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Reconstitution of cytoskeletal protein assemblies for large-scale membrane transformation
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ScienceDirect Reconstitution of cytoskeletal protein assemblies for large-scale membrane transformation Germa´n Rivas1, Sven K Vogel2 and Petra Schwille2 Membranes determine two-dimensional and threedimensional biochemical reaction spaces in living systems. Defining size and shape of surfaces and volumes encompassed by membrane is of key importance for cellular metabolism and homeostasis, and the maintenance and controlled transformation of membrane shapes are coordinated by a large number of different protein assemblies. The orchestration of spatial elements over distances orders of magnitudes larger than protein molecules, as required for cell division, is a particularly challenging task, requiring large-scale ordered protein filaments and networks. The structure and function of these networks, particularly of cytoskeletal elements, have been characterized extensively in cells and reconstituted systems. However, their co-reconstitution with membranes from the bottom-up under defined conditions, to elucidate their mode of action in detail, is still a relatively new field of research. In this short review, we discuss recent approaches and achievements with regard to the study of cytoskeletal protein assemblies on model membranes, with specific focus on contractile elements as those based on the bacterial division FtsZ protein and eukaryotic actomyosin structures. Addresses 1 Centro de Investigaciones Biolo´gicas, CSIC, 28040 Madrid, Spain 2 Max Planck Institute for Biochemistry, Martinsried, Germany Corresponding authors: Rivas, Germa´n ([email protected]), Schwille, Petra ([email protected], [email protected])
Current Opinion in Chemical Biology 2014, 22:18–26 This review comes from a themed issue on Synthetic biology Edited by Pier Luigi Luisi, Pasquale Stano and Cristiano Chiarabelli For a complete overview see the Issue and the Editorial
volumes. The boundaries of these volumes, mostly phospholipid membranes, are characterized by their enormous plasticity, requiring an elaborate meshwork of macromolecules for their transformation and maintenance [1]. During the last decades, our knowledge about the multitude of elements regulating cellular morphology has grown immensely, by the combined use of biochemical, biophysical, genetic and cell biological tools, providing new insights into their functional role [2]. However, despite this progress, many fundamental questions still remain unanswered. In particular, the large number of factors involved in key processes of cell and organelle division, and the many different realizations of cell division machineries in different organisms renders it difficult to identify common and unifying motifs, as would be desirable for a more than anecdotal understanding of the first principles of life [3]. In the context of Synthetic Biology, reconstituting minimal functional machineries from the bottom-up, and subjecting these systems to detailed quantitative analysis that would not be possible in the complexity of a living cell, has become quite attractive. Membranes belong to the cellular structures that have long been mimicked in simplified assays, due to the ease of inducing self-assembly of phospholipids. Thus, quantitative biophysical analysis of model membranes in many different topologies is a very established field, using light, but also electron, and atomic force microscopy and a multitude of spectroscopic techniques [4,5]. In the past years, many assays for functional reconstitution of protein machineries in and on these model membranes have been developed [6–8].
Available online 12th August 2014 http://dx.doi.org/10.1016/j.cbpa.2014.07.018 1367-5931/# 2014 Elsevier Ltd. All rights reserved.
Introduction: bottom-up synthetic biology of minimal membrane-transforming systems Biological systems are composed and maintained by the self-organization and self-assembly of macromolecules into ordered structures, continuously consuming chemical energy. One of the most fundamental tasks is to compartmentalize these macromolecular systems into cells and organelles, defining reaction spaces of conserved Current Opinion in Chemical Biology 2014, 22:18–26
Along the same lines, functional reconstitution of many force-inducing protein assemblies, such as molecular motors and their respective environments, has been accomplished. With respect to the transformation of membranes, the focus has been primarily on protein scaffolds and cages for small-scale fission, as in endocytosis and exocytosis, as well as secretion [9,10]. Only recently, with respect to vesicle-based proto-cells, questions on large-scale membrane transformation, such as in cytokinesis, have come into the view of reductionist approaches. Theory and experiments on lipid-only systems reveal that the forces required to transform membranes on micron scales are reasonably low [11,12]. However, in biology, there are hardly any free-standing membranes available on these scales, as they are all www.sciencedirect.com
Cellular reconstruction in minimal membrane systems Rivas, Vogel and Schwille 19
stabilized and kept in shape by other biopolymers. The mechanical task of division thus becomes rather involved, with cytoskeletal networks, cortices and cell walls, the viscoelastic nature of which are dependent on a multitude of factors.
assays have also been devised that rely on droplets only [22,23], assuming that the protein assemblies under study require only a single leaflet (or a lipid monolayer, respectively) to target the membrane and unfold their transformative potential.
Nevertheless, due to the increasing interest in proto-cells, it appears to be an attractive task to assemble, on basis of force-inducing systems known from different cellular systems, a minimal large-scale force-inducing element able to controllably split a membrane vesicle. Of particular interest are machineries that are supposed to assemble into contractile rings, as the ring structure appears to be particularly well suited for simple volume division. Contractile rings are described in bacteria, whose division appears to be greatly dependent on the so-called Z ring, but also in eukaryotic systems such as yeast, where the contraction is believed to be orchestrated by an actomyosin ring system [13,14].
Although free-standing membranes, as in GUVs, have the advantage of almost unlimited flexibility, and ideally support the activity of membrane-transforming machineries, they also comprise several disadvantages. First, they are extremely delicate and can be easily destroyed or transformed by rather unspecific factors, such as fluid convection or buffer-induced charge effects. Second, they do not allow to probe the influence of membrane topology on protein assembly although there are clear indications that membrane shapes play a role in the recruitment, assembly and positioning of cytoskeletal filaments and protein scaffolds (e.g., BAR proteins; [24]).
