DOI 10.1515/hsz-2013-0242 Biol. Chem. 2014; 395(3): 275–283
Review Mijo Simunovic and Patricia Bassereau*
Reshaping biological membranes in endocytosis: crossing the configurational space of membraneprotein interactions Abstract: Lipid membranes are highly dynamic. Over several decades, physicists and biologists have uncovered a number of ways they can change the shape of membranes or alter their phase behavior. In cells, the intricate action of membrane proteins drives these processes. Considering the highly complex ways proteins interact with biological membranes, molecular mechanisms of membrane remodeling still remain unclear. When studying membrane remodeling phenomena, researchers often observe different results, leading them to disparate conclusions on the physiological course of such processes. Here we discuss how combining research methodologies and various experimental conditions contributes to the understanding of the entire phase space of membrane-protein interactions. Using the example of clathrin-mediated endocytosis we try to distinguish the question ‘how can proteins remodel the membrane?’ from ‘how do proteins remodel the membrane in the cell?’ In particular, we consider how altering physical parameters may affect the way membrane is remodeled. Uncovering the full range of physical conditions under which membrane phenomena take place is key in understanding the way cells take advantage of membrane properties in carrying out their vital tasks. Keywords: BAR domain; clathrin; membrane remodeling; membrane tension; multiscale simulation. *Corresponding author: Patricia Bassereau, Institut Curie, Centre National de la Recherche Scientifique Unité Mixte de Recherche 168, Université Pierre et Marie Curie, F-75248 Paris, France, e-mail: [email protected] Mijo Simunovic: Institut Curie, Centre National de la Recherche Scientifique Unité Mixte de Recherche 168, Université Pierre et Marie Curie, F-75248 Paris, France; Department of Physics, Université Paris Diderot, 10, rue Alice Domon et Léonie Duquet, 75013 Paris, France; and Department of Chemistry, The University of Chicago, Chicago, IL 60637, USA
What is a membrane? Biological membranes are highly dynamic supramolecular assemblies. They are mainly made up of lipids, which form bilayers in water (Lipowsky, 1991). Structural features of such bilayers are essentially multiscale (Goetz et al., 1999). They can span microns in lateral dimensions to form elastic sheets governed by macroscopic mechanics. Conversely, the cross-section of a lipid bilayer is orders of magnitude smaller, with only two molecules making up its thickness (Lipowsky, 1995). This aspect demands a molecular point of view when studying any interactions with the membrane. Furthermore, lipids move relatively freely in a quasi two-dimensional space. Such mobility makes the membrane fluid, further contributing to its diverse behavior (Lipowsky, 1995). The interplay between the macroscopic mechanics and molecular interactions, in turn, determines their equilibrium shape. The hydrophobic repulsion at the interface of water molecules and lipid tails gives rise to line tension that minimizes the edge of a bilayer. This process competes with elastic energy induced by bending the membrane. As a result of these opposing forces, biological membranes form a remarkable range of geometries (Seifert, 1997). Membrane remodeling is essential for cellular function, as it is a key step in communication among cells, the formation of organelles, division, trafficking, and migration (Alberts, 2002). To understand the physics underlying these phenomena, it is important to investigate the way membranes can be reshaped. According to a theory introduced by Helfrich, bending energy will decrease as the membrane deforms in a way so that its curvature matches the spontaneous curvature (Helfrich, 1973). Spontaneous curvature of free and symmetric bilayers is zero, however binding of other molecules, such as proteins, may induce a non-zero value of this term (Farsad and De Camilli, 2003; McMahon and Gallop, 2005; Sens et al., 2008). Understanding how the inclusion or peripheral binding of proteins couples with the morphology of membranes
Brought to you by | Northern Illinois University Authenticated Download Date | 1/26/15 12:34 AM
276 M. Simunovic and P. Bassereau: Configurational space of endocytosis presents a significant scientific challenge for physicists and biologists. Over several decades, scientists have described the very rich phase behavior of lipid membranes, dependent mostly on their composition, surrounding media, and temperature (Cullis and de Kruijff, 1979). The presence of proteins adds to their already complex behavior. Researchers have uncovered a number of different ways proteins can interact with membranes, which induces a plethora of morphological transitions. Our main goal in this review is to discuss how the interpretation of this configurational diagram becomes important for biology. We believe that a leading problem in biophysics in years to come will be distinguishing the question ‘what can proteins do to cell membranes?’ from ‘what proteins do to cell membranes?’ A similar issue may be raised in the field of materials science. We can engineer alloys with remarkable phase behavior, with only fractions of the phase diagram having industrial importance. This observation does not imply that characterizing the wide range of capabilities of materials such as membranes is a futile task. On the contrary, understanding the full scope of membrane-protein interactions gives important insights into their behavior in the cells, as long as this behavior is carefully extrapolated from the studied conditions. This is an exceptional challenge for membrane physicists and biologists.
Are proteins needed for the remodeling of cell membranes? A significant source of diversity among membranes comes from a large variety of lipids that can assemble into bilayers. Some lipids, such as sphingomyelins, due to their structure prefer to form more ordered domains, whereas others, like phosphocholines, phosphoserines, etc., form disordered domains (Cullis and de Kruijff, 1979). It has been shown that the latter lipids can even, under certain conditions, be recruited to curved membrane regions without any help of proteins (Sorre et al., 2009). The presence of both kinds of lipids will result in phase separation, which may induce enough line tension between the phases to drive membrane remodeling, such as budding and vesiculation (Julicher and Lipowsky, 1993; Baumgart et al., 2003; Lipowsky and Dimova, 2003). Clearly, lipids alone comprise powerful machinery that can induce structural and phase changes in the membrane. However, cells often require membranes to be highly curved and that the remodeling occurs rapidly,
efficiently, in a controlled and reproducible manner, which makes proteins indispensible players in these processes (McMahon and Gallop, 2005). In fact, they involve a large number of proteins, often coupled in complex and unclear ways. Endocytosis is an important example in which membrane is remodeled to facilitate the entry of molecules and particles into the cell (Fotin et al., 2004; Kirchhausen, 2009; Saheki and De Camilli, 2012). According to the bestunderstood mechanism of this process, protein clathrin polymerizes into a rigid coat onto the membrane, mediating the formation of buds and vesicles (Kirchhausen, 2009; Saheki and De Camilli, 2012). At the same time, large families of proteins, that contain Bin/amphiphysin/ Rvs (BAR) or epsin N-terminal homology (ENTH) domains, play various roles in the course of endocytosis. Unlike clathrin, that binds to the membrane via its adaptor molecules, BAR and ENTH domain proteins directly interact with the membrane (Qualmann et al., 2011; Mim and Unger, 2012; Saheki and De Camilli, 2012). However, different experimental techniques have resulted in various and often contrary observations, leaving an open question how deeply involved these proteins are in membrane remodeling. From a physical point of view, bending a membrane requires energy. Binding of BAR and ENTH proteins may provide this energy via adhesion or inclusion into the bilayer (Campelo et al., 2008; Ayton and Voth, 2010; Mim and Unger, 2012). As a result, proteins will generate a non-zero spontaneous curvature, in which the magnitude will depend on the way the proteins interact with the membrane. It has been shown that overexpression of BAR proteins in cells will induce tubulation of their membranes (Peter et al., 2004). Tubule formation has also been observed in vitro with electron microscopy when incubating these proteins with highly charged liposomes (Peter et al., 2004; Frost et al., 2008; Mim et al., 2012a). Computer simulations at the atomic level have shown that a single N-BAR domain induces significant local deformation of the membrane, thus giving rise to spontaneous curvature (Blood and Voth, 2006). Simulating the collective behavior of proteins using coarsegrained models has shown that curvature-inducing mechanism become much more complex at larger scales. Anisotropic interactions with the membrane induce local curvaturem, which then mediates their aggregation and subsequent large-scale morphological changes, such as budding (Reynwar et al., 2007; Simunovic et al., 2013b). These results confirm the prediction that BAR and ENTH domain proteins have the capabilities to drive curvature generation in cells. Furthermore, BAR proteins
Brought to you by | Northern Illinois University Authenticated Download Date | 1/26/15 12:34 AM
M. Simunovic and P. Bassereau: Configurational space of endocytosis 277
preferentially bind to curved membranes, and thus potentially rapidly stabilize the tubular neck formed in endocytosis (Bhatia et al., 2009, 2010; Sorre et al., 2012; Zhu et al., 2012). At the same time, the polymerization of clathrin on giant liposomes has been demonstrated to induce the formation of vesicles upon heating (Dannhauser and Ungewickell, 2012). As demonstrated in these cases, electron microscopy imaging gives vital structural information on the interaction between proteins and remodeled membranes at very high resolution. However, from electron microscopy it is hard to deduce the temporal sequence of remodeling events and describe these processes in quantitative terms. Fluorescence microscopy allows a more quantitative observation of binding and remodeling, but at the cost of resolution. It is evident from both of these approaches that BAR proteins and clathrin have a marked effect on the membrane alone. However, do these observations give direct clues of their mode of action and dynamics in the cell? It is possible that the elevated protein concentration and membrane charge enabled the remodeling with the help of increased effective binding energy. Genetic studies on live cells can help elucidate the way these proteins affect cellular viability. It has been shown that the deletion of endophilin and amphiphysin (both N-BAR proteins) only changes the rate and efficiency of endocytosis (Saheki and De Camilli, 2012). Conversely, in other studies, knocking out endophilin or disrupting its membrane bending activity has a much more detrimental effect on functionality of synaptic vesicles (Verstreken et al., 2002; Bai et al., 2010). Live cell imaging with fluorescence microscopy provides an important link to understanding the single molecule dynamics in the course of endocytosis (Kirchhausen, 2009; Taylor et al., 2011; Weinberg and Drubin, 2012). Another approach – correlative fluorescence and electron microscopy – bridges the gap between temporal and spatial resolution in the two microscopies (Kukulski et al., 2011). Live cell imaging studies predict that FCHo (an F-BAR protein) is recruited to already curved membranes and thus most likely participates in the stabilization of membrane curvature, but not its generation (Cocucci et al., 2012). By studying endocytosis in yeast with the correlative microscopy approach, it has been shown that BAR and ENTH proteins are already present on flat membranes, however curvature is induced only after actin recruitment (Kukulski et al., 2012). In this study, amphiphysin is predicted to help elongate the initially formed membrane invagination; therefore, its membrane bending activity is only important for the efficiency of endocytosis and not its initiation.
