Fig. 5. Dynamics of ER luminal flow correlated with tubule contractions.

a. Analysis of particle trajectories’ directionality. Tubular junctions/nodes denoted by orange ellipses; grey lines denote all particle trajectories. Trajectories connecting two nodes indicated as A and B are colour coded according to their direction either in red, denoting travel from A to B, or blue for travel from B to A. Lower graph represents the temporal pattern of traversal-directionality. Shown is representative of n=108 node-pairs.

b. Distribution of time periods of unidirectional inter-node displacement.

c. Plots of instantaneous particle velocities fluctuations. Velocities of particles following departure from nodes and traveling along tubules (between nodes, red), and those of particles residing within nodes (black). Solid lines represent mean values for all trajectories, shaded regions represent SD of total sample size: n=111 internode and n=140 intranode trajectory displacements.

d. Analysis of time duration T_H of high-velocity (v > 20 μm/s) peaks (left) and time interval T_L between high-velocity peaks. Red line represents an exponential fit (R² = 0.998).

e. High-speed Structured Illumination Microscopy (SIM) super-resolved images of the tubular ER in live COS7 cells stained with an ER membrane dye (ER Tracker-Green). Images were acquired in 54 ms intervals and processed as described in Methods. The resulting SIM reconstructions were colour-coded according to intensity. The magnified area shows the contours of ER tubules at higher magnification. Arrows denote positions where transient contraction events occur repeatedly. Shown are frames from a time series measurement presented in full in supplementary Video S2. Tubule contractions are better visualized in COS7 cells, but detectable in HEK-293 cells too (Fig. S5). Shown is representative of n=5 independent experiments.

f. Box plot of tubule contraction frequencies extracted from high-speed SIM time series as shown in (e) before and after ATP depletion. Red line – median, boxes’ bottom/top edges – the 25^th and 75^th percentiles respectively, whiskers – extreme data points. ***: Two-sided Mann-Whitney U-test p=1.7019e-7, n=20 ER tubules.

g. Distributions of contraction duration, intervals and lengths from SIM videos as in (e) and Fig. S5. Red curves: exponential (left and middle) and Gaussian (right) fits (R² = 0.988, R² = 0.969, R² = 0.937 respectively).

h. Schematic representation of the model for estimating tubule contraction-induced particle velocity.

All values are given as AVG ± SD for noted n.