In this short review, we address very recent advances (covering the last two years) in the bottom-up reconstitution of machineries that could potentially be assembled into contractile rings able to transform large-scale volumes. We first highlight the work performed with respect to the reconstitution of the bacterial division Z ring, and then discuss the advances on minimal actomyosin cortices in conjunction with model membranes. For more a comprehensive review on biochemical and cellular reconstitution of membrane–cytoskeletal interactions and cell division in minimal systems covering the field before 2012, readers should consult [15].
Model systems for reconstitution studies of membrane transformations To investigate membrane-transforming protein assemblies under controlled conditions, it is most attractive to produce free-standing lipid bilayers of defined composition, which are ideally accessible from both sides, and can be observed by optical microscopy. The most attractive systems are giant unilamellar vesicles (GUVs), which because of their large size (with diameters from several to hundreds of micrometers) are well fitted to investigate the dynamics and spatial organization of membrane–protein interactions by high-resolution imaging and micro-spectroscopy (Figure 1) [16]. GUVs can be produced in several ways, the most prominent of which — electroformation — is however incompatible with many buffer conditions required for protein functionality [17]. Only recently, production methods that allow operation at high salt conditions have been described (reviewed in [18]). Many of them rely on reverse emulsions, transforming water-in-oil droplets into vesicles by forcing them through an oil–water interface doped with lipid molecules (Figure 2a) [19]. The force can be generated by microjetting [20] or sophisticated centrifugation [21]. Some www.sciencedirect.com
Thus, an attractive alternative to free-standing membranes in studies which require larger stability of the system, or pre-defined membrane topology, are supported membranes on polished glass or mica surfaces, microfabricated soft polymers, or even glass beads and rods (Figure 1) [8,25,26,27]. These supported membrane systems allow studying the membrane structural organization and the dynamics of proteins and lipids at the membrane by surface-sensitive techniques, for example, atomic force microscopy or fluorescence imaging [28,29]. In addition, supported bilayers are well suited to probe the interaction between soluble and membrane-bound proteins by microfluorescence-based assays, and acoustic and plasmoning sensing [30–32]. Finally, phospholipid bilayer nanodiscs, consisting of a scaffold membrane protein encircling a lipid mixture, may be also considered in reconstitution studies as they can incorporate membrane proteins while the system remains soluble, allowing the application of quantitative solution biochemical and biophysical tools to measure their properties and interactions with other companion elements in lipid environments (Figure 1) [33].
Reconstitution of the bacterial divisome elements on minimal membrane systems A very prominent candidate for a contractile ring potentially able to divide a membrane vesicle is the Z ring in Escherichia coli. It is formed as a result of the anchoring of the GTPase FtsZ (a self-assembling protein, homolog to cytoskeletal tubulin) to the cytoplasmic membrane by the action of FtsA, an amphitropic protein, and ZipA, a bitopic membrane protein (Figure 1, upper panel) [34]. Many recent studies have focused on reconstituting the activities and assembly properties of FtsZ, and its interactions with other proto-ring elements, in different minimal membrane systems, from nanodiscs and supported bilayers to the more complex giant vesicles (Figure 1). Current Opinion in Chemical Biology 2014, 22:18–26
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Figure 1
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Reconstitution of protein assemblies in minimal membrane systems. Upper panel: Diagram illustrating the essential elements of the divisome in E. coli cells, starting with the molecular assembly of the proto-ring elements (colored). Middle panel: Minimal membrane systems to reconstitute the activities and interaction properties of cytoskeletal and cell division assemblies (e.g., the proto-ring complex). Lower panel: Examples of successful reconstitution of proto-ring elements in membrane environments. Adapted from [38,18,37,22] (and unpublished observations from Rivas laboratory).
First evidence that FtsZ acts as a contractile element was provided by Erickson and co-workers, showing that polymers of an artificially membrane-targeted (mts)-FtsZYGP mutant were able to locally indent free-standing membranes of tubular liposomes [35]. Recently, the same group reported that FtsZ-YGP, when encapsulated inside liposomes together with a gain-of-function FtsA* variant, occasionally formed ring-like structures in constricted regions of the vesicles which, in few cases, suggest to complete vesicle division [36]. Permeable giant vesicles containing FtsZ and ZipA were found to shrink upon controlled FtsZ polymerization (Figure 2b,c) [37]. Shrinkage was dependent on the Current Opinion in Chemical Biology 2014, 22:18–26
surface density of ZipA, modulated by the rate of GTP hydrolysis and inhibited by peptides containing sequences of the C-terminus of FtsZ which also compete with FtsZ polymers for the binding to ZipA inserted in phospholipid bilayer nanodiscs (Figure 2d) [38]. Also, polymerization of FtsZ upon the release of caged GTP was found to modulate membrane plasticity in E. coli vesicles in which ZipA has been inserted at both sides of the membrane [39]. On structured membranes supported by micro-structured substrates, it was shown that assembly and orientation of copolymers of wild type and membrane-targeted FtsZ is strongly dependent on the curvature of the surface www.sciencedirect.com
Cellular reconstruction in minimal membrane systems Rivas, Vogel and Schwille 21
Figure 2
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Reconstitution of bacterial division elements in giant vesicles. Water-in-oil droplet transfer procedure (a) used to encapsulate and polymerize FtsZ inside permeable giant vesicles (b). (c) Progressive shrinking of permeable vesicles as a result of the interaction of FtsZ polymers with membraneassociated ZipA at elapsed times 0, 5 and 10 min. (d) Blocking vesicle shrinkage by the addition of an FtsZ-derived peptide that inhibits the interaction of membrane bound ZipA with FtsZ polymers. Scheme based on the one shown in [19]; confocal images adapted from [37].