Does physics matter? The observed discrepancies perfectly reflect the complexity of membrane remodeling processes. They may imply that endocytosis occurs in a heterogeneous way and that it depends on numerous factors, such as physical conditions, studied organism, cell type, or even the observed part of the cell. For example, the part of membrane attached to the microscopy coverslip shows different behavior than the free membrane, which the authors attribute to the difference in tension of those two membranes (Boulant et al., 2011). It has even been shown that the experimental way proteins are expressed in the cells may affect the endocytosis rate as well (Doyon et al., 2011). Combining fluorescence microscopy with mechanical measurements helps to describe, in a quantitative way, the underlying physics of membrane remodeling. Such experiments have shown that the density of BAR proteins bound to the membrane deeply affects their behavior (Heinrich et al., 2010; Sorre et al., 2012; Ramesh et al., 2013). In the case of amphiphysin, when the density is low enough that the proteins do not interact with each other, they sense curvature and enrich membrane nanotubes. At higher densities, their interaction provides mechanical force that stabilizes tubulation and induces curvature (Sorre et al., 2012). In this density regime, coarse-grained simulations and cryo-electron microscopy have shown that proteins arrange into a nematic-like assembly, driving the formation and stabilization of membrane tubes (Yin et al., 2009; Mim et al., 2012b; Ramakrishnan et al., 2013) and, at very high densities, even breaking the surface of the vesicle to form a closed-surface tubular network (Simunovic et al., 2013a). The surprising fissiogenic properties of N-BAR proteins have also been observed with electron microscopy (Boucrot et al., 2012). These results show that it is crucial to understand the complexity of protein-protein interactions and how they may alter the behavior of the membrane. Strikingly, membrane can be deformed just as a result of entropic pressure caused by clustering of macromolecules. This has been shown for polymers and proteins that do not specifically interact with the membrane (Breidenich et al., 2000; Bickel et al., 2001; Stachowiak et al., 2012). The important implication of these studies is that the cell may use the underlying physics to bend the membrane, while the complex protein machinery controls, what would otherwise be, a very inefficient process. Mechanical experiments also help in understanding the full scope of the phase space of membrane-protein interactions by accounting for and controlling physical parameters (Singh et al., 2012). One such parameter is membrane tension that plays a key role in protein-induced
Brought to you by | Northern Illinois University Authenticated Download Date | 1/26/15 12:34 AM
278 M. Simunovic and P. Bassereau: Configurational space of endocytosis remodeling (Lipowsky, 2013). For example, the protein dynamin, which cuts the membrane to release the endocytic vesicle, works more efficiently at higher tensions on tubular membrane structures (Morlot et al., 2012). It has been proposed that membrane tension can even control the course of endocytosis. High membrane tension requires the polymerization of actin that will then help in elongating the endocytic bud, a step absent at low tension (Boulant et al., 2011). Conversely, in yeast actin is required regardless of tension. High tension of vesicles may explain why sometimes BAR proteins do not remodel the membrane in vitro to as large an extent as in vivo (Peter et al., 2004). Possibly, the efficiency of remodeling by BAR or ENTH proteins in live cell studies was also precluded by high tension. Of note, we still do not know the precise origin of membrane tension in cells and how it is controlled, so its role in membrane remodeling phenomena remains elusive. It is evident, however, that fundamental physical parameters may affect the course of membrane remodeling. Therefore, it is important to keep in mind
their potential active role in experimental observations and whether similar conditions may take place in the cell. Figure 1 depicts how the configurational space manifests in the cell and how different conditions lead to different outcomes in endocytosis and related phenomena.
Is remodeling part of a microenvironment? Considering that there is a low concentration of BAR proteins in cells, based on the results of mechanical experiments we could conclude that they act only as curvature sensors (Sorre et al., 2012). However, it is reasonable to think of the endocytic site as a microenvironment separated from the bulk of the cell. In light of the observed high sorting of amphiphysin in the dilute protein regime (Sorre et al., 2012), the effective concentration of BAR proteins may thus increase well above the density required
F C
D
B BAR proteins Clathrin Actin
E
A
Unknown protein Macromolecules
Figure 1 Examples of the configurational space of membrane remodeling. (A) Low concentration of proteins: BAR proteins are only sensors of membrane curvature. (B) High concentration of proteins (e.g., through curvature recruitment): proteins induce budding and tubulation of the membrane. (C) Very high concentration (clustering) of macromolecules (including the proteins without specific affinity to the membrane): entropy-induced non-specific tubulation. (D) Endocytosis at high membrane tension: actin polymerization is required for vesicle formation. (E) Successive sorting of BAR proteins: first, low-curvature BAR proteins (red, e.g., BAR, F-BAR) sense buds of low radius. They then induce more curvature, increasing the affinity of BAR proteins of higher curvature (orange) and decreasing their own affinity to the membrane. High-curvature BAR proteins (orange) further increase curvature, recruiting very high-curvature BAR proteins (yellow, e.g., N-BAR from endophilin). (F) Endocytosis at the interface of two cells. The molecular mechanism of membrane remodeling in the context of tissues and whole organisms is yet to be discovered.
Brought to you by | Northern Illinois University Authenticated Download Date | 1/26/15 12:34 AM
M. Simunovic and P. Bassereau: Configurational space of endocytosis 279
for generating local curvature. The accumulation of BAR proteins may be mediated by the roughness of the membrane (Helfrich, 1989), curvature induced by endocytic cargo, clathrin and other proteins, including the ones that do not specifically interact with the membrane. Because of the uniquely high anisotropy in interactions between the BAR proteins and the membrane, we expect it would take less density to induce curvature than through clustering, giving them the potential as curvature generators. It is tempting to consider the existence of local membrane tension possibly caused by the heterogeneity in composition and the surface roughness of the membrane. Existing mechanical studies go against this thought, as they have predicted that membrane tension is in fact homogenous all around the cell, even between the apical and the basal part of the cell (Dai and Sheetz, 1999; Raucher and Sheetz, 1999). At the same time, it appears that the effective membrane tension is greatly influenced by the adhesive cytoskeleton-membrane interactions (Dai and Sheetz, 1999; Lieber et al., 2013). Effective membrane tension is typically deduced by the force measurement on tethers pulled from the cell, where small, but possibly important, fluctuations in the membrane tension may be overlooked, because of the curvature-driven protein sorting that reduces the force or the fact that the tether may tap into large lipid reservoirs, known to exist in membrane folds (Dai and Sheetz, 1999). It will be a challenge for the future researchers to find not only the precise source of membrane tension in cells, but also to elucidate if fluctuations in the tension may occur on such length and timescales to contribute to the formation of microenvironments on the membrane. It is still puzzling that there is a significant variability among BAR proteins, with respect to both the sign of their intrinsic curvature and its magnitude. For example, in humans there are 12 BAR proteins belonging to a group of sorting nexins, responsible for maintaining endosomal sorting. They are each recruited to membranes of different curvature (van Weering et al., 2012). Additional variability among BAR proteins comes from a different number of amphipathic helices that they may have. These subdomains are thought to shallowly insert into the bilayer and thus induce even higher spontaneous curvature (Drin and Antonny, 2010; Qualmann et al., 2011). The structural variability may attest to the finely tuned nature of membrane remodeling phenomena, directed toward achieving rapid local density increases. After all, it is still a major challenge to answer how proteins find each other in the right place at the right time. Coarse-grained simulations elucidated a novel self-assembly mechanism in which particles form linear aggregates on the membrane surface,
promoting their rapid accumulation on a localized region of the membrane. This mechanism was observed with N-BAR proteins (Simunovic et al., 2013b) and spherical colloids (Saric and Cacciuto, 2012), giving insights into the way proteins or viral particles interact in the cellular microenvironment. An even bigger challenge is to understand how the correct temporal sequence of the membrane bending machinery is achieved. One possibility is that there is a tight coupling between the protein’s affinity to the membrane, sensing of (specific) membrane curvature, and the spontaneous curvature it induces. One BAR protein may sense the curvature of certain magnitude and get recruited to it. It would then further bend the membrane, and thus increase the effective affinity of another BAR protein to the membrane, facilitating its recruitment. Finally, the variability among BAR and ENTH proteins comes from their non-bending domains, known to specifically target either lipids or proteins. For example, Pleckstrin homology (PH) domain targets phosphatidylinositol (4,5)-bisphosphate (PIP2) lipids, whereas the Src Homology 3 (SH3) domain targets dynamin (Mim and Unger, 2012; Meinecke et al., 2013), thus further contributing to the formation of microenvironments in membrane remodeling processes. It appears that the conversion from one phosphoinositide lipid to another determines the course of endocytosis, by selectively recruiting BAR proteins (Posor et al., 2013). Therefore even just identifying the components that comprise a biological process may give important insights into its spatiotemporal sequence.
