Organelle-specific thickness patterns in human cells, including leaflet asymmetry analysis. (A) Summary of mean membrane thicknesses across organelles in human embryonic kidney cells (HEK293). Box plots show median (center line), interquartile range (box), whiskers extending to 1.5× the interquartile range, and individual data points overlaid. Each point corresponds to the mean thickness value for a given membrane instance: NE (analyzed as one continuous membrane, n = 4 instances, 0.7 M point-pair measurements), ER (n = 6 instances, 0.4 M point-pair measurements), PM (n = 4 instances, 0.5 M point-pair measurements), OMM (n = 9 instances, 0.8 M point pair measurements), and IMM (n = 9 instances, 3.2 M point-pair measurements). Statistical comparisons of mean thickness values: NE vs ER (two-sided unpaired t test, ns: P = 0.076), NE vs PM (two-sided unpaired t test, *: P = 0.021), ER vs PM (two-sided unpaired t test, **: P = 0.003), and OMM vs IMM (two-sided paired t test, ***: P < 0.001). (B) Comparison of inner versus outer nuclear membrane (INM and ONM) thicknesses for individual NE instances. Two-sided paired t tests of the mean INM and ONM thicknesses: ns (P = 0.070). (C) Validation of the sensitivity of the thickness measurement algorithm using cholesterol depletion in HEK293 cells with MBCD (10 mM, 30 min). Normalized thickness distributions of PM instances from control (n = 4) and cholesterol-depleted (n = 4) cells. (D) Central slice from a tomogram showing two control HEK293 cells in contact, with two PMs (PM1 and PM2) at the cell–cell interface and NE membranes (INM, ONM) in cell 1. Scale bar, 100 nm. (E) Mean intensity profiles for PM1 (upper) and INM (lower), with intensity minima marking the headgroup regions and central maxima—the hydrophobic cores. Using the coordinates of the paired measurement points, one can trace back to the original tomogram to determine which cellular compartment each headgroup minima faces—the PM leaflet can be on the extracellular or cytoplasmic side; the INM leaflet—on the perinuclear or the nucleoplasmic side. Vertical lines mark the mean positions of paired measurement points used for thickness calculation. The asymmetry score is calculated as the ratio of the intensity value differences (ΔI1 and ΔI2) between each headgroup minimum and the central maximum, with the larger Δ in the nominator. To reduce noise from individual measurements, we binned and averaged the intensity profiles based on membrane thickness (see Materials and methods). (F) Thickness-binned asymmetry distributions for four control (green) and four cholesterol-depleted (red) PMs, with median values marked by vertical lines. Perfect symmetry corresponds to a score of 1.0 (or 0% asymmetry), while increasing asymmetry yields asymmetry scores >1.0, with percentage asymmetry derived as (score -1.0) × 100% (e.g., 1.05 = 5% asymmetry).
Figure 6.

Organelle-specific thickness patterns in human cells, including leaflet asymmetry analysis. (A) Summary of mean membrane thicknesses across organelles in human embryonic kidney cells (HEK293). Box plots show median (center line), interquartile range (box), whiskers extending to 1.5× the interquartile range, and individual data points overlaid. Each point corresponds to the mean thickness value for a given membrane instance: NE (analyzed as one continuous membrane, n = 4 instances, 0.7 M point-pair measurements), ER (n = 6 instances, 0.4 M point-pair measurements), PM (n = 4 instances, 0.5 M point-pair measurements), OMM (n = 9 instances, 0.8 M point pair measurements), and IMM (n = 9 instances, 3.2 M point-pair measurements). Statistical comparisons of mean thickness values: NE vs ER (two-sided unpaired t test, ns: P = 0.076), NE vs PM (two-sided unpaired t test, *: P = 0.021), ER vs PM (two-sided unpaired t test, **: P = 0.003), and OMM vs IMM (two-sided paired t test, ***: P < 0.001). (B) Comparison of inner versus outer nuclear membrane (INM and ONM) thicknesses for individual NE instances. Two-sided paired t tests of the mean INM and ONM thicknesses: ns (P = 0.070). (C) Validation of the sensitivity of the thickness measurement algorithm using cholesterol depletion in HEK293 cells with MBCD (10 mM, 30 min). Normalized thickness distributions of PM instances from control (n = 4) and cholesterol-depleted (n = 4) cells. (D) Central slice from a tomogram showing two control HEK293 cells in contact, with two PMs (PM1 and PM2) at the cell–cell interface and NE membranes (INM, ONM) in cell 1. Scale bar, 100 nm. (E) Mean intensity profiles for PM1 (upper) and INM (lower), with intensity minima marking the headgroup regions and central maxima—the hydrophobic cores. Using the coordinates of the paired measurement points, one can trace back to the original tomogram to determine which cellular compartment each headgroup minima faces—the PM leaflet can be on the extracellular or cytoplasmic side; the INM leaflet—on the perinuclear or the nucleoplasmic side. Vertical lines mark the mean positions of paired measurement points used for thickness calculation. The asymmetry score is calculated as the ratio of the intensity value differences (ΔI1 and ΔI2) between each headgroup minimum and the central maximum, with the larger Δ in the nominator. To reduce noise from individual measurements, we binned and averaged the intensity profiles based on membrane thickness (see Materials and methods). (F) Thickness-binned asymmetry distributions for four control (green) and four cholesterol-depleted (red) PMs, with median values marked by vertical lines. Perfect symmetry corresponds to a score of 1.0 (or 0% asymmetry), while increasing asymmetry yields asymmetry scores >1.0, with percentage asymmetry derived as (score -1.0) × 100% (e.g., 1.05 = 5% asymmetry).

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