(Figure 3A). FtsZ filaments preferentially align along curvatures of ca. 1 mm that replicate the dimensions of native E. coli cells [27]. The assembly of FtsZ tethered to supported lipid bilayers by a soluble variant of ZipA, or the mts-FtsZ, respectively, resulted in the formation of two-dimensional interconnected networks that dynamically reorganize by fragmentation, annealing and lateral condensation, as revealed by AFM [40,41] and optical microscopy [42]. Very recently, co-reconstitution of FtsZ and its native membrane anchor FtsA on supported membranes [43] revealed that FtsA drives the self-organization of FtsZ polymers into dynamic patterns such as collective streams and swirling rings with preferential directions, consistent with treadmilling of dynamic polymers (Figure 3B). Although the biochemical details of these dynamics remain to be clarified, they suggest a dual role of the FtsA, tethering FtsZ to the membrane, but at the same time destabilizing FtsZ filaments. Interestingly, this second activity of FtsA resembles the functionality of ADF/ cofilin, an acting binding protein that enhances the treadmilling of actin polymers in the cell and increases the rate of polymer disassembly in vitro [44]. In order to reveal the www.sciencedirect.com
biological implications of these findings, further quantitative studies on these proteins and how are they modulated by other division-related proteins are required.
Actin based cytoskeletal assemblies on minimal membrane systems The actin cytoskeleton does not only play pivotal roles in organizing the interior of eukaryotic cells, but also a major player in maintaining and changing their shape by interacting with the cell membrane. The mechanical interaction of the actin cytoskeleton with the cell membrane governs fundamental processes such as cytokinesis and cell migration with the aid of actin binding proteins and myosin motors. The main common themes for these processes are the quick reorganization and turnover of the actin cytoskeleton and the generation of force by cytoskeletal elements and myosin motors that are transmitted to the membrane (reviewed in [45]). The underlying mechanisms are far from being understood and very often difficult to determine from in vivo approaches as genetic and biochemical manipulations of the omnipotent players such as actin or myosin are often not feasible. Hence, based on pioneering efforts (reviewed in [15]) new bottom-up in vitro systems were recently designed Current Opinion in Chemical Biology 2014, 22:18–26
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Figure 3
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Reconstitution of bacterial division elements in supported lipid bilayers. (A) Using micro-fabricated supports for model membranes the curvature of FtsZ filaments within a groove or around a capillary was studied to understand its mechanical features. Adapted from [27]. (B) Array of dynamic rings of FtsZ polymers, resulting from its interaction with FtsA on a supported membrane, forming traveling streams and rotating swirls (top). Representative snapshots (middle) and schematic illustration (bottom) of treadmilling and fragmentation of FtsZ polymers observed when recruited to the membrane by FtsA. Adapted from [43].
where filamentous actin is either coupled to planar supported and free-standing lipid bilayers [46,47] or to the inside [48] and outside [49] of giant liposomes with the unifying theme of mimicking a cellular actin cortex (Figure 4). The contractile behavior of a cellular actomyosin cortex is provided with the addition of myosin motor assemblies in the presence of ATP to these systems. Recent studies on actin filaments coupled to supported bilayers revealed a possible mechanism of how compressive stress exerted by myosin motors on individual actin filaments may contribute to actin turnover [50] and how the degree of actin meshwork adhesion to the membrane may control the ratio between meshwork contraction and actin filament severing [51]. A first step to understand the physical basis for large-scale membrane shape transformations has been made by anchoring actin filaments inside and outside of synthetic liposomes (Figure 4a). Here, Current Opinion in Chemical Biology 2014, 22:18–26
myosin motor induced contraction of an outer membrane attached actin meshwork with a strong network connectivity showed occasional crushing events of liposomes (Figure 4a, right), while a weaker actin connectivity resulted in spontaneous rupture of the actin layer [49]. Biomimetic actin cortices inside liposomes were also used to investigate the contribution of the actin cytoskeleton to the effective membrane friction by performing nanotube extrusion experiments [50]. These experiments unveiled that the contribution of the actin cytoskeleton is additional but not dominant, and that the content of the membranes as well as the technique how the liposomes are prepared may play a major role. In summary, all these findings emphasize the power of bottom-up reconstitution approaches and may build a solid basis toward developing more complex contractile systems such as actomyosin ring structures that are www.sciencedirect.com
Cellular reconstruction in minimal membrane systems Rivas, Vogel and Schwille 23
Figure 4
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Reconstitution of actin-based assemblies on supported lipid membranes and giant vesicles. (a) Schematic representation of the assembly steps for a minimal actin cortex (MAC) (left). Small unilamellar vesicles are added to the support and result in the formation of a supported lipid bilayer. The bilayer contains functionalized lipids that with the aid of linker proteins can couple functionalized actin filaments to the lipid bilayer, eventually forming a MAC. TIRF images of a MAC before and after the addition of myosin motors (right); myosin motors (red) induce the contraction of the MAC and the formation of actomyosin clusters (right image). (b) Single confocal plane image and a reconstructed stack of confocal images of a GUV before and after coating with actin filaments (left). Adapted from [49]. Time-lapse images of an actin-coated liposome during myosin-induced contraction (right); the liposome crushed as a result of myosin contraction combined with strong actin network connectivity. (A), adapted from [50]. (B), adapted from [49]. All scale bars, 10 mm.
capable of driving force induced membrane shape changes to ultimately reconstitute a minimalistic version of eukaryotic cytokinesis.