How to resolve the mystery? Despite abundant studies, the precise role of all proteins in membrane remodeling phenomena, such as endocytosis, is still unclear. It is certain that these processes are tremendously complex and understanding their full scope demands a multifaceted view. Although sometimes the results obtained with one method seem contradictory to those obtained by another, their complementarity becomes evident when placed in a broader perspective. There is always the danger of prematurely excluding a potential important role of a protein, if its effect was in some conditions absent. The remarkable richness of the phase space of protein-membrane interactions indicates that a different set of physical conditions in membrane remodeling could alter the spatial or temporal sequence of its players. It is tempting to speculate that remodeling processes could have evolved to ensure that the cell may
Brought to you by | Northern Illinois University Authenticated Download Date | 1/26/15 12:34 AM
280 M. Simunovic and P. Bassereau: Configurational space of endocytosis employ alternative strategies during such changes. In the near future, we are tasked with demystifying this complex role of physical and experimental conditions in reshaping membranes and identifying how the cellular behavior fits into the configurational space of protein-membrane interactions. It will also be highly valuable to understand what happens outside the normal steady state. How do disease and infection shift membranes to a different part of the configurational space and thus take advantage of the large capacity of membrane for reshaping? Researchers also face a challenging task of determining whether membrane remodeling phenomena take different routes when cells are part of living tissues and organisms. New capabilities of key players in membrane remodeling continue to emerge. The need to understand the full scope of protein-membrane interactions drives the development of novel experimental and theoretical techniques. The development is directed toward increasing the complexity of reconstituted systems we can study in vitro, the spatial and temporal resolution in live cell studies, and toward improving the modeling of large biomolecular assemblies in computational studies (Ayton and Voth, 2010). A combination of these methodologies appears crucial in connecting all the remarkable ways proteins affect the membrane with how they are used by the cell to carry out its vital tasks.
Outlook 1: What is membrane remodeling? Lipid membranes prefer to have a closed structure with minimal surface area, but at the same time be as flat as possible. To curve, they need external energy. In endocytosis, all sorts of membrane curvatures may be found. When the bud enveloping external cargo emerges from the membrane, both mean and Gaussian curvatures are positive. This bud will gradually turn into a vesicle. Right before the vesicle is released into the cytosol, there will be very high negative Gaussian curvature at the neck of the bud. In some species, such as yeast, there is an intermediate state where the vesicle is tethered to the membrane by a tube, characterized as having positive mean, but zero Gaussian curvature. The two types of curvatures, mean vs. Gaussian, in addition to their mathematical definition can also be simply distinguished by the way they are perceived and measured. Mean curvature is only defined by external observation and its sign assigned by convention, while Gaussian curvature may be perceived if sitting on the object itself. Cellular machinery induces and maintains both types of curvature on the membrane, which determines the final shapes of cells and its compartments. It is very difficult to study shortscale geometric properties of membranes with experimental techniques, so analytical theory and computational simulations are the leading tools in elucidating the complex ways interactions with the membrane affect their large-scale morphology and the function membrane ultimately carries out.
Outlook 2: What is the configurational space of protein-membrane interactions? We define the configurational space of a membrane as a complete set of states a membrane may adopt as a function of conditions (temperature, lipid composition, concentration of the substrate, tension). Such states may comprise large-scale morphological changes, like budding or tubulation, or phase separation of lipids. Binding of proteins makes this diagram even more complex. To understand the full picture of membrane remodeling phenomena, we need to understand all the ways proteins may affect the membrane and under which conditions these transformations take place. For example, depending on physical conditions, BAR proteins may sense membrane curvature, they may induce buds and tubules, and according to most recent studies, they may even induce membrane fission. A future challenge for researchers will be finding ways of describing the full scope of interactions between proteins and membranes and the physical conditions under which they occur.
Outlook 3: How to connect the configurational space of membrane remodeling with the role of proteins in the cell? Membrane remodeling may be triggered in a number of different ways and is largely dependent on physical parameters, such as membrane tension, lipid composition, and the concentration of the bound substrate. Therefore we may have different experimental observations of the same phenomenon if studied under different conditions. A major challenge in the future will be relating what proteins are able to do with their actual role in remodeling. This means that we have to elucidate how cells have selected narrow parts of the configurational diagram to carry out function in normal conditions and how they can adjust to use other parts in pathogenic or stress conditions. To overcome this challenge, we will need to push the capabilities of experimental and theoretical techniques. It will be valuable to improve the complementarity between microscopy and multiscale simulations to access the structure and dynamics of remodeled membranes at very high temporal and spatial resolution. We will also need to find ways of combining in vitro with in vivo approaches, especially to control and describe the physical parameters while studying such processes in live cells.
Outlook 4: How do proteins find each other in the right place at the right time? Proteins control membrane remodeling phenomena to ensure that they take place correctly and efficiently. Live cell fluorescence imaging techniques help in elucidating the time sequence of proteins in the course of these processes. Correlating this data with electron microscopy will also answer how the recruitment of specific proteins leads to morphological changes in the membrane. We are still left with an important task of finding out
Brought to you by | Northern Illinois University Authenticated Download Date | 1/26/15 12:34 AM
M. Simunovic and P. Bassereau: Configurational space of endocytosis 281
the underlying mechanism that drives the rapid recruitment of proteins when and where they are needed. We need to understand if the places where membrane remodeling starts define a microenvironment, aided for example by crowding and the formation of nanodomains in the membrane. In such microenvironments, the cell could achieve conditions that are different from the bulk of the cell and thus access different parts of the configurational diagram. To study this problem, we will need to develop techniques that can characterize single molecule processes in living cells at high speeds and at high resolution.
Acknowledgments: The P.B. group belongs to the French research consortium ‘CellTiss’ and to the Labex CelTisPhyBio 11-LBX-0038. M.S. is funded by the Chateaubriand fellowship and the France and Chicago Collaborating in the Sciences grant.
Received August 20, 2013; accepted December 16, 2013; previously published online December 17, 2013
References Alberts, B. (2002). Molecular biology of the cell, 4th edition. (New York, USA: Garland Science). Ayton, G.S. and Voth, G.A. (2010). Multiscale simulation of protein mediated membrane remodeling. Semin. Cell Dev. Biol. 21, 357–362. Bai, J., Hu, Z., Dittman, J.S., Pym, E.C., and Kaplan, J.M. (2010). Endophilin functions as a membrane-bending molecule and is delivered to endocytic zones by exocytosis. Cell 143, 430–441. Baumgart, T., Hess, S.T., and Webb, W.W. (2003). Imaging coexisting fluid domains in biomembrane models coupling curvature and line tension. Nature 425, 821–824. Bhatia, V.K., Hatzakis, N.S., and Stamou, D. (2010). A unifying mechanism accounts for sensing of membrane curvature by BAR domains, amphipathic helices and membrane-anchored proteins. Semin. Cell Dev. Biol. 21, 381–390. Bhatia, V.K., Madsen, K.L., Bolinger, P.Y., Kunding, A., Hedegard, P., Gether, U., and Stamou, D. (2009). Amphipathic motifs in BAR domains are essential for membrane curvature sensing. EMBO J. 28, 3303–3314. Bickel, T., Jeppesen, C., and Marques, C.M. (2001). Local entropic effects of polymers grafted to soft interfaces. Eur. Phys. J. E. 4, 33–43. Blood, P.D. and Voth, G.A. (2006). Direct observation of Bin/ amphiphysin/Rvs (BAR). domain-induced membrane curvature by means of molecular dynamics simulations. Proc. Natl. Acad. Sci. USA 103, 15068–15072. Boucrot, E., Pick, A., Camdere, G., Liska, N., Evergren, E., McMahon, H.T., and Kozlov, M.M. (2012). Membrane fission is promoted by insertion of amphipathic helices and is restricted by crescent BAR domains. Cell 149, 124–136. Boulant, S., Kural, C., Zeeh, J.C., Ubelmann, F., and Kirchhausen, T. (2011). Actin dynamics counteract membrane tension during clathrin-mediated endocytosis. Nat. Cell Biol. 13, 1124–1131. Breidenich, M., Netz, R.R., and Lipowsky, R. (2000). The shape of polymer-decorated membranes. Europhys. Lett. 49, 431–437. Campelo, F., McMahon, H.T., and Kozlov, M.M. (2008). The hydrophobic insertion mechanism of membrane curvature generation by proteins. Biophys. J. 95, 2325–2339. Cocucci, E., Aguet, F., Boulant, S., and Kirchhausen, T. (2012). The first five seconds in the life of a clathrin-coated pit. Cell 150, 495–507. Cullis, P.R. and de Kruijff, B. (1979). Lipid polymorphism and the functional roles of lipids in biological membranes. Biochim. Biophys. Acta 559, 399–420.
Dai, J.W. and Sheetz, M.P. (1999). Membrane tether formation from blebbing cells. Biophys. J. 77, 3363–3370. Dannhauser, P.N. and Ungewickell, E.J. (2012). Reconstitution of clathrin-coated bud and vesicle formation with minimal components. Nat. Cell Biol. 14, 634–639. Doyon, J.B., Zeitler, B., Cheng, J., Cheng, A.T., Cherone, J.M., Santiago, Y., Lee, A.H., Vo, T.D., Doyon, Y., Miller, J.C., et al. (2011). Rapid and efficient clathrin-mediated endocytosis revealed in genome-edited mammalian cells. Nat. Cell Biol. 13, 331–337. Drin, G. and Antonny, B. (2010). Amphipathic helices and membrane curvature. FEBS Lett 584, 1840–1847. Farsad, K. and De Camilli, P. (2003). Mechanisms of membrane deformation. Curr. Opin. Cell Biol. 15, 372–381. Fotin, A., Cheng, Y., Sliz, P., Grigorieff, N., Harrison, S.C., Kirchhausen, T., and Walz, T. (2004). Molecular model for a complete clathrin lattice from electron cryomicroscopy. Nature 432, 573–579. Frost, A., Perera, R., Roux, A., Spasov, K., Destaing, O., Egelman, E.H., De Camilli, P., and Unger, V.M. (2008). Structural basis of membrane invagination by F-BAR domains. Cell 132, 807–817. Goetz, R., Gompper, G., and Lipowsky, R. (1999). Mobility and elasticity of self-assembled membranes. Phys. Rev. Lett. 82, 221–224. Heinrich, M.C., Capraro, B.R., Tian, A., Isas, J.M., Langen, R., and Baumgart, T. (2010). Quantifying membrane curvature generation of amphiphysin N-BAR domains. J. Phys. Chem. Lett. 1, 3401–3406. Helfrich, W. (1973). Elastic properties of lipid bilayers: theory and possible experiments. Z. Naturforsch. C 28, 693–703. Helfrich, W. (1989). Hats and saddles in lipid-membranes. Liquid Crystals 5, 1647–1658. Julicher, F. and Lipowsky, R. (1993). Domain-induced budding of vesicles. Phys. Rev. Lett. 70, 2964–2967. Kirchhausen, T. (2009). Imaging endocytic clathrin structures in living cells. Trends Cell Biol. 19, 596–605. Kukulski, W., Schorb, M., Kaksonen, M., and Briggs, J.A. (2012). Plasma membrane reshaping during endocytosis is revealed by time-resolved electron tomography. Cell 150, 508–520. Kukulski, W., Schorb, M., Welsch, S., Picco, A., Kaksonen, M., and Briggs, J.A. (2011). Correlated fluorescence and 3D electron microscopy with high sensitivity and spatial precision. J. Cell Biol. 192, 111–119.
