Membrane fission via transmembrane contact

Abstract

Division of intracellular organelles often correlates with additional membrane wrapping, e.g., by the endoplasmic reticulum or the outer mitochondrial membrane. Such wrapping plays a vital role in proteome and lipidome organization. However, how an extra membrane impacts the mechanics of the division has not been investigated. Here we combine fluorescence and cryo-electron microscopy experiments with self-consistent field theory to explore the stress-induced instabilities imposed by membrane wrapping in a simple double-membrane tubular system. We find that, at physiologically relevant conditions, the outer membrane facilitates an alternative pathway for the inner-tube fission through the formation of a transient contact (hemi-fusion) between both membranes. A detailed molecular theory of the fission pathways in the double membrane system reveals the topological complexity of the process, resulting both in leaky and leakless intermediates, with energies and topologies predicting physiological events.

Fig. 1. Osmotically driven scission of DM and SM tubes.

Time sequence of SM (a) and DM tube scission (b) upon constriction by osmotic stress. The membrane is shown in pseudocolor. Cartoons illustrate the steps in the scission process in each case, while stars mark scission events. See also Supplementary Movie 1. c Quantification of the radii of the SM and the inner membrane of the DM tubes right before scission (n = 3 independent experiments; each point represents a tube). Inner DM radii were estimated assuming a constant intermembrane (IM) distance between opposing hydrophobic heads region as 2 or 5 nm, respectively (see “Methods”). ** statistically different at the 0.01 level; *** statistically different at the 0.001 level (unpaired two-sided two sample t-test, equal variance assumed; Box plots indicate median (middle line), 25th and 75th percentile (box), and outliers (whiskers)). Source data are provided as a Source data file.

Fig. 2. CryoEM snapshots of tubes constricted by hydrodynamic flow.

a Representative CryoEM micrograph of a SM tube under constriction undergoing fission. The insets show the formation of WLMs immediately before (upper image, white arrowheads) and after membrane scission occurs (lower image, *). Pseudocolor (cyan hot) is used for clarity. See Supplementary Fig. 1d. Scission, WLM, and HF intermediates were detected in 16, 25, and 20% of SM tubes, respectively (94 SM tubes, 7 independent experiments). b CryoEM snapshot of SM and DM tubes under constriction. White arrowheads indicate SM thickness (5.1$\pm$0.5 nm (mean $\pm$SD), n = 111 random measurements, 18 tubes, 3 independent experiments). Green and magenta arrowheads point to proximal membranes of DM tubes under constriction. Pseudocolor is used for clarity. c IMC and fission in DM tubes. Left: representative image of a DM tube with an IMC (white arrowheads), i.e., with intermembrane distance below 0.5 nm. IMCs were detected in 9 out of 16 DM tubes (7 independent experiments). Right: DM tube fission. Simultaneous retraction of both membranes of a DM tube upon fission is indicated by (*). Fission was detected in 4 out of 16 DM tubes. Analysis is provided as a Source data file.

Fig. 3. Canonical and HF pathways of inner membrane fission in DM background.

a The canonical SM and DM fission pathways with tension set at ~1 Dyn/cm. The single tube has a radius r_s ~ 8.5 nm. The inner membrane in the DM tube has a radius of r_in ~ 8 nm while the outer membrane has a radius r_out ~ 16 nm. During constriction, the outer radius shrinks to r_out,c ~ 13.5 nm, as the outer tube is no longer expanded by hydration repulsion. Membrane configurations and free-energy barriers were calculated using SCFT. b Free energies as a function of the reaction coordinate, α, for canonical (red crosses) and HF (blue circles) pathways of inner-tube fission at a membrane tension of ~1 Dyn/cm. Each local minimum in the curves corresponds to a metastable state shown in (c). c The HF pathway for membrane fission in a DM tube. Free energies are given in units of the bending energy, κ. Reaction coordinates, α, correspond to values in (a). Source data are provided as a Source data file.

Fig. 4. Intermediates in the HF pathway.

The rate-limiting step in the HF pathway to inner tube fission at ~1 Dyn/cm. Upper and lower images show views rotated by 90^o. Arrows point to the transient pore. See also Supplementary Movie 2.