Conclusions and outlook Can a simplified cytoskeleton or divisome of complex cellular organisms be reconstituted from purified elements, with the perspective of introducing and activating it for the division of protocells? The reconstitution approaches described here have been exploited to study the properties of selected cytoskeletal and cell division elements, the bacterial FtsZ and the eukaryotic actomyosin, which are both potential candidates for large-scale force-inducing systems. So far, the bacterial Z ring reconstitution, in spite of its successful assembly into ring-like structures, has not yet unambiguously revealed force induction [52]. In contrast, the actomyosin reconstitution has been shown to induce contractile large-scale forces, but so far not be brought into a ring structure that is coupled to a membrane [14,53]. These observations suggest that some elements are still missing from the current attempts to reconstitute www.sciencedirect.com
these molecular assemblies in proto-cellular systems, and further experimental and theoretical efforts are required to integrate biochemical and mechanical information on the systems within the complex architectures that have evolved to function within the cell [7,8,54,55,56]. Thus, despite of the remarkable progress made in the past years with regard to functional reconstitution of protein machineries, a minimal set of modules for controlled proto-cell division has yet to be identified. In order to achieve this ambitious goal, a deeper quantitative biochemical understanding, in terms of equilibrium and kinetic properties, of how these proteins work together as a system of multiple interacting elements, including the lipid membrane, is required [55,56–58]. The behavior of a membrane-confined system of soluble reactive proteins that combine reversibly with each other and with membrane components to produce a directed mechanical force influencing the shape of the confining enclosure will in all probability depend upon several factors: (1) the shape or local curvature of the enclosing membrane, (2) local Current Opinion in Chemical Biology 2014, 22:18–26
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variations in the plasticity of the membrane arising from an inhomogeneous distribution of membrane components (lipids, proteins), (3) the distribution of sites on the membrane to which the reactive proteins and their complexes may be anchored, (4) the stoichiometry of each of the reactive species, and (5) the composition of other confined macromolecular and small molecule solutes which, even though not binding to the reactive species, may modulate the behavior of these species through nonspecific interaction. The complexity of the system as a whole requires that each of these factors be studied individually and in combination. As these systems have co-evolved together with the particular compartment shapes of their hosts, it is essential to be able to subject them to environments that display essential spatial features of these organisms [1,59]. Thus, flexible vesicle membranes without any mimicry of shapes induced by peptidoglycan walls or protein cytoskeleton will likely not be appropriate. If the assembly of these structures prove too complex to be functionally reconstituted, cutting-edge nano-fabrication and microfabrication procedures, in which volumes or surfaces can be precisely and reproducibly shaped, will have to be employed. As many of these minimal cellular modules, such as FtsZ, will be potentially derived from microorganisms and operate on extremely small dimensions, we will have to investigate them by advanced biophysical methods, particularly super resolution microscopy. The use of cell-free expression systems [60,61] or cells in which certain elements have been removed [62] will help us in capturing functional modules that are hard to be purified, as many membranetargeted proteins. Finally, after having understood the principles of a fundamental set of modules, we may even be able to construct minimal functional elements from non-protein-based macromolecules, for example, DNA nanostructures, known as DNA origami [63], and to engineer assemblies with novel functionalities in ways that cannot be easily achieved in the cell.
Acknowledgements We thank Allen Minton (NIH) for useful comments. This work was supported in part by Human Frontier Science Program through Grant RGP0050/2010-C012 (to PS and GR), by the DFG Leibniz Prize (to PS), and by the European Commission through Contract HEALTH-F3-2009223431 and by the Spanish Government through Grant BIO2011-28941C03-03 (both to GR).
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25. Zieske K, Schwille P: Reconstitution of pole-to-pole oscillations of min proteins in microengineered polydimethylsiloxane compartments. Angew Chem Int Ed Engl 2013, 52:459-462. 26. Zieske K, Schweizer J, Schwille P: Surface topology assisted alignment of Min protein waves. FEBS Lett 2014 http:// dx.doi.org/10.1016/j.febslet.2014.06.026. 27. Arumugam S, Chwastek G, Fischer-Friedrich E, Ehrig C, Mo¨nch I, Schwille P: Surface topology engineering of membranes for the mechanical investigation of the tubulin homologue FtsZ. Angew Chem Int Ed Engl 2012, 51:11858-11862. This paper describes the behavior of polymers of a membrane-targeted FtsZ variant on micro-structured substrates of different curvature supporting lipid bilayers. FtsZ polymers preferentially orient along curvatures close to the diameter of E. coli cells, suggesting that additional factors than FtsZ alone are required to facilitate cell constriction. 28. Herrig A, Janke M, Austermann J, Gerke V, Janshoff A, Steinem C: Cooperative adsorption of erzin on PIP2-containing membranes. Biochemistry 2006, 45:13025-13034. 29. Groves JT, Parthasarathy R, Forstner MB: Fluorescence imaging of membrane dynamics. Annu Rev Biomed Eng 2008, 10:311338. 30. Castellana ET, Cremer PS: Solid supported lipid bilayers: from biophysical studies to sensor design. Surf Sci Rep 2006, 61:429-444. 31. Baciu CL, Becker J, Janshoff A, So¨nnichsen C: Protein– membrane interaction probed by single plasmonic nanoparticles. Nano Lett 2008, 8:1724-1728. 32. Eisele NB, Frey S, Piehler J, Go¨rlich D, Richter RP: Ultrathin nucleoporin phenylalanine–glycine repeat films and their interaction with nuclear transport receptors. EMBO Rep 2010, 11:366-372. 33. Schu¨ler MA, Denisov IG, Sligar SG: Nanodiscs as a new tool to examine lipid-protein interactions. Methods Mol Biol 2013, 974:415-433. 34. Rico AI, Krupka M, Vicente M: In the beginning, Escherichia coli assembled the proto-ring: an initial phase of division. J Biol Chem 2013, 288:20830-20836. 