Extended Data Fig. 3: Molecular motion analysis of LiveDrop and mutant hairpins using MINFLUX nanoscopy.
From: Membrane bridges and nanodomain partitioning govern membrane protein targeting to lipid droplets
a. AlphaFold-predicted structure of GPAT4, with the engineered hairpin region LiveDrop highlighted in blue. b. Representative MINFLUX single-molecule tracks of LiveDrop in human cells, with individual trajectories shown in different colors. c. Sampling rate distribution of MINFLUX tracking for LiveDrop. d. Mean squared displacement (MSD) curves of LiveDrop single-molecule tracks, colored by subcellular location: ER (purple) and LD (orange). Shaded area represents 95% confidence interval. e. Jump distance distributions for LiveDrop tracks localized to the ER (purple) or LD (orange). Median values are indicated by dotted lines. f. Apparent diffusion coefficients were calculated using a 5-ms rolling window for ER- (purple) and LD-localized (orange) LiveDrop tracks. A two-sided Mann–Whitney test was used to assess the statistical significance of the difference of Dapp. Nsteps = 381,317. P = 9.1 × 10−71. g. Hairpin sequences of LiveDrop and its mutants used in this study. The hinge region (green) was preserved to maintain hairpin integrity. h. Subcellular distribution of trajectories for each construct, categorized as ER-localized, LD-localized or spanning both compartments. Table shows percentages of tracks found at each compartment. A two sided Welch’s t-test was performed between mutants to assess the significance of changes in localizations. Pinside: 0.13 (WT vs 3 W), 0.03 (WT vs 3 W + KRR), Poutside 0.21 (WT vs 3 W), 0.01 (WT vs 3 W + KRR), Pboth: 0.98 (WT vs 3 W), 0.67 (WT vs 3 W + KRR). i-l. Representative MINFLUX tracks and the apparent diffusion coefficients of 3 W mutant tracks (i, j) and 3 W + KRR (k, l). Apparent diffusion coefficients were calculated with a 5-ms rolling window; median values are indicated by dotted lines. A two-sided Mann–Whitney test was used to assess the statistical significance of the difference in Dapp. Nsteps = 377,221 (3 W), P < 10−70 (3 W), Nsteps = 441,999 (3 W + KRR), P = 0.002 (3 W + KRR). Source numerical data are available in source data.
