Fig. 5: Spatial analysis of hepatocyte or interaction and organization patterns.

A Representative scanning electron microscopy (SEM) and reconstructed organelle interaction network and nodes within 500 nanometers (nm) of each other. Data from a mouse fasted overnight. B Sankey graphs displaying the relative proportion of ER, LD, mitochondria (Mito), and glycogen (Gly) nodes that are within 500 nm range of each other in fasting or after continuous infusion with 40 mg/min/kg of [U-¹³C]-glucose for 1 or 4 hours. C Scatter plots showing the results of Monte Carlo simulation analysis to determine randomness of LD, glycogen, and mitochondria nodes in each reconstructed hepatocyte spatial network. Each dot represents a cell. Lines at 2 and -2 mark the range where organelle positioning is determined to be random. Dots below -2 indicate cells where the positioning of organelles is clustered and not random. D Close up of mitochondria (green) and ER (white) organelles segmented using trained 2D U-nets applied to SEM data. Inset, SEM data where thin magenta lines annotate the location of Mitochondria-ER contact sites. Yellow arrows point to mitochondria-ER contact sites. E–G Relative frequency of ER or Mitochondria organelle-contact types after overnight-fasting, 1 or 4 hours of [U-¹³C]-glucose infusion, or random fed states at mouse, single cell, or organelle level averages – respectively. H Representative SEM panels showing annotated hepatocyte mitochondria-ER contacts in fasting, glucose, or fed conditions shown in E–G. Yellow arrowheads and lines mark the position of identified ER-mitochondria contact sites. I Relative fraction of mitochondria and ER that are isolated, or connected to LD, ER, glycogen (Gly), or mitochondria (Mito). J ¹³C/¹²C levels by different types of mitochondria classified by the identity of their closest interacting partner after 4 hours of 40 mg/min/kg [U-¹³C₆]-glucose infusion. K Scatter plots showing the results of our Monte Carlo simulation analysis to determine randomness of isolated mitochondria (Mito) or mitochondria connected to ER (Mito-ER) or LD (Mito-LD) nodes in each reconstructed hepatocyte spatial network. Each dot represents a cell. All data shown with error bars and 95% confidence interval of the data. In E–G and J, One-way ANOVA with Dunns or Kruskal-Wallis tests where used, respectively. In E–G, ***p < 0.001, and data from individual animals are shown in different colors, and each dot = 1 animal (E) or = 1 cell (F). In J, p values are shown in the figure.