35. Osawa M, Anderson DE, Erickson HP: Reconstitution of contractile FtsZ rings in liposomes. Science 2008, 320:792-794. 36. Osawa M, Erickson HP: Liposome division by a simple bacterial division machinery. Proc Natl Acad Sci U S A 2013, 110:1100011004. In this study, the authors tether native FtsZ to liposome membranes by the action of a gain-of-function FtsA* variant. FtsZ–FtsA* ring-like structures are formed inside vesicles and, in few cases, are able to constrict vesicles. 37. Cabre´ EJ, Sa´nchez-Gorostiaga A, Carrara P, Ropero N, Casanova M, Palacios P, Stano P, Jime´nez M, Rivas G, Vicente M: Bacterial division proteins FtsZ and ZipA induce vesicle shrinkage and cell membrane invagination. J Biol Chem 2012, 288:26625-26634. A minimal division machinery assembly was reconstructed in permeable giant vesicles containing FtsZ and ZipA. These vesicles shrank upon FtsZ polymerization, partly mimicking membrane constriction. 38. Herna´ndez-Rocamora VM, Reija B, Garcı´a C, Natale P, Alfonso C, Minton AP, Zorrilla S, Rivas G, Vicente M: Dynamic interaction of the Escherichia coli cell division ZipA and FtsZ proteins evidenced in nanodiscs. J Biol Chem 2012, 287:30097-30104. 39. Lo´pez-Montero I, Lo´pez-Navajas P, Mingorance J, Ve´lez M, Vicente M, Monroy F: Membrane reconstitution of FtsZ–ZipA complex inside giant spherical vesicles made of E. coli lipids: large membrane dilation and analysis of membrane plasticity. Biochim Biophys Acta 2013, 1828:687-698. 40. Mateos-Gil P, Ma´rquez I, Lo´pez-Navajas P, Jime´nez M, Vicente M, Mingorance J, Rivas G, Ve´lez M: FtsZ polymers bound to lipid bilayers through ZipA form dynamic two-dimensional networks. Biochim Biophys Acta 2012, 1818:806-813. This atomic force microscopy study shows that FtsZ polymers bound to lipid bilayers through ZipA form dynamic two-dimensional networks that retain their capacity to dynamically restructure, to fragment, to anneal and to condense laterally. www.sciencedirect.com
41. Encinar M, Kralicek AV, Martos A, Krupka M, Cid S, Alonso A, Rico AI, Jime´nez M, Ve´lez M: Polymorphism of FtsZ filaments on lipid surfaces: role of monomer orientation. Langmuir 2013, 29:9436-9446. 42. Arumugam S, Petrasˇek Z, Schwille P: MinCDE exploits the dynamic nature of FtsZ filaments for its spatial regulation. Proc Natl Acad Sci U S A 2014, 111:E1192-E1200. This paper proposes a new mechanism of spatial positioning of FtsZ filaments by the Min protein system, building on a constant dynamic turnover of FtsZ monomers all along the filaments that it is enhanced in the presence of MinC. 43. Loose M, Mitchison TJ: The bacterial cell division proteins FtsA and FtsZ self-organize into dynamic cytoskeletal patterns. Nat Cell Biol 2014, 16:38-46. This paper describes the co-reconstitution of FtsZ and FtsA in supported bilayers, showing that FtsA (in the presence of ATP) not only recruits FtsZ polymers to the membrane but also the formation of FtsA–FtsZ polymer networks that undergo rapid chiral treadmilling. 44. Carlier MF, Laurent V, Santolini J, Melki R, Didry D, Xia GX, Hong Y, Chua NH, Pantaloni D: Actin depolymerizing factor (ADF/cofilin) enhances the rate of filament turnover: implication in actinbased motility. J Cell Biol 1997, 136:1307-1322. 45. Clark AG, Wartlick O, Salbreux G, Paluch EK: Stresses at the cell surface during animal cell morphogenesis. Curr Biol 2014, 24:R484-R494. Elegant review about the actomyosin cortex and how myosin generated stresses on the molecular level control actin cortex and membrane tension. 46. Vogel SK, Heinemann F, Chwastek G, Schwille P: The design of MACs (minimal actin cortices). Cytoskeleton (Hoboken) 2013, 70:706-717. 47. Heinemann F, Vogel SK, Schwille P: Lateral membrane diffusion modulated by a minimal actin cortex. Biophys J 2013, 104:14651475. 48. Campillo C, Sens P, Ko¨ster D, Pontani LL, Levy D, Bassereau P, Nassoy P, Sykes C: Unexpected membrane dynamics unveiled by membrane nanotube extrusion. Biophys J 2013, 104:12481256. 49. Carvalho K, Tsai FC, Lees E, Voituriez R, Koenderink GH, Sykes C: Cell-sized liposomes reveal how actomyosin cortical tension drives shape change. Proc Natl Acad Sci U S A 2013, 110:1645616461. In this study, the authors present actin coated lipid vesicles that upon myosin contraction either burst in the case of strong actin connectivity or display rupture and peeling away of the actin coat when the actin connectivity is weak. 50. Vogel SK, Petrasek Z, Heinemann F, Schwille P: Myosin motors fragment and compact membrane-bound actin filaments. Elife 2013, 2:e00116. This article gives the first direct evidence that myosin motors can break and compact membrane coupled actin filaments in a reconstituted actomyosin cortex. Based on the experimental data and a theoretical description the authors provide a mechanism for actin breakage by myosin motors that could potentially contribute to actin turnover in vivo. 51. Murrell MP, Gardel ML: F-actin buckling coordinates contractility and severing in a biomimetic actomyosin cortex. Proc Natl Acad Sci U S A 2012, 109:20820-20825. 52. Meier EL, Goley ED: Form and function of the bacterial cytokinetic ring. Curr Opin Cell Biol 2014, 26:19-27. 53. Mishra M, Huang J, Balasubramanian MK: The yeast actin cytoskeleton. FEMS Microbiol Rev 2014, 38:213-227. 54. Vicente M: Will test tubes ever undergo division? In Curtis T, Daran JM, Pronk JT, Frey J, Jansson JK, Robbins-Pianka A, et al.: Crystal ball – 2013. Microb Biotechnol 2013, 6:3-16. Commentary on the state of the art of bacterial division, with focus on central issues that are still unresolved. 55. Dogterom M, Surrey T: Microtubule organization in vitro. Curr Opin Cell Biol 2013, 25:23-29. Comprehensive review on bottom-up reconstitution approaches to study microtubule cytoskeleton self-organization and function. Current Opinion in Chemical Biology 2014, 22:18–26
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56. Hyman A: Whither systems biology. Philos Trans R Soc B 2011, 366:3635-3637. 57. Pollard TD: No questions about exciting questions in cell biology. PLoS Biol 2013, 11:e1001734. 58. Rivas G, Alfonso C, Jime´nez M, Monterroso B, Zorrila S: Macromolecular interactions of the bacterial division FtsZ protein: from quantitative biochemistry and crowding to reconstructing minimal divisomes in the test tube. Biophys Rev 2013, 5:63-77. 59. Stachowiak MR, Laplante C, Chin HF, Guirao B, Karatekin E, Pollard TD, O’Shaughnessy B: Mechanism of cytokinetic contractile ring constriction in fission yeast. Dev Cell 2014, 29:547-561.
