How Curvature-Generating Proteins Build Scaffolds on Membrane Nanotubes

How curvature-generating proteins build scaffolds on membrane nanotubes Mijo Simunovica,b,c,d,e,1,2, Emma Evergrenf,3, Ivan Golushkog, Coline Prévosta,4, Henri-François Renardh,5, Ludger Johannesh, Harvey T. McMahonf, Vladimir Lormang, Gregory A. Vothb,c,d,e, and Patricia Bassereaua,i,1 aLaboratoire Physico Chimie Curie, Institut Curie, PSL Research University, CNRS UMR168, F-75005 Paris, France; bDepartment of Chemistry, The University of Chicago, Chicago, IL 60637; cInstitute for Biophysical Dynamics, The University of Chicago, Chicago, IL 60637; dJames Franck Institute, The University of Chicago, Chicago, IL 60637; eComputation Institute, The University of Chicago, Chicago, IL 60637; fLaboratory of Molecular Biology, Medical Research Council, Cambridge CB2 0QH, United Kingdom; gLaboratoire Charles Coulomb, UMR 5221 CNRS, Université de Montpellier, F-34095 Montpellier, France; hChemical Biology of Membranes and Therapeutic Delivery Unit, Institut Curie, PSL Research University, CNRS UMR3666, INSERM U1143, F-75005 Paris, France; and iSorbonne Universités, Université Pierre et Marie Curie, Université Paris 6, F-75005, Paris, France Edited by James H. Hurley, University of California, Berkeley, CA, and accepted by Editorial Board Member K. C. Garcia August 9, 2016 (received for review May 2, 2016) Bin/Amphiphysin/Rvs (BAR) domain proteins control the curvature protein density, they affect the mechanical properties of the of lipid membranes in endocytosis, trafficking, cell motility, the membrane and stabilize membrane nanotubes (7, 10, 17–20). formation of complex subcellular structures, and many other cellular An assembly of BAR proteins on cylindrical membranes has so phenomena. They form 3D assemblies that act as molecular far only been visualized using EM (e.g., refs. 8, 9, and 21). Although scaffolds to reshape the membrane and alter its mechanical these studies provide important and detailed assessments of how properties. It is unknown, however, how a protein scaffold forms BAR domains may interact with one another on curved membranes and how BAR domains interact in these assemblies at protein as a packed protein arrangement, membrane tubules in those ex- densities relevant for a cell. In this work, we use various experi- periments were generated typically from highly charged liposomes mental, theoretical, and simulation approaches to explore how BAR exposed to very high protein concentrations. In the cell, especially in proteins organize to form a scaffold on a membrane nanotube. By combining quantitative microscopy with analytical modeling, we the context of endocytosis, protein concentration is not high enough demonstrate that a highly curving BAR protein endophilin nucleates to induce appreciable spontaneous tubulation, nor would such a its scaffolds at the ends of a membrane tube, contrary to a weaker mechanism be beneficial to the cell. Importantly, a tightly packed curving protein centaurin, which binds evenly along the tube’s assembly of BAR proteins would preclude the recruitment of many length. Our work implies that the nature of local protein–membrane other proteins required in endocytosis and trafficking. interactions can affect the specific localization of proteins on membrane-remodeling sites. Furthermore, we show that amphipathic Significance helices are dispensable in forming protein scaffolds. Finally, we explore a possible molecular structure of a BAR-domain scaffold using Lipid membranes are dynamic assemblies, changing shape on coarse-grained molecular dynamics simulations. Together with fluo- nano- to micron-sized scales. Some proteins can sculpt membranes rescence microscopy, the simulations show that proteins need only by organizing into a molecular scaffold, dictating the membrane’s – ’ to cover 30 40% of a tube s surface to form a rigid assembly. Our shape and properties. We combine microscopy, mathematical work provides mechanical and structural insights into the way BAR modeling, and simulations to explore how Bin/Amphiphysin/Rvs proteins may sculpt the membrane as a high-order cooperative as- proteins assemble to form scaffolds on nanotubes. We show that sembly in important biological processes. the way protein locally deforms the membrane affects where it will nucleate before making a scaffold. In this process, the pro- protein scaffold | BAR proteins | membrane curvature | self-assembly | tein’s amphipathic helices—which shallowly insert into the mem- endocytosis brane—seem dispensable. Surprisingly, the scaffold forms at low protein density on the nanotube. We simulate a structure of urvature of lipid membranes plays important roles in the cell. protein scaffolds at molecular resolution, shedding light on how CIt allows dynamic cellular phenomena, such as trafficking or these proteins may sculpt the membrane to facilitate important cell division, and it can also mediate the interactions among many dynamic events in cells. membrane-bound proteins (1, 2). Proteins containing a Bin/ Amphiphysin/Rvs (BAR) domain participate in numerous mem- Author contributions: M.S., G.A.V., and P.B. designed research; M.S., I.G., and V.L. per- formed research; E.E., I.G., H.