Journal of General Virology (2005), 86, 3357–3363
DOI 10.1099/vir.0.81129-0
Membrane deformations induced by the matrix protein of vesicular stomatitis virus in a minimal system Je´roˆme Solon,13 Olivier Gareil,1 Patricia Bassereau1 and Yves Gaudin2 Correspondence Yves Gaudin
[email protected]
Received 25 April 2005 Accepted 25 August 2005
1
Institut Curie, UMR CNRS 168, 26 Rue d’Ulm, F75248 Paris Cedex 5, France
2
Unite´ Mixte de Virologie Mole´culaire et Structurale, UMR CNRS 2472, UMR INRA 1157, CNRS, 91198 Gif sur Yvette Cedex, France
The matrix (M) protein of vesicular stomatitis virus plays a key role in both assembly and budding of progeny virions. In vitro experiments have shown a strong propensity of M protein to bind to vesicles containing negatively charged phospholipids. In vivo, it has also been demonstrated that recruitment of some cellular proteins by M protein is required for efficient virus budding and release of newly synthesized virions in the extracellular medium. The ability of M protein to deform target membranes in vitro was investigated in this study. It was shown that incubation of purified M protein with giant unilamellar vesicles results in the formation of patches of M protein at their surface, followed by deformations of the membrane toward the inside of the vesicle, which could be observed in phase-contrast microscopy. This provides the first evidence that M protein alone is able to impose the correct budding curvature on the membrane. Using confocal microscopy, patches of M protein that colocalized with negatively charged lipid domains a few minutes after vesicle injection were observed. After a longer incubation period, membrane deformations appeared in these domains. At this time, a strict colocalization of M protein, negatively charged lipids and membrane deformation was observed. The influence on this process of the basic N-terminal part of the protein and of the previously identified hydrophobic loop has also been investigated. Interestingly, the final fission event has never been observed in our experimental system, indicating that other partners are required for this step.
INTRODUCTION Enveloped viruses acquire their membrane by budding at a membrane of their host cell. In many viral families, including filoviruses (Timmins et al., 2001), orthomyxoviruses (Gomez-Puertas et al., 2000) and rhabdoviruses (Mebatsion et al., 1999), a viral protein, the matrix protein (M), plays a major role in the budding process. Although neither the amino acid sequence nor the crystal structure (Sha & Luo, 1997; Dessen et al., 2000; Gaudier et al., 2002) reveal any homology between M proteins from viruses belonging to these different families, these proteins have many common characteristics. First, all M proteins have been shown to interact with cellular membranes (Chong & Rose, 1993; Zhang & Lamb, 1996; Timmins et al., 2001). In vitro experiments have shown a strong propensity of these proteins to bind to vesicles containing negatively charged phospholipids (Zakowski et al., 1981; Luan & Glaser, 1994; Luan et al., 3Present address: Institute for Medicine and Engineering, University of Pennsylvania, 1010 Vagelos Research Laboratories, 3340 Smith Walk, Philadelphia, PA 19104, USA.