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60. Maeda YT, Nakadai T, Shin J, Uryu K, Noireaux V, Libchaber A: Assembly of MreB filaments on liposome membranes: a synthetic biology approach. ACS Synth Biol 2012, 1:53-59. 61. Nourina Z, Scott A, Danelon C: Toward the assembly of a minimal divisome. Syst Synth Biol 2014 http://dx.doi.org/ 10.1007/s11693-014-9150-x. 62. Pazos M, Casanova M, Palacios P, Margolin W, Natale P, Vicente M: FtsZ placement in nucleoid-free bacteria. PLOS ONE 2014, 9:e91984. 63. Langecker M, Arnaut V, Martin TG, List J, Renner S, Mayer M, Dietz H, Simmel FC: Synthetic lipid membrane channels formed by designed DNA nanostructures. Science 2012, 338:932-936.
www.sciencedirect.com
ScienceDirect Reconstitution of cytoskeletal protein assemblies for large-scale membrane transformation Germa´n Rivas1, Sven K Vogel2 and Petra Schwille2 Membranes determine two-dimensional and threedimensional biochemical reaction spaces in living systems. Defining size and shape of surfaces and volumes encompassed by membrane is of key importance for cellular metabolism and homeostasis, and the maintenance and controlled transformation of membrane shapes are coordinated by a large number of different protein assemblies. The orchestration of spatial elements over distances orders of magnitudes larger than protein molecules, as required for cell division, is a particularly challenging task, requiring large-scale ordered protein filaments and networks. The structure and function of these networks, particularly of cytoskeletal elements, have been characterized extensively in cells and reconstituted systems. However, their co-reconstitution with membranes from the bottom-up under defined conditions, to elucidate their mode of action in detail, is still a relatively new field of research. In this short review, we discuss recent approaches and achievements with regard to the study of cytoskeletal protein assemblies on model membranes, with specific focus on contractile elements as those based on the bacterial division FtsZ protein and eukaryotic actomyosin structures. Addresses 1 Centro de Investigaciones Biolo´gicas, CSIC, 28040 Madrid, Spain 2 Max Planck Institute for Biochemistry, Martinsried, Germany Corresponding authors: Rivas, Germa´n ([email protected]), Schwille, Petra ([email protected], [email protected])
Current Opinion in Chemical Biology 2014, 22:18–26 This review comes from a themed issue on Synthetic biology Edited by Pier Luigi Luisi, Pasquale Stano and Cristiano Chiarabelli For a complete overview see the Issue and the Editorial
volumes. The boundaries of these volumes, mostly phospholipid membranes, are characterized by their enormous plasticity, requiring an elaborate meshwork of macromolecules for their transformation and maintenance [1]. During the last decades, our knowledge about the multitude of elements regulating cellular morphology has grown immensely, by the combined use of biochemical, biophysical, genetic and cell biological tools, providing new insights into their functional role [2]. However, despite this progress, many fundamental questions still remain unanswered. In particular, the large number of factors involved in key processes of cell and organelle division, and the many different realizations of cell division machineries in different organisms renders it difficult to identify common and unifying motifs, as would be desirable for a more than anecdotal understanding of the first principles of life [3]. In the context of Synthetic Biology, reconstituting minimal functional machineries from the bottom-up, and subjecting these systems to detailed quantitative analysis that would not be possible in the complexity of a living cell, has become quite attractive. Membranes belong to the cellular structures that have long been mimicked in simplified assays, due to the ease of inducing self-assembly of phospholipids. Thus, quantitative biophysical analysis of model membranes in many different topologies is a very established field, using light, but also electron, and atomic force microscopy and a multitude of spectroscopic techniques [4,5]. In the past years, many assays for functional reconstitution of protein machineries in and on these model membranes have been developed [6–8].
Available online 12th August 2014 http://dx.doi.org/10.1016/j.cbpa.2014.07.018 1367-5931/# 2014 Elsevier Ltd. All rights reserved.