Brought to you by | Northern Illinois University Authenticated Download Date | 1/26/15 12:34 AM
282 M. Simunovic and P. Bassereau: Configurational space of endocytosis Lieber, A.D., Yehudai-Resheff, S., Barnhart, E.L., Theriot, J.A., and Keren, K. (2013). membrane tension in rapidly moving cells is determined by cytoskeletal forces. Curr. Biol., 23, 1409–1417. Lipowsky, R. (1991). The conformation of membranes. Nature 349, 475–481. Lipowsky, R. (2013). Spontaneous tubulation of membranes and vesicles reveals membrane tension generated by spontaneous curvature. Farad. Discuss. 161, 305–331. Lipowsky, R. and Dimova, R. (2003). Domains in membranes and vesicles. J. Phys. Condens. Matter. 15, S31–S45. Lipowsky, R. and Sackmann, E. (ed). (1995). Structure and Dynamics of Membranes: From Cells to Vesicles. Elsevier: North Holland, Amsterdam. McMahon, H.T. and Gallop, J.L. (2005). Membrane curvature and mechanisms of dynamic cell membrane remodelling. Nature 438, 590–596. Meinecke, M., Boucrot, E., Camdere, G., Hon, W.C., Mittal, R., and McMahon, H.T. (2013). Cooperative Recruitment of Dynamin and BIN/Amphiphysin/Rvs (BAR). Domain-containing Proteins Leads to GTP-dependent Membrane Scission. J. Biol. Chem. 288, 6651–6661. Mim, C., Cui, H., Gawronski-Salerno, J.A., Frost, A., Lyman, E., Voth, G.A., and Unger, V.M. (2012a). Structural basis of membrane bending by the N-BAR protein endophilin. Cell 149, 137–145. Mim, C., Cui, H.S., Gawronski-Salerno, J.A., Frost, A., Lyman, E., Voth, G.A., and Unger, V.M. (2012b). Structural basis of membrane bending by the N-BAR protein endophilin. Cell 149, 137–145. Mim, C. and Unger, V.M. (2012). Membrane curvature and its generation by BAR proteins. Trends Biochem. Sci. 37, 526–533. Morlot, S., Galli, V., Klein, M., Chiaruttini, N., Manzi, J., Humbert, F., Dinis, L., Lenz, M., Cappello, G., and Roux, A. (2012). Membrane shape at the edge of the dynamin helix sets location and duration of the fission reaction. Cell 151, 619–629. Peter, B.J., Kent, H.M., Mills, I.G., Vallis, Y., Butler, P.J., Evans, P.R., and McMahon, H.T. (2004). BAR domains as sensors of membrane curvature: the amphiphysin BAR structure. Science 303, 495–499. Posor, Y., Eichhorn-Gruenig, M., Puchkov, D., Schoneberg, J., Ullrich, A., Lampe, A., Muller, R., Zarbakhsh, S., Gulluni, F., Hirsch, E., et al. (2013). Spatiotemporal control of endocytosis by phosphatidylinositol-3,4-bisphosphate. Nature 499, 233–237. Qualmann, B., Koch, D., and Kessels, M.M. (2011). Let’s go bananas: revisiting the endocytic BAR code. EMBO J. 30, 3501–3515. Ramakrishnan, N., Kumar, P.B.S., and Ipsen, J.H. (2013). Membranemediated aggregation of curvature-inducing nematogens and membrane tubulation. Biophys. J. 104, 1018–1028. Ramesh, P., Baroji, Y.F., Reihani, S.N., Stamou, D., Oddershede, L.B., and Bendix, P.M. (2013). FBAR syndapin 1 recognizes and stabilizes highly curved tubular membranes in a concentration dependent manner. Sci. Rep. 3, 1565. Raucher, D. and Sheetz, M.P. (1999). Characteristics of a membrane reservoir buffering membrane tension. Biophys. J. 77, 1992–2002.
Reynwar, B.J., Illya, G., Harmandaris, V.A., Muller, M.M., Kremer, K., and Deserno, M. (2007). Aggregation and vesiculation of membrane proteins by curvature-mediated interactions. Nature 447, 461–464. Saheki, Y. and De Camilli, P. (2012). Synaptic vesicle endocytosis. Cold Spring Harb. Perspect. Biol. 4, a005645. Saric, A. and Cacciuto, A. (2012). Fluid membranes can drive linear aggregation of adsorbed spherical nanoparticles. Phys. Rev. Lett. 108, 118101. Seifert, U. (1997). Configurations of fluid membranes and vesicles. Adv. Phys. 46, 13–137. Sens, P., Johannes, L., and Bassereau, P. (2008). Biophysical approaches to protein-induced membrane deformations in trafficking. Curr. Opin. Cell Biol. 20, 476–482. Simunovic, M., Mim, C., Marlovits, T.C., Resch, G., Unger, V.M., and Voth, G.A. (2013a). Protein-mediated transformation of lipid vesicles into tubular networks. Biophys J. 105, 711–719. Simunovic, M., Srivastava, A., and Voth, G.A. (2013b). Linear aggregation of proteins on the membrane as a prelude to membrane remodeling. Proc. Natl. Acad. Sci. USA, 110, 20396–20401. Singh, P., Mahata, P., Baumgart, T., and Das, S.L. (2012). Curvature sorting of proteins on a cylindrical lipid membrane tether connected to a reservoir. Phys. Rev. E, 85. Sorre, B., Callan-Jones, A., Manneville, J.B., Nassoy, P., Joanny, J.F., Prost, J., Goud, B., and Bassereau, P. (2009). Curvaturedriven lipid sorting needs proximity to a demixing point and is aided by proteins. Proc. Natl. Acad. Sci. USA 106, 5622–5626. Sorre, B., Callan-Jones, A., Manzi, J., Goud, B., Prost, J., Bassereau, P., and Roux, A. (2012). Nature of curvature coupling of amphiphysin with membranes depends on its bound density. Proc. Natl. Acad. Sci. USA 109, 173–178. Stachowiak, J.C., Schmid, E.M., Ryan, C.J., Ann, H.S., Sasaki, D.Y., Sherman, M.B., Geissler, P.L., Fletcher, D.A., and Hayden, C.C. (2012). Membrane bending by protein-protein crowding. Nat. Cell Biol. 14, 944–949. Taylor, M.J., Perrais, D., and Merrifield, C.J. (2011). A High precision survey of the molecular dynamics of mammalian clathrinmediated endocytosis. Plos Biology, 9, e1000604. van Weering, J.R.T., Sessions, R.B., Traer, C.J., Kloer, D.P., Bhatia, V.K., Stamou, D., Carlsson, S.R., Hurley, J.H., and Cullen, P.J. (2012). Molecular basis for SNX-BAR-mediated assembly of distinct endosomal sorting tubules. EMBO J. 31, 4466–4480. Verstreken, P., Kjaerulff, O., Lloyd, T.E., Atkinson, R., Zhou, Y., Meinertzhagen, I.A., and Bellen, H.J. (2002). Endophilin mutations block clathrin-mediated endocytosis but not neurotransmitter release. Cell 109, 101–112. Weinberg, J. and Drubin, D.G. (2012). Clathrin-mediated endocytosis in budding yeast. Trends Cell Biol, 22, 1–13. Yin, Y., Arkhipov, A., and Schulten, K. (2009). Simulations of Membrane Tubulation by Lattices of Amphiphysin N-BAR Domains. Structure 17, 882–892. Zhu, C., Das, S.L., and Baumgart, T. (2012). Nonlinear sorting, curvature generation, and crowding of endophilin N-BAR on tubular membranes. Biophys. J. 102, 1837–1845.
Brought to you by | Northern Illinois University Authenticated Download Date | 1/26/15 12:34 AM
M. Simunovic and P. Bassereau: Configurational space of endocytosis 283
Mijo Simunovic is a PhD student in chemistry at the University of Chicago and in physics at the Curie Institute in Paris. In his research, he combines coarse-grained theoretical techniques in the Voth group with experimental biophysical methods at the Bassereau group to study the physics underlying protein-induced membrane remodeling phenomena. Before joining these two groups, he received his BS and MS in physical chemistry from the University of Zagreb, where he employed theoretical and experimental approaches in investigating problems in synthetic and quantum chemistry.
Patricia Bassereau is currently Directrice de Recherche, CNRS at the Curie Institute in Paris. After spending 7 years in Montpellier (GDPC) working on the structure of surfactant-based phases, and a year as a visiting scientist at the Almaden IBM Center (San Jose, USA) on the structure of thin polymer films, she moved in 1993 to the Curie Institute. She initially investigated the interactions of soluble proteins with polymer monolayers. In the last 15 years, she has been working in the field of ‘physics for cell biology’. She has developed a multidisciplinary approach to understand the role of lipid membranes in important cellular functions such as intracellular trafficking, endo/ exocytosis, transmembrane ion transport (‘active membranes’), or cell adhesion.
