Biological and Medicinal Chemistry

Membrane fusion mediated by non-covalent binding of re-engineered cholera toxin assemblies to glycolipids

Authors

Abstract

Membrane fusion is essential for the transport of macromolecules and viruses across membranes. While glycan-binding proteins (lectins) often initiate cellular adhesion, subsequent fusion events require additional protein machinery. No mechanism for membrane fusion arising from simply a protein binding to membrane glycolipids has been described thus far. Herein we report that a biotinylated protein derived from cholera toxin, becomes a fusogenic lectin upon crosslinking with streptavidin. This novel reengineered protein brings about hemifusion and fusion of vesicles as demonstrated by mixing of fluorescently labelled lipids between vesicles as well as content mixing of liposomes filled with fluorescently labelled dextran. Exclusion of the complex at vesicle-vesicle interfaces could also be observed indicating the formation of hemifusion diaphragms. We propose that negative membrane curvature, caused by binding of the cholera toxin to the membrane surface, induces formation of a fusion stalk as a result of high bending energies building up between multiple inverted membrane dimples aligned on opposing membranes at the vesicle-vesicle interface. Discovery of this fusogenic lectin complex demonstrates that new emergent properties can arise from simple changes in protein architecture and provides insights towards new mechanisms of lipid-driven fusion

Content

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Supplementary material

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Supplementary Information
supplementary figures, supplementary video captions, supplementary experimental methods

Supplementary weblinks

Supplementary Video SV1: Biotinylated AB5 protein induces membrane tubules
video showing 400 nM biotinylated AB5 protein was added to vesicles functionalised with 1 mol% GM1 and 0.5 mol% BodipyFL-C5-HPC (green) and resulted in mobile membrane tubules.
Supplementary Video SV2: Tubules distant from crosslinked interfaces can be induced by Strep-(AB5)n, related to Supplementary Fig. S1
video showing Vesicles functionalized with 1 mol% GM1 and 0.5 mol% BodipyFL-C5-HPC (green) were crosslinked with 120 nM AB5-biotin ‒ streptavidin-AF555 (red). White arrows point towards elongated interfaces while purple arrows indicate regions with tubules in the merge channel.
Supplementary Video SV3: Hemifusion, fusion, or vesicle rupture can be induced by Strep-(AB5)n, related to Fig. 3.
video showing Two vesicle populations containing 2.5 mol% GM1 and either no membrane dye or 0.5 mol% DHPE-TxRed (red) were incubated with 100 nM AB5-biotin ‒ streptavidin-AF488 (green) complex. Yellow arrows in the red channel indicate hemifusion by the transfer of fluorescently labelled lipids between vesicles. The turquois arrow in the merge channel indicates a possible hemifusion diaphragm from which the complex becomes excluded which first increases in size until it subsequently decreases shortly before fusion illustrated by white arrows at the distal ends of the interface. The white circle in all channels indicates the complete fusion of two vesicles. Blue arrows in the merge channel point illustrate mobile domains of protein exclusion within one interface. The pink arrow in the merge channel points towards a vesicle which ruptures during the course of the time-lapse.
Supplementary Video SV4: Content mixing as a result of fusion induced by Strep-(AB5)n, related to Fig. 4.
video showing Two vesicle populations (5 mol% GM1 without membrane staining) filled with either dextran-AF488 (green) or dextran-647 (blue) were incubated with 200 nM AB5-biotin ‒ streptavidin-AF555 (red) complex. White circles in all channels indicate the fusion of two vesicles inscribed as fusion event 1 or 2. The yellow arrow in the blue channel points towards a vesicle with a slight increase of blue fluorescence even before the first fusion event, which then increases after fusion event 1 (light orange arrow) and even further after fusion event 2 (orange arrow), resulting in a turquoise appearing vesicle. Turquoise arrows in the red channel indicate the exclusion of the complex from interfaces between the vesicles i, ii, and iii, which did not result in content mixing.
Supplementary Video SV5: Vesicle fusion does not necessarily follow nor require extensive vesicle crosslinking with complex exclusion, related to Fig. 5.
video showing Two vesicle populations containing 5 mol% GM1 and either no membrane dye or 0.5 mol% DOPE-Atto488 (green) were incubated with 200 nM AB5-biotin ‒ streptavidin-AF555 (red) complex. Purple arrows in the merge channel illustrate the rupture of vesicles during the course of the time-lapse. Turquois arrows in the red channel indicate interfaces from which the complex became excluded. Yellow arrows in the green channel point towards vesicles which demonstrate transfer of fluorescently labelled lipids. White circles in all channels indicate the fusion of two vesicles during fusion event 1 and 2.
Supplementary Video SV6: Content leakage of dextran filled vesicles, related to Supplementary Fig. S3
Video showing Two vesicles populations (5 mol% GM1 without membrane staining) filled with either dextran-AF488 (green) or dextran-647 (blue) were incubated with 200 nM AB5-biotin ‒ streptavidin-AF555 (red) complex. The white arrow in the merge channel points towards a vesicle which loses its dextran-AF488 content during the course of the time-lapse. The pink arrow in the merge channel points towards a rupturing vesicle.
Supplementary Video SV7: Vesicle rupturing induced by Strep-(AB5)n
Video showing Two vesicle populations containing 2.5 mol% GM1 and either no membrane dye or 0.5 mol% DHPE-TxRed (red) were incubated with 100 nM AB5-biotin ‒ streptavidin-AF488 (green) complex. Rupture of vesicles started after approximately 60 min and resulted in remains of lipid debris.
Supplementary Video SV8: Hemifusion of liquid-ordered phase vesicles by Strep-(AB5)n
Video showing GUVs were constituted from lipid bilayers with a rigid liquid-ordered (Lo) phase (5 mol% GM1, 0.5 mol% Atto 647N-DOPE). When 20 μl Lo GUVs was incubated with 200 nM Strep-(AB5)n tubular invaginations were not visible. Lo GUVs were still observed to undergo crosslinking and HD formation, but no fusion.