Introduction: bottom-up synthetic biology of minimal membrane-transforming systems Biological systems are composed and maintained by the self-organization and self-assembly of macromolecules into ordered structures, continuously consuming chemical energy. One of the most fundamental tasks is to compartmentalize these macromolecular systems into cells and organelles, defining reaction spaces of conserved Current Opinion in Chemical Biology 2014, 22:18–26
Along the same lines, functional reconstitution of many force-inducing protein assemblies, such as molecular motors and their respective environments, has been accomplished. With respect to the transformation of membranes, the focus has been primarily on protein scaffolds and cages for small-scale fission, as in endocytosis and exocytosis, as well as secretion [9,10]. Only recently, with respect to vesicle-based proto-cells, questions on large-scale membrane transformation, such as in cytokinesis, have come into the view of reductionist approaches. Theory and experiments on lipid-only systems reveal that the forces required to transform membranes on micron scales are reasonably low [11,12]. However, in biology, there are hardly any free-standing membranes available on these scales, as they are all www.sciencedirect.com
Cellular reconstruction in minimal membrane systems Rivas, Vogel and Schwille 19
stabilized and kept in shape by other biopolymers. The mechanical task of division thus becomes rather involved, with cytoskeletal networks, cortices and cell walls, the viscoelastic nature of which are dependent on a multitude of factors.
assays have also been devised that rely on droplets only [22,23], assuming that the protein assemblies under study require only a single leaflet (or a lipid monolayer, respectively) to target the membrane and unfold their transformative potential.
Nevertheless, due to the increasing interest in proto-cells, it appears to be an attractive task to assemble, on basis of force-inducing systems known from different cellular systems, a minimal large-scale force-inducing element able to controllably split a membrane vesicle. Of particular interest are machineries that are supposed to assemble into contractile rings, as the ring structure appears to be particularly well suited for simple volume division. Contractile rings are described in bacteria, whose division appears to be greatly dependent on the so-called Z ring, but also in eukaryotic systems such as yeast, where the contraction is believed to be orchestrated by an actomyosin ring system [13,14].
Although free-standing membranes, as in GUVs, have the advantage of almost unlimited flexibility, and ideally support the activity of membrane-transforming machineries, they also comprise several disadvantages. First, they are extremely delicate and can be easily destroyed or transformed by rather unspecific factors, such as fluid convection or buffer-induced charge effects. Second, they do not allow to probe the influence of membrane topology on protein assembly although there are clear indications that membrane shapes play a role in the recruitment, assembly and positioning of cytoskeletal filaments and protein scaffolds (e.g., BAR proteins; [24]).
In this short review, we address very recent advances (covering the last two years) in the bottom-up reconstitution of machineries that could potentially be assembled into contractile rings able to transform large-scale volumes. We first highlight the work performed with respect to the reconstitution of the bacterial division Z ring, and then discuss the advances on minimal actomyosin cortices in conjunction with model membranes. For more a comprehensive review on biochemical and cellular reconstitution of membrane–cytoskeletal interactions and cell division in minimal systems covering the field before 2012, readers should consult [15].
Model systems for reconstitution studies of membrane transformations To investigate membrane-transforming protein assemblies under controlled conditions, it is most attractive to produce free-standing lipid bilayers of defined composition, which are ideally accessible from both sides, and can be observed by optical microscopy. The most attractive systems are giant unilamellar vesicles (GUVs), which because of their large size (with diameters from several to hundreds of micrometers) are well fitted to investigate the dynamics and spatial organization of membrane–protein interactions by high-resolution imaging and micro-spectroscopy (Figure 1) [16]. GUVs can be produced in several ways, the most prominent of which — electroformation — is however incompatible with many buffer conditions required for protein functionality [17]. Only recently, production methods that allow operation at high salt conditions have been described (reviewed in [18]). Many of them rely on reverse emulsions, transforming water-in-oil droplets into vesicles by forcing them through an oil–water interface doped with lipid molecules (Figure 2a) [19]. The force can be generated by microjetting [20] or sophisticated centrifugation [21]. Some www.sciencedirect.com
Thus, an attractive alternative to free-standing membranes in studies which require larger stability of the system, or pre-defined membrane topology, are supported membranes on polished glass or mica surfaces, microfabricated soft polymers, or even glass beads and rods (Figure 1) [8,25,26,27]. These supported membrane systems allow studying the membrane structural organization and the dynamics of proteins and lipids at the membrane by surface-sensitive techniques, for example, atomic force microscopy or fluorescence imaging [28,29]. In addition, supported bilayers are well suited to probe the interaction between soluble and membrane-bound proteins by microfluorescence-based assays, and acoustic and plasmoning sensing [30–32]. Finally, phospholipid bilayer nanodiscs, consisting of a scaffold membrane protein encircling a lipid mixture, may be also considered in reconstitution studies as they can incorporate membrane proteins while the system remains soluble, allowing the application of quantitative solution biochemical and biophysical tools to measure their properties and interactions with other companion elements in lipid environments (Figure 1) [33].
Reconstitution of the bacterial divisome elements on minimal membrane systems A very prominent candidate for a contractile ring potentially able to divide a membrane vesicle is the Z ring in Escherichia coli. It is formed as a result of the anchoring of the GTPase FtsZ (a self-assembling protein, homolog to cytoskeletal tubulin) to the cytoplasmic membrane by the action of FtsA, an amphitropic protein, and ZipA, a bitopic membrane protein (Figure 1, upper panel) [34]. Many recent studies have focused on reconstituting the activities and assembly properties of FtsZ, and its interactions with other proto-ring elements, in different minimal membrane systems, from nanodiscs and supported bilayers to the more complex giant vesicles (Figure 1). Current Opinion in Chemical Biology 2014, 22:18–26
20 Synthetic biology
Figure 1
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Reconstitution of protein assemblies in minimal membrane systems. Upper panel: Diagram illustrating the essential elements of the divisome in E. coli cells, starting with the molecular assembly of the proto-ring elements (colored). Middle panel: Minimal membrane systems to reconstitute the activities and interaction properties of cytoskeletal and cell division assemblies (e.g., the proto-ring complex). Lower panel: Examples of successful reconstitution of proto-ring elements in membrane environments. Adapted from [38,18,37,22] (and unpublished observations from Rivas laboratory).