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1995; Baudin et al., 2001) and it has been proposed that patches of basic residues at the surface of the protein are directly involved in membrane interactions (Lenard & Vanderoef, 1990; Sha & Luo, 1997; Dessen et al., 2000; Baudin et al., 2001; Gaudier et al., 2002). It has to be noted that the structure of M proteins also reveals hydrophobic bulks or loops exposed at the protein surface that may play a role in this association (Dessen et al., 2000; Gaudier et al., 2002). Second, M proteins are able to self-associate. The M protein (VP40) of Ebola virus (a filovirus) forms defined ringlike oligomers (Scianimanico et al., 2000) and VP40 of Marburg virus (another filovirus) polymerizes into rods composed of stacked rings (Timmins et al., 2003a). M proteins of other members of Mononegavirales have also been shown to polymerize both in vitro (Heggeness et al., 1982; Gaudin et al., 1995) and in vivo with the observation of a crystalline lattice on the plasma membrane (Buechi & Bachi, 1982). Polymer formation may increase the affinity of M proteins for the membrane and/or for the internal virus components (i.e. the nucleocapsid) through an avidity effect. It may also constitute a driving force for the budding process. 3357
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Third, many M proteins have the ability to recruit cellular partners that seem to be involved in the ultimate step of the budding process. In particular, Ebola VP40 and rhabdovirus M protein share PPxY and P(S/T)AP motifs. The PPxY motif has been shown to be recognized by NEDD4, an E3 ubiquitin ligase (Harty et al., 1999; Timmins et al., 2003b). Mutations in this motif affect budding efficiency (Jayakar et al., 2000). The P(S/T)AP motif recruits the protein TSG101, which is part of the machinery involved in the formation of vesicles that bud into the lumen of late endosomal compartments called multivesicular bodies (MVB) (Timmins et al., 2003b). It is noteworthy that the same machinery is also diverted by the GAG protein of retroviruses (Craven et al., 1999; Garrus et al., 2001). Nevertheless, the precise step of the budding process in which this machinery is involved has not been identified so far and may depend on the viral family. Vesicular stomatitis virus (VSV), the prototype rhabdovirus, has been used as a model for the budding process. Its M protein is a small 229 aa protein (26?6 kDa) comprising a positively charged N-terminal part (there are eight lysine residues in the first 20 residues) that can be removed by trypsin treatment to yield the Mt fragment (residues 44–229). As mentioned above, the M protein is able to interact with membranes both in vitro (Zakowski et al., 1981; Luan & Glaser, 1994; Gaudier et al., 2002) and in vivo (Chong & Rose, 1993). It also self-associates into large multimers at physiological NaCl concentration, a process initiated by trypsin-sensitive nucleation sites (Gaudin et al., 1995). Recently, the structure of a soluble thermolysinresistant core of VSV M protein (Mth) has been determined at 2 A˚ resolution (Gaudier et al., 2002). Mth is composed of two non-covalently-associated fragments (48–121 and 124–229). Its structure has revealed a single-globular domain with a new fold. The N-terminal part consists of a large five-stranded anti-parallel b-sheet packed against two a-helices; the C-terminal part comprises a small twostranded anti-parallel b-sheet and an a-helix. Cleavage by thermolysin between residues 121 and 124 takes place in a surface hydrophobic loop delimited by prolines 120 and 129 and surrounded by basic residues. This region has been proposed to mediate the interaction between the M protein and the membrane. In this study, using a minimal system, we have investigated the ability of VSV M protein to deform target membranes in vitro. We show that incubation of purified M protein with giant unilamellar vesicles (GUVs) results in the formation of patches of M protein at their surface, followed by deformation of the membrane toward the inside of the vesicle. This provides the first evidence that a viral M protein alone (i.e. in absence of any cellular proteins) is able to impose the correct budding curvature to the membrane. The influence on this process of the membrane composition together with the role of the basic N-terminal domain of the M protein (residues 1–43) and of the loop (residues 120–129) has also been investigated. 3358
METHODS Reagents. 1,2 Dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2 dioleoyl-sn-glycero-3-phospho-L-serine (DOPS) and fluorescent lipid 1-oleoyl-2-[12-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]dodecanoyl]sn-glycero-3-phospho-L-serine (NBD-PS) were purchased from Avanti Polar Lipid. Fluorescent lipid, 2-(12-(7-nitrobenz-2-oxa-1,3-diazol4-yl)amino)dodecanoyl-1-hexadecanoyl-sn-glycero-3-phosphocholine (NBD-PC) and fluorescent probe (Alexa 568) were purchased from Molecular Probes. All other chemicals were purchased from SigmaAldrich, except for the mercaptopropyltrimethoxysilane which was purchased from ABCR and the methoxypolyethylene glycolmaleimide (Mal-PEG, relative molecular mass 5000) which was purchased from Shearwater Polymers. Virus and cells. Wild-type VSV (Orsay strain, Indiana serotype) was propagated in BSR cells, a clone of baby hamster kidney 21 (BHK 21). After infection (24 h), virus particles were pelleted from the culture fluid through 30 % glycerol (v/v) in 10 mM Tris/HCl pH 7?5, 50 mM NaCl, 1 mM EDTA and resuspended in TD buffer (137 mM NaCl, 5 mM KCl, 0?7 mM Na2HPO4, 25 mM Tris/HCl, pH 7?5). VSV M protein purification. VSV M protein was purified as pre-