Brought to you by | Northern Illinois University Authenticated Download Date | 1/26/15 12:34 AM
Review Mijo Simunovic and Patricia Bassereau*
Reshaping biological membranes in endocytosis: crossing the configurational space of membraneprotein interactions Abstract: Lipid membranes are highly dynamic. Over several decades, physicists and biologists have uncovered a number of ways they can change the shape of membranes or alter their phase behavior. In cells, the intricate action of membrane proteins drives these processes. Considering the highly complex ways proteins interact with biological membranes, molecular mechanisms of membrane remodeling still remain unclear. When studying membrane remodeling phenomena, researchers often observe different results, leading them to disparate conclusions on the physiological course of such processes. Here we discuss how combining research methodologies and various experimental conditions contributes to the understanding of the entire phase space of membrane-protein interactions. Using the example of clathrin-mediated endocytosis we try to distinguish the question ‘how can proteins remodel the membrane?’ from ‘how do proteins remodel the membrane in the cell?’ In particular, we consider how altering physical parameters may affect the way membrane is remodeled. Uncovering the full range of physical conditions under which membrane phenomena take place is key in understanding the way cells take advantage of membrane properties in carrying out their vital tasks. Keywords: BAR domain; clathrin; membrane remodeling; membrane tension; multiscale simulation. *Corresponding author: Patricia Bassereau, Institut Curie, Centre National de la Recherche Scientifique Unité Mixte de Recherche 168, Université Pierre et Marie Curie, F-75248 Paris, France, e-mail: [email protected] Mijo Simunovic: Institut Curie, Centre National de la Recherche Scientifique Unité Mixte de Recherche 168, Université Pierre et Marie Curie, F-75248 Paris, France; Department of Physics, Université Paris Diderot, 10, rue Alice Domon et Léonie Duquet, 75013 Paris, France; and Department of Chemistry, The University of Chicago, Chicago, IL 60637, USA
What is a membrane? Biological membranes are highly dynamic supramolecular assemblies. They are mainly made up of lipids, which form bilayers in water (Lipowsky, 1991). Structural features of such bilayers are essentially multiscale (Goetz et al., 1999). They can span microns in lateral dimensions to form elastic sheets governed by macroscopic mechanics. Conversely, the cross-section of a lipid bilayer is orders of magnitude smaller, with only two molecules making up its thickness (Lipowsky, 1995). This aspect demands a molecular point of view when studying any interactions with the membrane. Furthermore, lipids move relatively freely in a quasi two-dimensional space. Such mobility makes the membrane fluid, further contributing to its diverse behavior (Lipowsky, 1995). The interplay between the macroscopic mechanics and molecular interactions, in turn, determines their equilibrium shape. The hydrophobic repulsion at the interface of water molecules and lipid tails gives rise to line tension that minimizes the edge of a bilayer. This process competes with elastic energy induced by bending the membrane. As a result of these opposing forces, biological membranes form a remarkable range of geometries (Seifert, 1997). Membrane remodeling is essential for cellular function, as it is a key step in communication among cells, the formation of organelles, division, trafficking, and migration (Alberts, 2002). To understand the physics underlying these phenomena, it is important to investigate the way membranes can be reshaped. According to a theory introduced by Helfrich, bending energy will decrease as the membrane deforms in a way so that its curvature matches the spontaneous curvature (Helfrich, 1973). Spontaneous curvature of free and symmetric bilayers is zero, however binding of other molecules, such as proteins, may induce a non-zero value of this term (Farsad and De Camilli, 2003; McMahon and Gallop, 2005; Sens et al., 2008). Understanding how the inclusion or peripheral binding of proteins couples with the morphology of membranes
Brought to you by | Northern Illinois University Authenticated Download Date | 1/26/15 12:34 AM
276 M. Simunovic and P. Bassereau: Configurational space of endocytosis presents a significant scientific challenge for physicists and biologists. Over several decades, scientists have described the very rich phase behavior of lipid membranes, dependent mostly on their composition, surrounding media, and temperature (Cullis and de Kruijff, 1979). The presence of proteins adds to their already complex behavior. Researchers have uncovered a number of different ways proteins can interact with membranes, which induces a plethora of morphological transitions. Our main goal in this review is to discuss how the interpretation of this configurational diagram becomes important for biology. We believe that a leading problem in biophysics in years to come will be distinguishing the question ‘what can proteins do to cell membranes?’ from ‘what proteins do to cell membranes?’ A similar issue may be raised in the field of materials science. We can engineer alloys with remarkable phase behavior, with only fractions of the phase diagram having industrial importance. This observation does not imply that characterizing the wide range of capabilities of materials such as membranes is a futile task. On the contrary, understanding the full scope of membrane-protein interactions gives important insights into their behavior in the cells, as long as this behavior is carefully extrapolated from the studied conditions. This is an exceptional challenge for membrane physicists and biologists.
Are proteins needed for the remodeling of cell membranes? A significant source of diversity among membranes comes from a large variety of lipids that can assemble into bilayers. Some lipids, such as sphingomyelins, due to their structure prefer to form more ordered domains, whereas others, like phosphocholines, phosphoserines, etc., form disordered domains (Cullis and de Kruijff, 1979). It has been shown that the latter lipids can even, under certain conditions, be recruited to curved membrane regions without any help of proteins (Sorre et al., 2009). The presence of both kinds of lipids will result in phase separation, which may induce enough line tension between the phases to drive membrane remodeling, such as budding and vesiculation (Julicher and Lipowsky, 1993; Baumgart et al., 2003; Lipowsky and Dimova, 2003). Clearly, lipids alone comprise powerful machinery that can induce structural and phase changes in the membrane. However, cells often require membranes to be highly curved and that the remodeling occurs rapidly,
efficiently, in a controlled and reproducible manner, which makes proteins indispensible players in these processes (McMahon and Gallop, 2005). In fact, they involve a large number of proteins, often coupled in complex and unclear ways. Endocytosis is an important example in which membrane is remodeled to facilitate the entry of molecules and particles into the cell (Fotin et al., 2004; Kirchhausen, 2009; Saheki and De Camilli, 2012). According to the bestunderstood mechanism of this process, protein clathrin polymerizes into a rigid coat onto the membrane, mediating the formation of buds and vesicles (Kirchhausen, 2009; Saheki and De Camilli, 2012). At the same time, large families of proteins, that contain Bin/amphiphysin/ Rvs (BAR) or epsin N-terminal homology (ENTH) domains, play various roles in the course of endocytosis. Unlike clathrin, that binds to the membrane via its adaptor molecules, BAR and ENTH domain proteins directly interact with the membrane (Qualmann et al., 2011; Mim and Unger, 2012; Saheki and De Camilli, 2012). However, different experimental techniques have resulted in various and often contrary observations, leaving an open question how deeply involved these proteins are in membrane remodeling. From a physical point of view, bending a membrane requires energy. Binding of BAR and ENTH proteins may provide this energy via adhesion or inclusion into the bilayer (Campelo et al., 2008; Ayton and Voth, 2010; Mim and Unger, 2012). As a result, proteins will generate a non-zero spontaneous curvature, in which the magnitude will depend on the way the proteins interact with the membrane. It has been shown that overexpression of BAR proteins in cells will induce tubulation of their membranes (Peter et al., 2004). Tubule formation has also been observed in vitro with electron microscopy when incubating these proteins with highly charged liposomes (Peter et al., 2004; Frost et al., 2008; Mim et al., 2012a). Computer simulations at the atomic level have shown that a single N-BAR domain induces significant local deformation of the membrane, thus giving rise to spontaneous curvature (Blood and Voth, 2006). Simulating the collective behavior of proteins using coarsegrained models has shown that curvature-inducing mechanism become much more complex at larger scales. Anisotropic interactions with the membrane induce local curvaturem, which then mediates their aggregation and subsequent large-scale morphological changes, such as budding (Reynwar et al., 2007; Simunovic et al., 2013b). These results confirm the prediction that BAR and ENTH domain proteins have the capabilities to drive curvature generation in cells. Furthermore, BAR proteins
Brought to you by | Northern Illinois University Authenticated Download Date | 1/26/15 12:34 AM
M. Simunovic and P. Bassereau: Configurational space of endocytosis 277
preferentially bind to curved membranes, and thus potentially rapidly stabilize the tubular neck formed in endocytosis (Bhatia et al., 2009, 2010; Sorre et al., 2012; Zhu et al., 2012). At the same time, the polymerization of clathrin on giant liposomes has been demonstrated to induce the formation of vesicles upon heating (Dannhauser and Ungewickell, 2012). As demonstrated in these cases, electron microscopy imaging gives vital structural information on the interaction between proteins and remodeled membranes at very high resolution. However, from electron microscopy it is hard to deduce the temporal sequence of remodeling events and describe these processes in quantitative terms. Fluorescence microscopy allows a more quantitative observation of binding and remodeling, but at the cost of resolution. It is evident from both of these approaches that BAR proteins and clathrin have a marked effect on the membrane alone. However, do these observations give direct clues of their mode of action and dynamics in the cell? It is possible that the elevated protein concentration and membrane charge enabled the remodeling with the help of increased effective binding energy. Genetic studies on live cells can help elucidate the way these proteins affect cellular viability. It has been shown that the deletion of endophilin and amphiphysin (both N-BAR proteins) only changes the rate and efficiency of endocytosis (Saheki and De Camilli, 2012). Conversely, in other studies, knocking out endophilin or disrupting its membrane bending activity has a much more detrimental effect on functionality of synaptic vesicles (Verstreken et al., 2002; Bai et al., 2010). Live cell imaging with fluorescence microscopy provides an important link to understanding the single molecule dynamics in the course of endocytosis (Kirchhausen, 2009; Taylor et al., 2011; Weinberg and Drubin, 2012). Another approach – correlative fluorescence and electron microscopy – bridges the gap between temporal and spatial resolution in the two microscopies (Kukulski et al., 2011). Live cell imaging studies predict that FCHo (an F-BAR protein) is recruited to already curved membranes and thus most likely participates in the stabilization of membrane curvature, but not its generation (Cocucci et al., 2012). By studying endocytosis in yeast with the correlative microscopy approach, it has been shown that BAR and ENTH proteins are already present on flat membranes, however curvature is induced only after actin recruitment (Kukulski et al., 2012). In this study, amphiphysin is predicted to help elongate the initially formed membrane invagination; therefore, its membrane bending activity is only important for the efficiency of endocytosis and not its initiation.
