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Reconstitution and real-time quantification of membrane remodeling by single proteins and protein complexes

Abstract

Cellular membrane processes, from signal transduction to membrane fusion and fission, depend on acute membrane deformations produced by small and short-lived protein complexes working in conditions far from equilibrium. Real-time monitoring and quantitative assessment of such deformations are challenging; hence, mechanistic analyses of the protein action are commonly based on ensemble averaging, which masks important mechanistic details of the action. In this protocol, we describe how to reconstruct and quantify membrane remodeling by individual proteins and small protein complexes in vitro, using an ultra-short (80- to 400-nm) lipid nanotube (usNT) template. We use the luminal conductance of the usNT as the real-time reporter of the protein interaction(s) with the usNT. We explain how to make and calibrate the usNT template to achieve subnanometer precision in the geometrical assessment of the molecular footprints on the nanotube membrane. We next demonstrate how membrane deformations driven by purified proteins implicated in cellular membrane remodeling can be analyzed at a single-molecule level. The preparation of one usNT takes ~1 h, and the shortest procedure yielding the basic geometrical parameters of a small protein complex takes 10 h.

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Fig. 1: Main geometric and electrical parameters of usNT.
Fig. 2: Experimental setup for usNT production and the protein delivery system.
Fig. 3: Step-by-step procedure for the preparation of the experimental chamber.
Fig. 4: The nonlinear current–voltage (I/U) characteristics of the usNT lumen.
Fig. 5: Box plot of the bending rigidity moduli (k) obtained by the procedure described in Box 2.
Fig. 6: Signal-to-noise ratio in the usNT conductance measurements.
Fig. 7: Representative examples of the usNT conductance behaviors in different experimental conditions.
Fig. 8: Representative examples of elementary constriction events produced by individual protein species.
Fig. 9: Bending rigidity of the usNT membrane with bound proteins.
Fig. 10: Self-limited character of membrane deformations produced by Dyn1 in the presence of GTP.

Data availability

The data that support the findings of this study are available from the corresponding authors upon reasonable request. The source data underlying Figs. 5, 7b, 8d, 9 and 10 are provided as Source Data files.

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Acknowledgements

This work was partially supported by NIH R01GM121725 to V.A.F.; Spanish Ministry of Science, Innovation and Universities and FEDER grant BFU2015-70552-P to V.A.F. and A.V.S.; Russian Science Foundation grant 17-75-30064 to P.V.B.; and the Fundación Biofísica Bizkaia and the Basque Excellence Research Centre (BERC) program of the Basque Government and the Ministry of Science and Higher Education of the Russian Federation.

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Conceptualization, V.A.F., P.V.B., A.V.S.; methodology, V.A.F., P.V.B.; investigation, K.C., P.A., J.V.L., P.V.B.; theoretical analysis, V.A.F., P.I.K., P.V.B.; writing of original draft, V.A.F., P.V.B., A.V.S.; review and editing, all authors; funding acquisition, V.A.F., P.V.B., A.V.S.; resources, V.A.F., P.V.B., A.V.S.; supervision, V.A.F., P.V.B., A.V.S.

Corresponding authors

Correspondence to Pavel V. Bashkirov or Vadim A. Frolov.

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The authors declare no competing interests.

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Peer review information Nature Protocols thanks Patricia Bassereau and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Related links

Key references using this protocol

Frolov, V. A. et al. Proc. Natl Acad. Sci. USA 100, 8698–8703 (2003): https://www.pnas.org/content/100/15/8698

Bashkirov, P. V. et al. Cell 135, 1276–1286 (2008): https://www.cell.com/cell/fulltext/S0092-8674(08)01503-1

Shnyrova, A. V. et al. Science. 339, 1433–1436 (2013): https://science.sciencemag.org/content/339/6126/1433.full

Mattila, J.-P. et al. Nature 524, 109–113 (2015): https://www.nature.com/articles/nature14509

Supplementary information

Source data

Source Data Fig. 5

Statistical source data.

Source Data Fig. 7

Source data (two columns x and y axes).

Source Data Fig. 8

Source data (two columns x and y axes).

Source Data Fig. 9

Statistical source data (two columns for gray and black histograms).

Source Data Fig. 10

Statistical source data (for the histograms in Fig. 10a and b).

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Bashkirov, P.V., Kuzmin, P.I., Chekashkina, K. et al. Reconstitution and real-time quantification of membrane remodeling by single proteins and protein complexes. Nat Protoc 15, 2443–2469 (2020). https://doi.org/10.1038/s41596-020-0337-1

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