Extended Data Fig. 1: MINFLUX single-molecule analysis of metabolic enzymes GPAT4 and HSD17B13 on the ER and LDs.
From: Membrane bridges and nanodomain partitioning govern membrane protein targeting to lipid droplets
a. The sampling rate of MINFLUX was determined by calculating the time interval between consecutive localizations of individual GPAT4 and HSD17B13 molecules. b. The difference between consecutive localizations within single-molecule MINFLUX tracks for GPAT4 (left) and HSD17B13 (right). Standard deviation along each axis was calculated as a proxy for localization precision. c. Representative image showing classification of single-molecule tracks based on subcellular localization. Molecules localized within the LD mask were classified as LD-associated (orange), and those outside the mask were assigned to the ER (purple). d. Proportion of single-molecule tracks for GPAT4 (left) and HSD17B13 (right) localized to LDs (orange), ER (purple), or spanning both compartments (gray). e. Distribution of single-molecule jump distances for GPAT4 (left) and HSD17B13 (right) on the ER (purple) and LD surface (orange). Median values are indicated. f. Diffusion coefficients for GPAT4 (left) and HSD17B13 (right), calculated using a 5-ms rolling-window mean squared displacement (MSD) analysis. Values are shown separately for ER-localized (purple) and LD-localized (salmon) trajectories. Dotted lines indicate medians. Nsteps = 551,673 (GPAT4), 1,460,156 (HSD17B13). A two-sided Mann–Whitney test was used to assess the statistical significance of the difference of Dapp. P = 9.77 × 10−79 (GPAT4), 2.1 × 10−144 (HSD17B13). g. Mean-square displacement (MSD) curves of individual ER- and LD-localized tracks for GPAT4 (left) and HSD17B13 (right), plotted as a function of time interval in linear (top) and log-log scale (bottom). Shaded area represents 95% confidence interval. h. Ensemble averaged (E-MSD) and time averaged (t-MSD) were plotted in log-log scale to display the differences in ergodicity for GPAT4 (left) and HSD17B13 (right) at the LD (top) and ER (bottom) membranes. Shaded area represents 95% confidence interval. i. 2D correlation map of mean Dapp and alpha values for GPAT4 (left) and HSD17B13 (right). ER and LD localized tracks were colored with purple and salmon, respectively. Mean Dapp values were calculated by taking the average of 5 ms rolling window Dapp values for each track. Pearson’s correlation analysis revealed no significant relationship between α and Dapp (GPAT4: r = −0.09, P = 0.15; HSD17B13: r = −0.03, P = 0.53). j. Velocity–velocity autocorrelation function (VVACF) plots for GPAT4 (left) and HSD17B13 (right). VVACF was calculated by taking the dot product of velocity vectors separated by defined lag times (Δt = 5, 25, and 50 ms) to quantify directional persistence of single-molecule trajectories. Source numerical data are available in source data.