Does physics matter? The observed discrepancies perfectly reflect the complexity of membrane remodeling processes. They may imply that endocytosis occurs in a heterogeneous way and that it depends on numerous factors, such as physical conditions, studied organism, cell type, or even the observed part of the cell. For example, the part of membrane attached to the microscopy coverslip shows different behavior than the free membrane, which the authors attribute to the difference in tension of those two membranes (Boulant et al., 2011). It has even been shown that the experimental way proteins are expressed in the cells may affect the endocytosis rate as well (Doyon et al., 2011). Combining fluorescence microscopy with mechanical measurements helps to describe, in a quantitative way, the underlying physics of membrane remodeling. Such experiments have shown that the density of BAR proteins bound to the membrane deeply affects their behavior (Heinrich et al., 2010; Sorre et al., 2012; Ramesh et al., 2013). In the case of amphiphysin, when the density is low enough that the proteins do not interact with each other, they sense curvature and enrich membrane nanotubes. At higher densities, their interaction provides mechanical force that stabilizes tubulation and induces curvature (Sorre et al., 2012). In this density regime, coarse-grained simulations and cryo-electron microscopy have shown that proteins arrange into a nematic-like assembly, driving the formation and stabilization of membrane tubes (Yin et al., 2009; Mim et al., 2012b; Ramakrishnan et al., 2013) and, at very high densities, even breaking the surface of the vesicle to form a closed-surface tubular network (Simunovic et al., 2013a). The surprising fissiogenic properties of N-BAR proteins have also been observed with electron microscopy (Boucrot et al., 2012). These results show that it is crucial to understand the complexity of protein-protein interactions and how they may alter the behavior of the membrane. Strikingly, membrane can be deformed just as a result of entropic pressure caused by clustering of macromolecules. This has been shown for polymers and proteins that do not specifically interact with the membrane (Breidenich et al., 2000; Bickel et al., 2001; Stachowiak et al., 2012). The important implication of these studies is that the cell may use the underlying physics to bend the membrane, while the complex protein machinery controls, what would otherwise be, a very inefficient process. Mechanical experiments also help in understanding the full scope of the phase space of membrane-protein interactions by accounting for and controlling physical parameters (Singh et al., 2012). One such parameter is membrane tension that plays a key role in protein-induced
Brought to you by | Northern Illinois University Authenticated Download Date | 1/26/15 12:34 AM
278 M. Simunovic and P. Bassereau: Configurational space of endocytosis remodeling (Lipowsky, 2013). For example, the protein dynamin, which cuts the membrane to release the endocytic vesicle, works more efficiently at higher tensions on tubular membrane structures (Morlot et al., 2012). It has been proposed that membrane tension can even control the course of endocytosis. High membrane tension requires the polymerization of actin that will then help in elongating the endocytic bud, a step absent at low tension (Boulant et al., 2011). Conversely, in yeast actin is required regardless of tension. High tension of vesicles may explain why sometimes BAR proteins do not remodel the membrane in vitro to as large an extent as in vivo (Peter et al., 2004). Possibly, the efficiency of remodeling by BAR or ENTH proteins in live cell studies was also precluded by high tension. Of note, we still do not know the precise origin of membrane tension in cells and how it is controlled, so its role in membrane remodeling phenomena remains elusive. It is evident, however, that fundamental physical parameters may affect the course of membrane remodeling. Therefore, it is important to keep in mind
their potential active role in experimental observations and whether similar conditions may take place in the cell. Figure 1 depicts how the configurational space manifests in the cell and how different conditions lead to different outcomes in endocytosis and related phenomena.
Is remodeling part of a microenvironment? Considering that there is a low concentration of BAR proteins in cells, based on the results of mechanical experiments we could conclude that they act only as curvature sensors (Sorre et al., 2012). However, it is reasonable to think of the endocytic site as a microenvironment separated from the bulk of the cell. In light of the observed high sorting of amphiphysin in the dilute protein regime (Sorre et al., 2012), the effective concentration of BAR proteins may thus increase well above the density required
F C
D
B BAR proteins Clathrin Actin
E
A
Unknown protein Macromolecules
Figure 1 Examples of the configurational space of membrane remodeling. (A) Low concentration of proteins: BAR proteins are only sensors of membrane curvature. (B) High concentration of proteins (e.g., through curvature recruitment): proteins induce budding and tubulation of the membrane. (C) Very high concentration (clustering) of macromolecules (including the proteins without specific affinity to the membrane): entropy-induced non-specific tubulation. (D) Endocytosis at high membrane tension: actin polymerization is required for vesicle formation. (E) Successive sorting of BAR proteins: first, low-curvature BAR proteins (red, e.g., BAR, F-BAR) sense buds of low radius. They then induce more curvature, increasing the affinity of BAR proteins of higher curvature (orange) and decreasing their own affinity to the membrane. High-curvature BAR proteins (orange) further increase curvature, recruiting very high-curvature BAR proteins (yellow, e.g., N-BAR from endophilin). (F) Endocytosis at the interface of two cells. The molecular mechanism of membrane remodeling in the context of tissues and whole organisms is yet to be discovered.
Brought to you by | Northern Illinois University Authenticated Download Date | 1/26/15 12:34 AM
M. Simunovic and P. Bassereau: Configurational space of endocytosis 279
for generating local curvature. The accumulation of BAR proteins may be mediated by the roughness of the membrane (Helfrich, 1989), curvature induced by endocytic cargo, clathrin and other proteins, including the ones that do not specifically interact with the membrane. Because of the uniquely high anisotropy in interactions between the BAR proteins and the membrane, we expect it would take less density to induce curvature than through clustering, giving them the potential as curvature generators. It is tempting to consider the existence of local membrane tension possibly caused by the heterogeneity in composition and the surface roughness of the membrane. Existing mechanical studies go against this thought, as they have predicted that membrane tension is in fact homogenous all around the cell, even between the apical and the basal part of the cell (Dai and Sheetz, 1999; Raucher and Sheetz, 1999). At the same time, it appears that the effective membrane tension is greatly influenced by the adhesive cytoskeleton-membrane interactions (Dai and Sheetz, 1999; Lieber et al., 2013). Effective membrane tension is typically deduced by the force measurement on tethers pulled from the cell, where small, but possibly important, fluctuations in the membrane tension may be overlooked, because of the curvature-driven protein sorting that reduces the force or the fact that the tether may tap into large lipid reservoirs, known to exist in membrane folds (Dai and Sheetz, 1999). It will be a challenge for the future researchers to find not only the precise source of membrane tension in cells, but also to elucidate if fluctuations in the tension may occur on such length and timescales to contribute to the formation of microenvironments on the membrane. It is still puzzling that there is a significant variability among BAR proteins, with respect to both the sign of their intrinsic curvature and its magnitude. For example, in humans there are 12 BAR proteins belonging to a group of sorting nexins, responsible for maintaining endosomal sorting. They are each recruited to membranes of different curvature (van Weering et al., 2012). Additional variability among BAR proteins comes from a different number of amphipathic helices that they may have. These subdomains are thought to shallowly insert into the bilayer and thus induce even higher spontaneous curvature (Drin and Antonny, 2010; Qualmann et al., 2011). The structural variability may attest to the finely tuned nature of membrane remodeling phenomena, directed toward achieving rapid local density increases. After all, it is still a major challenge to answer how proteins find each other in the right place at the right time. Coarse-grained simulations elucidated a novel self-assembly mechanism in which particles form linear aggregates on the membrane surface,
promoting their rapid accumulation on a localized region of the membrane. This mechanism was observed with N-BAR proteins (Simunovic et al., 2013b) and spherical colloids (Saric and Cacciuto, 2012), giving insights into the way proteins or viral particles interact in the cellular microenvironment. An even bigger challenge is to understand how the correct temporal sequence of the membrane bending machinery is achieved. One possibility is that there is a tight coupling between the protein’s affinity to the membrane, sensing of (specific) membrane curvature, and the spontaneous curvature it induces. One BAR protein may sense the curvature of certain magnitude and get recruited to it. It would then further bend the membrane, and thus increase the effective affinity of another BAR protein to the membrane, facilitating its recruitment. Finally, the variability among BAR and ENTH proteins comes from their non-bending domains, known to specifically target either lipids or proteins. For example, Pleckstrin homology (PH) domain targets phosphatidylinositol (4,5)-bisphosphate (PIP2) lipids, whereas the Src Homology 3 (SH3) domain targets dynamin (Mim and Unger, 2012; Meinecke et al., 2013), thus further contributing to the formation of microenvironments in membrane remodeling processes. It appears that the conversion from one phosphoinositide lipid to another determines the course of endocytosis, by selectively recruiting BAR proteins (Posor et al., 2013). Therefore even just identifying the components that comprise a biological process may give important insights into its spatiotemporal sequence.