viously described (Gaudin et al., 1995). Briefly, 1 vol. virus in TD buffer (viral protein concentration of about 10 mg ml21) was added to 1 vol. solubilization buffer (1 M NaCl, 40 mM Tris/HCl pH 8?0, 1?2 % CHAPS). After a 3 min incubation at 37 uC, the mixture was overlaid onto 20 % sucrose in 0?5 M NaCl, 40 mM Tris/HCl pH 8?0, 0?5 % CHAPS and centrifuged for 50 min at 35 000 r.p.m. in an SW41 rotor (Beckman) to separate solubilized proteins from insoluble material. The supernatant, mainly containing VSV M and G proteins but also viral lipids, was diluted four times with 20 mM Tris/HCl pH 8?0, 0?5 % CHAPS and loaded onto a phosphocellulose column (P11; Whatman); G protein and residual viral contaminants flowed through. After a wash with 200 mM NaCl, M protein was eluted in 0?7 M NaCl, 50 mM Tris/HCl pH 7?5. At this stage, SDS-PAGE analysis indicated that M protein was essentially free from contaminants. Labelling of VSV M protein with Alexa 568. We used Alexa
568, which contains a maleimide group, to selectively label the protein on the thiol group of its unique cysteine (cys 135). M protein (1 mg ml21) was incubated for 2 h in the dark with 1 mM Alexa 568 in 0?7 M NaCl and 50 mM Tris/HCl pH 7?5. The protein was then diluted four times in 50 mM NaCl Tris/HCl pH 7?5 and loaded onto a phosphocellulose column; free reagent flowed through. After a wash with 200 mM NaCl, 50 mM Tris/HCl pH 7?5, the protein was eluted in 0?7 M NaCl, 50 mM Tris/HCl pH 7?5. The degree of labelling was estimated to be about 90 % (using an eM of 92 000 for Alexa 568 at 575 nm). Proteolysis. M protein (1 mg ml21) was incubated in 0?7 M NaCl,
50 mM Tris/HCl pH 7?5 at 37 uC for 20 min with either trypsin (50 mg ml21) or thermolysin (200 mg ml21) to obtain the fragments Mt and Mth. The reaction was stopped by either adding an excess of soya bean tryptic inhibitor or 5 mM EDTA for thermolysin. GUV preparation and observation chamber. GUVs were pre-
pared from pure DOPC or from a mixture of DOPC : DOPS (9 : 1 molar ratio) with the electroformation technique according to Mathivet et al. (1996). The method consisted of first depositing a thin layer of a lipid solution in chloroform onto two indium tin oxide-coated glass slides (PGO) and then second completely evaporating the solvent under vacuum for at least 2 h. A chamber was prepared with the two slides and filled with a 300 mM sucrose solution. GUVs were obtained after applying an alternative tension (1 V, 10 Hz) between the two slides during 3 h. The observation chamber was composed of two glass coverslips (Fisher) held together by a spacer of melted Parafilm. In order to limit Journal of General Virology 86
Membrane deformations induced by VSV M protein unspecific adhesion of the vesicles on the glass, the bottom slide was coated beforehand with a layer of Mal-PEG covalently bound to the glass. The slides were first activated by grafting mercaptopropyltrimethoxysilane and further incubated for 1 or 2 h with a Mal-PEG solution in PBS at pH 7?0 (Cuvelier et al., 2003). Purified M (or Mt) proteins were diluted in an Eppendorf tube (concentration of 1–5 mM in a final volume of 100 ml) with a glucose solution at the appropriate concentration to fix the osmolarity at 300 mOsM (salt brought by M protein was taken into account). This protein solution was transferred to the chamber and 20 ml GUV solution was injected. All the experiments were performed at 22 uC.
details). The evolution of the shape of the GUVs was followed using phase-contrast microscopy. Two consecutive phenomena were observed. First, dark patches a few micrometres in diameter appeared at the surface of the vesicles (Fig. 2a). In phase-contrast, if the membrane
(a)
Microscopy. Fluorescence experiments were performed with an
inverted confocal microscope Zeiss Meta (Zeiss) equipped with a Zeiss 663 Plan Apo. (numerical aperture, 1?4) oil immersion lens. An argon ion laser operating at 470 and 533 nm was used to excite the fluorescence of NBD and of Alexa, respectively. The epifluorescence was converted into a static beam by an x-y scanner device and focused onto a photomultiplier. The fluorescence emission, after passing a 505–530 nm band pass filter, or a 560 high pass filter for the red emission, was focused into a pinhole in front of the photomultiplier to eliminate all the out-of-focus light. The pinhole blocking aperture was at 120 mm for the green emission and at 90 and 320 mm for the red emission. Commercial confocal software (Zeiss) was used for image analysis.
