Understanding the formation of vesicles as close to membranes as possible

In cellular membranes, protein complexes known as ESCRTs (“escorts”) serve to detach vesicles in cell compartments or remove viruses from the cell. Patricia Bassereau’s team at the Physico-Chimie Curie lab (UMR 168, Institut Curie, CNRS, Sorbonne university) observed the assembly of some of these “escorts” and their effects on the deformation or otherwise of the membranes. In Nature Communications on May 29, 2020, the team revealed some entirely unexpected results... helping to better understand the basic biophysical phenomena that are involved in viral infections, cell divisions or embryonic development.

Endocytosis and exocytosis are phenomena by which cells are able to move molecules or particles in or out. This is the case for example with viruses such as HIV, capable of producing in the cells their viral capsid which must be wrapped in the cellular membrane before exiting the cells to continue infecting their host (for other viruses such as SARS-CoV-2, other mechanisms are involved). 

The formation of a vesicle involves different phases: deformation of the membrane, strong constriction of the vesicle “neck” and cutting (also known as fission) which detaches it. Membrane fission involves protein complexes known as ESCRTs. These are highly conserved proteins that are found from archaebacteria to human cells. They are involved in a number of cell functions, including intracellular traffic, cytokinesis, exocytosis and membrane repair. Although these “escorts” seem to specialize in membrane deformation and fission by their assembly inside the “neck”, the way these proteins operate has yet to be established.

Patricia Bassereau’s team at the Physico-Chimie Curie lab (UMR 168, Institut Curie, CNRS, Sorbonne Université) sought to observe the assembly of some of these ESCRT proteins on deformable membranes and to examine the effect of membrane curvature on their recruitment. Until now, the work had been carried out on non-deformable membranes (supported bilayers on a solid substrate).

“In our laboratory our major specialty is the preparation of membrane nanotubes, i.e. very thin lipid structures, thus strongly curved, formed from giant liposomes (see figure below). Thanks to this unique experimental system, we have been able to observe the assembly of some ESCRT proteins, added either outside or inside nanotubes. In collaboration with Aurélie Bertin from the “Molecular Microscopy of Membranes” team, we were also able to analyze their effect on the shape of liposomes and nanotubes using electronic cryo-microscopy,” explains Patricia Bassereau, CNRS research director and head of the “Membranes and Cellular Functions” team at Institut Curie. “Our results are surprising since we thought that these proteins only assemble in specific geometries reminiscent of the vesicle “necks”.”

Conducted in collaboration with Winfried Weissenhorn of the Institute of Structural Biology (Grenoble Alpes university, CEA, CNRS), this study showed that the ESCRT CHMP4 - which comes first at the time of membrane fission - has no affinity on its own for curved membranes. It forms a spiral on a flat surface and is not capable, alone, of deforming a membrane. Surprisingly, the proteins CHMP2 and CHMP3 which polymerize together, have no affinity for the inside of nanotubes and tend to assemble outside the tubes in a geometry which is opposite to that expected, i.e. wrapping around the tube. Another surprise is that by adding CHMP4 and CHMP2/CHMP3 together, the electronic cryo-microscopy images revealed that the proteins assembled parallel to the tubes’ axis (see figure below), with probably small perpendicular structures holding the filaments together. In addition, these proteins are able to transform a spherical liposome into a “corkscrew-like” structure (see figure below).

These results highlight the versatility of ESCRT proteins on membranes. The next step is to understand how they induce fission in the presence of the Vps4 ATPase. Understanding these basic phenomena is essential to elucidate and act on viral infections for example, on cell division processes and thus on cancers, on the development or repair of the nucleus.

© Institut Curie - P. Bassereau, A. Bertin, N. de Francheschi, E. de la Mora, S. Mangenot

This study combined the following techniques: electron microscopy, micro-manipulations on a confocal microscope using optical tweezers, high-speed AFM microscopy in collaboration with W. Roos (Groningen, Netherlands). The figures above show the recruitment of ESCRTs on membrane nanotubes as well as the assembly and the specific geometry of the nanotubes after addition of the ESCRT proteins CHMP4, 2 and 3.

A: confocal microscope image of a nanotube formed from a giant liposome, showing the recruitment of fluorescently labeled CHMP2/3 (left) on the nanotube (right, fluorescent lipids). Bar: 5 mm

B: electron cryo-microscopy tomogram showing the 3D structure of helical tubes induced by the ESCRTs. Bar: 200 nm

C: high-resolution reconstruction of a set of electron tomography images showing the parallel assembly of proteins on the membrane tube.

D: diagram showing the characteristic shape of the helical tubes, with the parallel assembly of ESCRTs in gray.

Reference:

Bertin, A., de Franceschi, N., de la Mora, E. et al. Human ESCRT-III polymers assemble on positively curved membranes and induce helical membrane tube formation. Nat Commun 11, 2663 (2020). https://doi.org/10.1038/s41467-020-16368-5