How to resolve the mystery? Despite abundant studies, the precise role of all proteins in membrane remodeling phenomena, such as endocytosis, is still unclear. It is certain that these processes are tremendously complex and understanding their full scope demands a multifaceted view. Although sometimes the results obtained with one method seem contradictory to those obtained by another, their complementarity becomes evident when placed in a broader perspective. There is always the danger of prematurely excluding a potential important role of a protein, if its effect was in some conditions absent. The remarkable richness of the phase space of protein-membrane interactions indicates that a different set of physical conditions in membrane remodeling could alter the spatial or temporal sequence of its players. It is tempting to speculate that remodeling processes could have evolved to ensure that the cell may
Brought to you by | Northern Illinois University Authenticated Download Date | 1/26/15 12:34 AM
280 M. Simunovic and P. Bassereau: Configurational space of endocytosis employ alternative strategies during such changes. In the near future, we are tasked with demystifying this complex role of physical and experimental conditions in reshaping membranes and identifying how the cellular behavior fits into the configurational space of protein-membrane interactions. It will also be highly valuable to understand what happens outside the normal steady state. How do disease and infection shift membranes to a different part of the configurational space and thus take advantage of the large capacity of membrane for reshaping? Researchers also face a challenging task of determining whether membrane remodeling phenomena take different routes when cells are part of living tissues and organisms. New capabilities of key players in membrane remodeling continue to emerge. The need to understand the full scope of protein-membrane interactions drives the development of novel experimental and theoretical techniques. The development is directed toward increasing the complexity of reconstituted systems we can study in vitro, the spatial and temporal resolution in live cell studies, and toward improving the modeling of large biomolecular assemblies in computational studies (Ayton and Voth, 2010). A combination of these methodologies appears crucial in connecting all the remarkable ways proteins affect the membrane with how they are used by the cell to carry out its vital tasks.
Outlook 1: What is membrane remodeling? Lipid membranes prefer to have a closed structure with minimal surface area, but at the same time be as flat as possible. To curve, they need external energy. In endocytosis, all sorts of membrane curvatures may be found. When the bud enveloping external cargo emerges from the membrane, both mean and Gaussian curvatures are positive. This bud will gradually turn into a vesicle. Right before the vesicle is released into the cytosol, there will be very high negative Gaussian curvature at the neck of the bud. In some species, such as yeast, there is an intermediate state where the vesicle is tethered to the membrane by a tube, characterized as having positive mean, but zero Gaussian curvature. The two types of curvatures, mean vs. Gaussian, in addition to their mathematical definition can also be simply distinguished by the way they are perceived and measured. Mean curvature is only defined by external observation and its sign assigned by convention, while Gaussian curvature may be perceived if sitting on the object itself. Cellular machinery induces and maintains both types of curvature on the membrane, which determines the final shapes of cells and its compartments. It is very difficult to study shortscale geometric properties of membranes with experimental techniques, so analytical theory and computational simulations are the leading tools in elucidating the complex ways interactions with the membrane affect their large-scale morphology and the function membrane ultimately carries out.
Outlook 2: What is the configurational space of protein-membrane interactions? We define the configurational space of a membrane as a complete set of states a membrane may adopt as a function of conditions (temperature, lipid composition, concentration of the substrate, tension). Such states may comprise large-scale morphological changes, like budding or tubulation, or phase separation of lipids. Binding of proteins makes this diagram even more complex. To understand the full picture of membrane remodeling phenomena, we need to understand all the ways proteins may affect the membrane and under which conditions these transformations take place. For example, depending on physical conditions, BAR proteins may sense membrane curvature, they may induce buds and tubules, and according to most recent studies, they may even induce membrane fission. A future challenge for researchers will be finding ways of describing the full scope of interactions between proteins and membranes and the physical conditions under which they occur.
Outlook 3: How to connect the configurational space of membrane remodeling with the role of proteins in the cell? Membrane remodeling may be triggered in a number of different ways and is largely dependent on physical parameters, such as membrane tension, lipid composition, and the concentration of the bound substrate. Therefore we may have different experimental observations of the same phenomenon if studied under different conditions. A major challenge in the future will be relating what proteins are able to do with their actual role in remodeling. This means that we have to elucidate how cells have selected narrow parts of the configurational diagram to carry out function in normal conditions and how they can adjust to use other parts in pathogenic or stress conditions. To overcome this challenge, we will need to push the capabilities of experimental and theoretical techniques. It will be valuable to improve the complementarity between microscopy and multiscale simulations to access the structure and dynamics of remodeled membranes at very high temporal and spatial resolution. We will also need to find ways of combining in vitro with in vivo approaches, especially to control and describe the physical parameters while studying such processes in live cells.
Outlook 4: How do proteins find each other in the right place at the right time? Proteins control membrane remodeling phenomena to ensure that they take place correctly and efficiently. Live cell fluorescence imaging techniques help in elucidating the time sequence of proteins in the course of these processes. Correlating this data with electron microscopy will also answer how the recruitment of specific proteins leads to morphological changes in the membrane. We are still left with an important task of finding out
Brought to you by | Northern Illinois University Authenticated Download Date | 1/26/15 12:34 AM
M. Simunovic and P. Bassereau: Configurational space of endocytosis 281
the underlying mechanism that drives the rapid recruitment of proteins when and where they are needed. We need to understand if the places where membrane remodeling starts define a microenvironment, aided for example by crowding and the formation of nanodomains in the membrane. In such microenvironments, the cell could achieve conditions that are different from the bulk of the cell and thus access different parts of the configurational diagram. To study this problem, we will need to develop techniques that can characterize single molecule processes in living cells at high speeds and at high resolution.
Acknowledgments: The P.B. group belongs to the French research consortium ‘CellTiss’ and to the Labex CelTisPhyBio 11-LBX-0038. M.S. is funded by the Chateaubriand fellowship and the France and Chicago Collaborating in the Sciences grant.
Received August 20, 2013; accepted December 16, 2013; previously published online December 17, 2013
References Alberts, B. (2002). Molecular biology of the cell, 4th edition. (New York, USA: Garland Science). Ayton, G.S. and Voth, G.A. (2010). Multiscale simulation of protein mediated membrane remodeling. Semin. Cell Dev. Biol. 21, 357–362. Bai, J., Hu, Z., Dittman, J.S., Pym, E.C., and Kaplan, J.M. (2010). Endophilin functions as a membrane-bending molecule and is delivered to endocytic zones by exocytosis. Cell 143, 430–441. Baumgart, T., Hess, S.T., and Webb, W.W. (2003). Imaging coexisting fluid domains in biomembrane models coupling curvature and line tension. Nature 425, 821–824. Bhatia, V.K., Hatzakis, N.S., and Stamou, D. (2010). A unifying mechanism accounts for sensing of membrane curvature by BAR domains, amphipathic helices and membrane-anchored proteins. Semin. Cell Dev. Biol. 21, 381–390. Bhatia, V.K., Madsen, K.L., Bolinger, P.Y., Kunding, A., Hedegard, P., Gether, U., and Stamou, D. (2009). Amphipathic motifs in BAR domains are essential for membrane curvature sensing. EMBO J. 28, 3303–3314. Bickel, T., Jeppesen, C., and Marques, C.M. (2001). Local entropic effects of polymers grafted to soft interfaces. Eur. Phys. J. E. 4, 33–43. Blood, P.D. and Voth, G.A. (2006). Direct observation of Bin/ amphiphysin/Rvs (BAR). domain-induced membrane curvature by means of molecular dynamics simulations. Proc. Natl. Acad. Sci. USA 103, 15068–15072. Boucrot, E., Pick, A., Camdere, G., Liska, N., Evergren, E., McMahon, H.T., and Kozlov, M.M. (2012). Membrane fission is promoted by insertion of amphipathic helices and is restricted by crescent BAR domains. Cell 149, 124–136. Boulant, S., Kural, C., Zeeh, J.C., Ubelmann, F., and Kirchhausen, T. (2011). Actin dynamics counteract membrane tension during clathrin-mediated endocytosis. Nat. Cell Biol. 13, 1124–1131. Breidenich, M., Netz, R.R., and Lipowsky, R. (2000). The shape of polymer-decorated membranes. Europhys. Lett. 49, 431–437. Campelo, F., McMahon, H.T., and Kozlov, M.M. (2008). The hydrophobic insertion mechanism of membrane curvature generation by proteins. Biophys. J. 95, 2325–2339. Cocucci, E., Aguet, F., Boulant, S., and Kirchhausen, T. (2012). The first five seconds in the life of a clathrin-coated pit. Cell 150, 495–507. Cullis, P.R. and de Kruijff, B. (1979). Lipid polymorphism and the functional roles of lipids in biological membranes. Biochim. Biophys. Acta 559, 399–420.
Dai, J.W. and Sheetz, M.P. (1999). Membrane tether formation from blebbing cells. Biophys. J. 77, 3363–3370. Dannhauser, P.N. and Ungewickell, E.J. (2012). Reconstitution of clathrin-coated bud and vesicle formation with minimal components. Nat. Cell Biol. 14, 634–639. Doyon, J.B., Zeitler, B., Cheng, J., Cheng, A.T., Cherone, J.M., Santiago, Y., Lee, A.H., Vo, T.D., Doyon, Y., Miller, J.C., et al. (2011). Rapid and efficient clathrin-mediated endocytosis revealed in genome-edited mammalian cells. Nat. Cell Biol. 13, 331–337. Drin, G. and Antonny, B. (2010). Amphipathic helices and membrane curvature. FEBS Lett 584, 1840–1847. Farsad, K. and De Camilli, P. (2003). Mechanisms of membrane deformation. Curr. Opin. Cell Biol. 15, 372–381. Fotin, A., Cheng, Y., Sliz, P., Grigorieff, N., Harrison, S.C., Kirchhausen, T., and Walz, T. (2004). Molecular model for a complete clathrin lattice from electron cryomicroscopy. Nature 432, 573–579. Frost, A., Perera, R., Roux, A., Spasov, K., Destaing, O., Egelman, E.H., De Camilli, P., and Unger, V.M. (2008). Structural basis of membrane invagination by F-BAR domains. Cell 132, 807–817. Goetz, R., Gompper, G., and Lipowsky, R. (1999). Mobility and elasticity of self-assembled membranes. Phys. Rev. Lett. 82, 221–224. Heinrich, M.C., Capraro, B.R., Tian, A., Isas, J.M., Langen, R., and Baumgart, T. (2010). Quantifying membrane curvature generation of amphiphysin N-BAR domains. J. Phys. Chem. Lett. 1, 3401–3406. Helfrich, W. (1973). Elastic properties of lipid bilayers: theory and possible experiments. Z. Naturforsch. C 28, 693–703. Helfrich, W. (1989). Hats and saddles in lipid-membranes. Liquid Crystals 5, 1647–1658. Julicher, F. and Lipowsky, R. (1993). Domain-induced budding of vesicles. Phys. Rev. Lett. 70, 2964–2967. Kirchhausen, T. (2009). Imaging endocytic clathrin structures in living cells. Trends Cell Biol. 19, 596–605. Kukulski, W., Schorb, M., Kaksonen, M., and Briggs, J.A. (2012). Plasma membrane reshaping during endocytosis is revealed by time-resolved electron tomography. Cell 150, 508–520. Kukulski, W., Schorb, M., Welsch, S., Picco, A., Kaksonen, M., and Briggs, J.A. (2011). Correlated fluorescence and 3D electron microscopy with high sensitivity and spatial precision. J. Cell Biol. 192, 111–119.