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(ii)
(iii)
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RESULTS VSV M protein associates with and deforms the GUV membrane GUVs are particularly adapted to investigate membrane deformations induced by mechanical constraints imposed, for instance, by colloids (Aranda-Espinoza et al., 1999) or polymer adsorption (Denisov et al., 1998). Their size, ranging from 10 to 100 mm in diameter, is well suited for optical microscopy studies. After preparation, their shape is generally spherical and their membrane surface is smooth (Fig. 1).
(c)
Negatively charged GUVs composed of DOPC : DOPS (9 : 1) were injected into the observation chamber, which had been filled beforehand with the M protein at a concentration of 1–5 mM (see Methods for experimental
Fig. 1. Phase-contrast microscopy of a DOPC : DOPS vesicle in the absence of proteins. Bar, 5 mm. http://vir.sgmjournals.org
Fig. 2. Deformation of GUVs by VSV M protein. (a) Kinetics of deformation of a GUV (DOPC : DOPS, 9 : 1) by M protein (5 mM) observed by phase-contrast microscopy. The images correspond to the same part of the vesicle at 3 (i), 6 (ii) and 9 min (iii) after vesicle injection. A two-dimensional patch appears (i) on the membrane leading to an invagination after 9 min (iii, arrow). Bar, 5 mm. (b) Different types of deformation on different negatively charged vesicles induced by VSV M protein, observed by phase-contrast microscopy between 10 and 20 min after injection. Bar, 5 mm. (c) No deformation was observed when the GUVs were incubated for 20 min in a polylysin solution (150–300 kDa) at 0?1 mg ml”1, whereas large bidimensional patches were visible. Bar, 10 mm. 3359
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appears darker, it implies that the membrane is locally thicker (or that the refraction index is higher) (Gu, 2000). Dark patches thus correspond to zones where a greater quantity of protein is adsorbed and consequently indicate the formation of two-dimensional protein aggregates. Then, these patches create large membrane deformations towards the interior of the GUVs. The size of these invaginations was homogeneous with a depth of 2–3 mm (Fig. 2b). As VSV M protein was added on the outside of the vesicles, it should be noted that these deformations have the orientation as expected in the presence of proteins involved in virus budding. In order to investigate the specificity of M-induced bending, we incubated GUVs with polylysin in similar conditions (Fig. 2c). As previously reported in experiments performed with positively charged polymers (Denisov et al., 1998) or colloids (Aranda-Espinoza et al., 1999), two-dimensional aggregates but no macroscopic deformations of the membrane were observed with these systems. This indicates that the invaginations are characteristic of the interaction of M protein with the membrane. Kinetics of aggregation and deformation decreased with M protein concentration. In the presence of 5 mM M protein and 50 mM NaCl, patches were clearly detected after 1 min and deformations were observed a few minutes later (5–10 min). Moreover, in the range of M protein concentrations used, no patches and thus no deformations were formed for salt concentrations above 150 mM NaCl. We further performed similar experiments using pure DOPC GUVs. Patches and invaginations were also observed, but the kinetics of their appearance were considerably slower: it typically took 45–60 min at 5 mM M protein concentration to deform the membrane. Colocalization of VSV M protein and negatively charged phospholipids We next analysed a possible colocalization of VSV M protein with negatively charged DOPS, using confocal microscopy. GUVs [DOPC : DOPS (9 : 1)] containing 1 % fluorescent NBD-PS were injected into a chamber filled with VSV M protein, covalently bound to Alexa 568, at a similar concentration as above. After a fast initial homogeneous adsorption of M proteins on the GUV surface (revealed by an increase of Alexa fluorescence on the membrane), fluorescent domains of M proteins were observed, which colocalized with NBD-PS-enriched domains (Fig. 3). After longer incubation, invaginations appeared in these domains (Fig. 4a). Apart from these domains, very low concentrations of both M protein and NBD-PS were detected and the membrane remained devoid of deformation (Fig. 4b). The large apparent thickness of the fluorescent NBD-PS patches suggests a complex structure of the lipid bilayer, which is probably highly crumpled and/or multilamellar in these domains. Nevertheless, the absence of fluorescent M protein inside the GUV bulk (even after a long incubation) 3360
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(b)
Fig. 3. Colocalization of VSV M protein and negatively charged lipids. The images were acquired 3 min after vesicle injection into the chamber containing 5 mM Alexa 568-labelled M protein. The vesicle contained DOPC : DOPS : NBD-PS (90 : 9 : 1). Left image (a) corresponds to the negatively charged lipid fluorescence and the right image (b) corresponds to the fluorescence of the M protein. Bar, 5 mm.
