows reduced diffusion within both classes of hotspots as compared to freely diffusing in the surrounding ER. n = 89 and 99 hotspots for the MitoHS and OtherHS, respectively. Statistical analysis was performed using two-sided Mann–Whitney test for comparing % reduction in 2D D_eff in MitoHS between OtherHS and one sample Wilcoxon signed-rank test for comparing the hypothetical median (0) and the median of % reduction in 2D D_eff within MitoHS or OtherHS. ns: p > 0.05, ****p < 0.0001. The data are presented as individual points on box plots, with the center indicating the median, and the 25th and 75th percentiles represented by the box. Whiskers extend to the minimum and maximum values. f Sizes of MitoHS are significantly larger than those of OtherHS in the same cells. n = 89 and 99 hotspots for the MitoHS and OtherHS, respectively. The data are presented as individual points on box plots, with the center indicating the median, and the 25th and 75th percentiles represented by the box. Whiskers extend to the minimum and maximum values.Statistical analysis was performed using two-sided Mann–Whitney test. **p = 0.0065. g PDZD8 dwell times in individual MitoHS and OtherHS. Inset shows the leaving frequency (k_out) of individual PDZD8 molecules from probability hotspots associated or unassociated with mitochondria. Scale bar: a 5 µm, b, c 100 nm. Source data are provided as a Source Data file.