Brought to you by | Northern Illinois University Authenticated Download Date | 1/26/15 12:34 AM
282 M. Simunovic and P. Bassereau: Configurational space of endocytosis Lieber, A.D., Yehudai-Resheff, S., Barnhart, E.L., Theriot, J.A., and Keren, K. (2013). membrane tension in rapidly moving cells is determined by cytoskeletal forces. Curr. Biol., 23, 1409–1417. Lipowsky, R. (1991). The conformation of membranes. Nature 349, 475–481. Lipowsky, R. (2013). Spontaneous tubulation of membranes and vesicles reveals membrane tension generated by spontaneous curvature. Farad. Discuss. 161, 305–331. Lipowsky, R. and Dimova, R. (2003). Domains in membranes and vesicles. J. Phys. Condens. Matter. 15, S31–S45. Lipowsky, R. and Sackmann, E. (ed). (1995). Structure and Dynamics of Membranes: From Cells to Vesicles. Elsevier: North Holland, Amsterdam. McMahon, H.T. and Gallop, J.L. (2005). Membrane curvature and mechanisms of dynamic cell membrane remodelling. Nature 438, 590–596. Meinecke, M., Boucrot, E., Camdere, G., Hon, W.C., Mittal, R., and McMahon, H.T. (2013). Cooperative Recruitment of Dynamin and BIN/Amphiphysin/Rvs (BAR). Domain-containing Proteins Leads to GTP-dependent Membrane Scission. J. Biol. Chem. 288, 6651–6661. Mim, C., Cui, H., Gawronski-Salerno, J.A., Frost, A., Lyman, E., Voth, G.A., and Unger, V.M. (2012a). Structural basis of membrane bending by the N-BAR protein endophilin. Cell 149, 137–145. Mim, C., Cui, H.S., Gawronski-Salerno, J.A., Frost, A., Lyman, E., Voth, G.A., and Unger, V.M. (2012b). Structural basis of membrane bending by the N-BAR protein endophilin. Cell 149, 137–145. Mim, C. and Unger, V.M. (2012). Membrane curvature and its generation by BAR proteins. Trends Biochem. Sci. 37, 526–533. Morlot, S., Galli, V., Klein, M., Chiaruttini, N., Manzi, J., Humbert, F., Dinis, L., Lenz, M., Cappello, G., and Roux, A. (2012). Membrane shape at the edge of the dynamin helix sets location and duration of the fission reaction. Cell 151, 619–629. Peter, B.J., Kent, H.M., Mills, I.G., Vallis, Y., Butler, P.J., Evans, P.R., and McMahon, H.T. (2004). BAR domains as sensors of membrane curvature: the amphiphysin BAR structure. Science 303, 495–499. Posor, Y., Eichhorn-Gruenig, M., Puchkov, D., Schoneberg, J., Ullrich, A., Lampe, A., Muller, R., Zarbakhsh, S., Gulluni, F., Hirsch, E., et al. (2013). Spatiotemporal control of endocytosis by phosphatidylinositol-3,4-bisphosphate. Nature 499, 233–237. Qualmann, B., Koch, D., and Kessels, M.M. (2011). Let’s go bananas: revisiting the endocytic BAR code. EMBO J. 30, 3501–3515. Ramakrishnan, N., Kumar, P.B.S., and Ipsen, J.H. (2013). Membranemediated aggregation of curvature-inducing nematogens and membrane tubulation. Biophys. J. 104, 1018–1028. Ramesh, P., Baroji, Y.F., Reihani, S.N., Stamou, D., Oddershede, L.B., and Bendix, P.M. (2013). FBAR syndapin 1 recognizes and stabilizes highly curved tubular membranes in a concentration dependent manner. Sci. Rep. 3, 1565. Raucher, D. and Sheetz, M.P. (1999). Characteristics of a membrane reservoir buffering membrane tension. Biophys. J. 77, 1992–2002.
Reynwar, B.J., Illya, G., Harmandaris, V.A., Muller, M.M., Kremer, K., and Deserno, M. (2007). Aggregation and vesiculation of membrane proteins by curvature-mediated interactions. Nature 447, 461–464. Saheki, Y. and De Camilli, P. (2012). Synaptic vesicle endocytosis. Cold Spring Harb. Perspect. Biol. 4, a005645. Saric, A. and Cacciuto, A. (2012). Fluid membranes can drive linear aggregation of adsorbed spherical nanoparticles. Phys. Rev. Lett. 108, 118101. Seifert, U. (1997). Configurations of fluid membranes and vesicles. Adv. Phys. 46, 13–137. Sens, P., Johannes, L., and Bassereau, P. (2008). Biophysical approaches to protein-induced membrane deformations in trafficking. Curr. Opin. Cell Biol. 20, 476–482. Simunovic, M., Mim, C., Marlovits, T.C., Resch, G., Unger, V.M., and Voth, G.A. (2013a). Protein-mediated transformation of lipid vesicles into tubular networks. Biophys J. 105, 711–719. Simunovic, M., Srivastava, A., and Voth, G.A. (2013b). Linear aggregation of proteins on the membrane as a prelude to membrane remodeling. Proc. Natl. Acad. Sci. USA, 110, 20396–20401. Singh, P., Mahata, P., Baumgart, T., and Das, S.L. (2012). Curvature sorting of proteins on a cylindrical lipid membrane tether connected to a reservoir. Phys. Rev. E, 85. Sorre, B., Callan-Jones, A., Manneville, J.B., Nassoy, P., Joanny, J.F., Prost, J., Goud, B., and Bassereau, P. (2009). Curvaturedriven lipid sorting needs proximity to a demixing point and is aided by proteins. Proc. Natl. Acad. Sci. USA 106, 5622–5626. Sorre, B., Callan-Jones, A., Manzi, J., Goud, B., Prost, J., Bassereau, P., and Roux, A. (2012). Nature of curvature coupling of amphiphysin with membranes depends on its bound density. Proc. Natl. Acad. Sci. USA 109, 173–178. Stachowiak, J.C., Schmid, E.M., Ryan, C.J., Ann, H.S., Sasaki, D.Y., Sherman, M.B., Geissler, P.L., Fletcher, D.A., and Hayden, C.C. (2012). Membrane bending by protein-protein crowding. Nat. Cell Biol. 14, 944–949. Taylor, M.J., Perrais, D., and Merrifield, C.J. (2011). A High precision survey of the molecular dynamics of mammalian clathrinmediated endocytosis. Plos Biology, 9, e1000604. van Weering, J.R.T., Sessions, R.B., Traer, C.J., Kloer, D.P., Bhatia, V.K., Stamou, D., Carlsson, S.R., Hurley, J.H., and Cullen, P.J. (2012). Molecular basis for SNX-BAR-mediated assembly of distinct endosomal sorting tubules. EMBO J. 31, 4466–4480. Verstreken, P., Kjaerulff, O., Lloyd, T.E., Atkinson, R., Zhou, Y., Meinertzhagen, I.A., and Bellen, H.J. (2002). Endophilin mutations block clathrin-mediated endocytosis but not neurotransmitter release. Cell 109, 101–112. Weinberg, J. and Drubin, D.G. (2012). Clathrin-mediated endocytosis in budding yeast. Trends Cell Biol, 22, 1–13. Yin, Y., Arkhipov, A., and Schulten, K. (2009). Simulations of Membrane Tubulation by Lattices of Amphiphysin N-BAR Domains. Structure 17, 882–892. Zhu, C., Das, S.L., and Baumgart, T. (2012). Nonlinear sorting, curvature generation, and crowding of endophilin N-BAR on tubular membranes. Biophys. J. 102, 1837–1845.
Brought to you by | Northern Illinois University Authenticated Download Date | 1/26/15 12:34 AM
M. Simunovic and P. Bassereau: Configurational space of endocytosis 283
Mijo Simunovic is a PhD student in chemistry at the University of Chicago and in physics at the Curie Institute in Paris. In his research, he combines coarse-grained theoretical techniques in the Voth group with experimental biophysical methods at the Bassereau group to study the physics underlying protein-induced membrane remodeling phenomena. Before joining these two groups, he received his BS and MS in physical chemistry from the University of Zagreb, where he employed theoretical and experimental approaches in investigating problems in synthetic and quantum chemistry.
Patricia Bassereau is currently Directrice de Recherche, CNRS at the Curie Institute in Paris. After spending 7 years in Montpellier (GDPC) working on the structure of surfactant-based phases, and a year as a visiting scientist at the Almaden IBM Center (San Jose, USA) on the structure of thin polymer films, she moved in 1993 to the Curie Institute. She initially investigated the interactions of soluble proteins with polymer monolayers. In the last 15 years, she has been working in the field of ‘physics for cell biology’. She has developed a multidisciplinary approach to understand the role of lipid membranes in important cellular functions such as intracellular trafficking, endo/ exocytosis, transmembrane ion transport (‘active membranes’), or cell adhesion.
Brought to you by | Northern Illinois University Authenticated Download Date | 1/26/15 12:34 AM