indicates that the membrane remains impermeable to the protein (Fig. 4b). This also excludes the formation of microscopic pores larger than a few nanometres resulting from these deformations. As a control, similar experiments were performed on DOPC : DOPS (9 : 1) vesicles, but labelled with neutral NBD-PC. In regions where the membrane was not deformed, patches of fluorescent NBD-PC were not observed (Fig. 4c). Nevertheless, at the position where the membrane was strongly deformed, the fluorescence of NBD-PC was enhanced (Fig. 4c), reflecting the considerable accumulation of lipids of all types, in good agreement with our above suggestion that the protein induces the formation of complex lipid structures. Effect of Mt and Mth on the GUV membrane In order to investigate the role of the highly positively charged N-terminal part of the protein, we used Mt (a trypsin-truncated form of the M protein, corresponding to residues 44–229) in the same series of experiments. Using a similar range of protein concentration and salinity as for M protein, we found that adsorption of Mt onto membranes was much less efficient, as judged by the low Alexa intensity on the vesicle. This low affinity of Mt for the membrane has been previously described (Gaudier et al., 2002). Nevertheless, few two-dimensional clusters were observed (Fig. 5), showing that a significant local protein concentration can be achieved. These clusters never led to deformations, indicating that either protein–protein interactions or protein–membrane interactions were affected when the Nterminal part of the M protein had been removed. Finally, when Mth (thermolysin-cleaved M protein) was used, no association with the membrane was observed; this result is in good agreement with previously published results (Gaudier et al., 2002). Journal of General Virology 86
Membrane deformations induced by VSV M protein
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(b)
(c)
Fig. 4. VSV M protein aggregates are localized at the position of the membrane invagination. (a and b) Colocalization of M protein aggregates and negatively charged lipids at the position of the deformation. The images were acquired 20 min after vesicle injection into the chamber containing 5 mM Alexa 568labelled M protein. The vesicle contained DOPC : DOPS : NBDPS (90 : 9 : 1). Left image corresponds to the negatively charged lipid fluorescence and the right image corresponds to the fluorescence of the M protein. The bars represent 5 mm in (a) and 10 mm in (b). (c) Localization of the neutral lipids in GUV incubated with M protein. The images were acquired 20 min after vesicle injection into the chamber containing 5 mM Alexa 568-labelled M protein. The vesicle contained DOPC : DOPS : NBD-PC (90 : 9 : 1). Left image corresponds to the neutral lipid fluorescence and the right image corresponds to the fluorescence of the M protein. Bar, 10 mm.
DISCUSSION Despite extensive work on many viral families, the molecular mechanisms of viral budding are actually unknown. Clearly, the identification of cellular partners, required for MVB biogenesis, which bind M proteins of other members of Mononegavirales and small proteins derived from retroviral Gag, has constituted a milestone in our understanding of this process. Nevertheless, the exact role of these proteins http://vir.sgmjournals.org
Fig. 5. Mt induces few two-dimensional protein clusters, but no membrane deformation. Colocalization of Mt and negatively charged lipids. The images were acquired 25 min after vesicle injection into the chamber containing 5 mM Alexa 568-labelled Mt protein. The vesicle contained DOPC : DOPS : NBD-PC (90 : 9 : 1). Left image corresponds to the negatively charged lipid fluorescence and the right image corresponds to the fluorescence of the Mt protein. Segregation but no deformation was observed, as previously shown with the complete form of the protein. Bar, 5 mm.
