Insights into Spontaneous Curvature in Complex Membranes from Dual-Tether Pulling Experiments

Authors

Geiger, B. J., Hamel Ascanio, L. E., Drakouli, E., Thusgaard Ruhoff, V., Klasner, N., Baroojii, Y. F., Moreno-Pescador, G., Schjoldager, K. T., Joshi, H. J., Narimatsu, Y., Mathiasen, S., Nylandsted, J., Bendix, P. M., Pezeshkian, W.

Abstract

Spontaneous curvature characterizes the propensity of a membrane to bend in a specific direction. It is therefore crucial in the multitude of cellular processes that involve membrane shape remodelling. Yet, experimentally quantifying the spontaneous curvature remains a significant challenge in complex biological membranes, as their heterogeneity causes ambiguities in spontaneous curvature's physical interpretation. Here, we introduce a general experiment-simulation framework to measure an effective spontaneous curvature using dual-direction tether pulling from cell-attached giant plasma membrane vesicles (GPMVs) and mesoscale simulations. For homogeneous membranes, the force difference between inward and outward pulls yields a tension-independent readout of spontaneous curvature. We show that this continuum observable can be generalized to the mean of the spontaneous curvature in a heterogeneous membrane, independent of the underlying microscopic spontaneous curvature distribution. Applied to HEK-derived GPMVs, a baseline negative spontaneous curvature of the plasma membrane is revealed. Sucrose treatment and extracellular addition of Annexin A5 systematically shift the effective spontaneous curvature, while mucin reporter overexpression does not measurably alter it under the conditions tested. We also measure the curvature imprint of individual fluorescently tagged proteins through a sorting index. Benchmarked with Annexin A5, our scheme recovers curvature imprints very similar to previous atomistic molecular dynamics simulations. Taken together this makes spontaneous curvature accessible as a directly measurable material property of native membranes and membrane-proteins, enabling quantitative studies of membrane remodelling across diverse cellular processes.

Preprint server: bioRxiv
The authors list and abstract were imported from bioRxiv on 05 Aug 2026.

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