-F.R., L.J., H.T.M., and V.L. contributed new reagents/analytic brane-curving processes, such as endocytosis, trafficking, motility, tools; M.S., E.E., C.P., H.T.M., G.A.V., and P.B. analyzed data; and M.S., E.E., G.A.V., and the formation of T-tubules, cytokinesis, and so on (3, 4). BAR P.B. wrote the paper. domains are characterized by a crescent shape whose curvature, The authors declare no conflict of interest. length, and binding affinity to the membrane are distinct among This article is a PNAS Direct Submission. J.H.H. is a Guest Editor invited by the Editorial different members (4–6). Many BAR proteins also contain am- Board. 1 phipathic helices that shallowly insert into the bilayer. To whom correspondence may be addressed. Email: [email protected] or i [email protected]. BAR proteins generate curvature as a combination of ( )ad- 2 ii Present address: Center for Studies in Physics and Biology, The Rockefeller University, hesive electrostatic interactions via their BAR domain and ( )the New York, NY 10065. insertion of amphipathic helices. Additionally, BAR proteins can 3Present address: Centre for Cancer Research and Cell Biology, Queen’s University Belfast, associate into highly ordered assemblies on the membrane, thus Belfast BT7 1NN, United Kingdom. collectively altering its shape and mechanics (7–10). Precisely how 4Present address: Department of Genetics and Complex Diseases, T. H. Chan School of they assemble and affect the membrane is argued to depend on Public Health, and Department of Cell Biology, Harvard Medical School, Boston, MA 02115. the surface density of proteins, membrane tension, and membrane 5Present address: Institut des Sciences de la Vie, Université Catholique de Louvain, B-1348 shape (11). On a flat membrane at a low surface density, BAR Louvain-la-Neuve, Belgium. proteins can form strings and a meshlike network, which can give This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. rise to budding and tubulation (12–16). At a sufficiently high 1073/pnas.1606943113/-/DCSupplemental. 11226–11231 | PNAS | October 4, 2016 | vol. 113 | no. 40 www.pnas.org/cgi/doi/10.1073/pnas.1606943113 Downloaded by guest on October 1, 2021 To achieve close packing, protein–protein interactions were protein at 0.5–2.5 μM (dimeric concentration in the pipette). implicated to be important, namely the lateral interactions be- Note that due to diffusion the concentration of the protein near tween neighboring BAR domains in F-BAR proteins (8) or be- the GUV is approximately half that in the pipette (31). Endo- tween N-terminal amphipathic helices in N-BAR proteins (9). philin showed a remarkable specificity for the base of a pulled It is unclear whether BAR proteins in endocytosis and trafficking nanotube, binding first either at the interface with the vesicle or cooperatively shape the membrane by virtue of specific protein– with the trapped bead (Fig. 1A). Note that the two interfaces are protein interactions or whether they assemble as a result of a morphologically equivalent, having the same saddle-like mem- more general membrane-mediated mechanism. Moreover, it is brane geometry. Out of 59 experiments, endophilin first bound important to understand how BAR proteins assemble at much to the GUV–tube interface in 53 of them, while also simulta- lower protein surface densities and on membrane compositions neously binding to the interface with the bead in 27 experiments. that much more likely resemble those found within the cell. In four cases, endophilin seemed to bind homogeneously along We hypothesize that BAR proteins can oligomerize on a the tube where, possibly, the initial binding was not recorded membrane nanotube at densities much lower than close packing sufficiently fast. Only in the two remaining cases considered as owing to membrane-mediated attractions. We refer to this struc- negative the protein first bound to a region other than the ture as a protein scaffold. It is to be noted that the term “scaffold” interface. is often used to describe a single BAR domain, imprecisely termed Shortly after binding, the region covered by endophilin con- the scaffolding domain. Here, a scaffold represents a 3D rigid tinuously grew along the tube, eventually partially or fully cov- assembly of multiple proteins that adheres to the membrane and ering it (Fig. 1 A and B;

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