during viral budding and the step of the process for which they are required (determination of the budding site in the cell, initial bud formation, pinching off the virion, etc.) are not known and may depend on the virus family (Blot et al., 2004). These questions are all the more difficult to answer because the precise role of the M protein in the budding process is far from being completely understood. In this study, we have developed a minimal system using GUVs in order to investigate membrane deformations induced by VSV M protein. We made two major observations. First, in agreement with previous observations made on large unilamellar vesicles (LUV) (Luan & Glaser, 1994; Luan et al., 1995), M protein forms patches at the surface of the GUVs and these patches are enriched in negatively charged phospholipids (when such lipids are present inside the membrane). Second, these patches create large invaginations toward the inside of the vesicles. Such deformations were not observed at the LUV surface (Luan & Glaser, 1994; Luan et al., 1995). The kinetics of the interaction of M protein with the membrane are much faster when the GUVs contained negatively charged phosphatidylserine. Nevertheless, consistent with previous observations that M protein also binds membranes containing only neutral lipids (M. Gaudier & Y. Gaudin, unpublished results), M-induced deformations are also observed when pure phosphatidylcholine vesicles are used. As a very small residual negative charge has been previously measured on pure phosphatidylcholine vesicles (Pincet et al., 1999), this result does not exclude a role for electrostatic interactions in the whole process. Indeed, high salt concentration decreases M-binding efficiency and Mt lacking the positively charged N-terminal part of the protein has less affinity for the membranes. 3361
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Non-specific aggregation of positively charged colloids on negatively charged membranes has been previously reported (Aranda-Espinoza et al., 1999). This behaviour, although counterintuitive, can be explained by theoretical considerations (Aranda-Espinoza et al., 1999). It has to be noted that, in general, this aggregation does not lead to membrane deformation. Thus, the invaginations we observed are specifically induced by the M protein. As it is known that M protein has the ability to self-associate even in the absence of a target membrane, we suggest that it is this specific self-association together with M protein interaction with lipids that shapes the membrane. Indeed, patches but no deformations are observed with Mt protein that is unable to self-associate in solution (Gaudin et al., 1995). The deformations we observed, although having the expected orientation, are an order of magnitude larger than the virus size (2–3 mm versus 200 nm). Clearly, in the infected cells either cellular (such as the cytoskeleton and the machinery involved in MVB formation) or viral (such as the nucleocapsid or the viral glycoproteins) partners regulate the size and the shape of the budding particle. Furthermore, as described in the Results section, lipid organization inside these deformations appears to be complex at the resolution of fluorescence microscopy. Interestingly, it has been reported that in some cell types, budding of rhabdoviruses induces the formation of dense branched tubular structures up to 1 mm long at the surface of the cell (Hummeler et al., 1967; Birdwell & Strauss, 1974). These observations were made by electron microscopy. At the optical microscopy resolution, this type of structure would appear similar to the lipid–M protein complex structure at the surface of the GUVs. Unfortunately, observation of GUVs by negative staining or cryo-electron microscopy is technically not possible. Finally, this minimal system has provided us with a characterization of the role of VSV M protein in the budding process. The protein alone is able to strongly deform a lipid membrane in the absence of any other protein, either viral or cellular. However, the final fission event has never been observed with this system, suggesting that other partners are required for this step, in agreement with previous results (Jayakar et al., 2000). Alternatively, we cannot exclude that more complex lipid composition leading to domain formations (Roux et al., 2005; Baumgart et al., 2003) would favour pinching-off events. Membrane deformation properties of other M proteins (e.g. those of influenza virus, filoviruses and retroviruses) might be investigated using this system for a further generalization of these results.
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ACKNOWLEDGEMENTS We thank the microscopy platform team of the Curie Institute for their valuable technical support and Rob Ruigrok for critical reading and helpful comments on the manuscript. This work is supported by CNRS (ACI ‘dynamique et re´activite´ des assemblages biologiques’). 3362
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