First evidence that FtsZ acts as a contractile element was provided by Erickson and co-workers, showing that polymers of an artificially membrane-targeted (mts)-FtsZYGP mutant were able to locally indent free-standing membranes of tubular liposomes [35]. Recently, the same group reported that FtsZ-YGP, when encapsulated inside liposomes together with a gain-of-function FtsA* variant, occasionally formed ring-like structures in constricted regions of the vesicles which, in few cases, suggest to complete vesicle division [36]. Permeable giant vesicles containing FtsZ and ZipA were found to shrink upon controlled FtsZ polymerization (Figure 2b,c) [37]. Shrinkage was dependent on the Current Opinion in Chemical Biology 2014, 22:18–26
surface density of ZipA, modulated by the rate of GTP hydrolysis and inhibited by peptides containing sequences of the C-terminus of FtsZ which also compete with FtsZ polymers for the binding to ZipA inserted in phospholipid bilayer nanodiscs (Figure 2d) [38]. Also, polymerization of FtsZ upon the release of caged GTP was found to modulate membrane plasticity in E. coli vesicles in which ZipA has been inserted at both sides of the membrane [39]. On structured membranes supported by micro-structured substrates, it was shown that assembly and orientation of copolymers of wild type and membrane-targeted FtsZ is strongly dependent on the curvature of the surface www.sciencedirect.com
Cellular reconstruction in minimal membrane systems Rivas, Vogel and Schwille 21
Figure 2
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Reconstitution of bacterial division elements in giant vesicles. Water-in-oil droplet transfer procedure (a) used to encapsulate and polymerize FtsZ inside permeable giant vesicles (b). (c) Progressive shrinking of permeable vesicles as a result of the interaction of FtsZ polymers with membraneassociated ZipA at elapsed times 0, 5 and 10 min. (d) Blocking vesicle shrinkage by the addition of an FtsZ-derived peptide that inhibits the interaction of membrane bound ZipA with FtsZ polymers. Scheme based on the one shown in [19]; confocal images adapted from [37].
(Figure 3A). FtsZ filaments preferentially align along curvatures of ca. 1 mm that replicate the dimensions of native E. coli cells [27]. The assembly of FtsZ tethered to supported lipid bilayers by a soluble variant of ZipA, or the mts-FtsZ, respectively, resulted in the formation of two-dimensional interconnected networks that dynamically reorganize by fragmentation, annealing and lateral condensation, as revealed by AFM [40,41] and optical microscopy [42]. Very recently, co-reconstitution of FtsZ and its native membrane anchor FtsA on supported membranes [43] revealed that FtsA drives the self-organization of FtsZ polymers into dynamic patterns such as collective streams and swirling rings with preferential directions, consistent with treadmilling of dynamic polymers (Figure 3B). Although the biochemical details of these dynamics remain to be clarified, they suggest a dual role of the FtsA, tethering FtsZ to the membrane, but at the same time destabilizing FtsZ filaments. Interestingly, this second activity of FtsA resembles the functionality of ADF/ cofilin, an acting binding protein that enhances the treadmilling of actin polymers in the cell and increases the rate of polymer disassembly in vitro [44]. In order to reveal the www.sciencedirect.com
biological implications of these findings, further quantitative studies on these proteins and how are they modulated by other division-related proteins are required.
Actin based cytoskeletal assemblies on minimal membrane systems The actin cytoskeleton does not only play pivotal roles in organizing the interior of eukaryotic cells, but also a major player in maintaining and changing their shape by interacting with the cell membrane. The mechanical interaction of the actin cytoskeleton with the cell membrane governs fundamental processes such as cytokinesis and cell migration with the aid of actin binding proteins and myosin motors. The main common themes for these processes are the quick reorganization and turnover of the actin cytoskeleton and the generation of force by cytoskeletal elements and myosin motors that are transmitted to the membrane (reviewed in [45]). The underlying mechanisms are far from being understood and very often difficult to determine from in vivo approaches as genetic and biochemical manipulations of the omnipotent players such as actin or myosin are often not feasible. Hence, based on pioneering efforts (reviewed in [15]) new bottom-up in vitro systems were recently designed Current Opinion in Chemical Biology 2014, 22:18–26
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Figure 3
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Reconstitution of bacterial division elements in supported lipid bilayers. (A) Using micro-fabricated supports for model membranes the curvature of FtsZ filaments within a groove or around a capillary was studied to understand its mechanical features. Adapted from [27]. (B) Array of dynamic rings of FtsZ polymers, resulting from its interaction with FtsA on a supported membrane, forming traveling streams and rotating swirls (top). Representative snapshots (middle) and schematic illustration (bottom) of treadmilling and fragmentation of FtsZ polymers observed when recruited to the membrane by FtsA. Adapted from [43].
where filamentous actin is either coupled to planar supported and free-standing lipid bilayers [46,47] or to the inside [48] and outside [49] of giant liposomes with the unifying theme of mimicking a cellular actin cortex (Figure 4). The contractile behavior of a cellular actomyosin cortex is provided with the addition of myosin motor assemblies in the presence of ATP to these systems. Recent studies on actin filaments coupled to supported bilayers revealed a possible mechanism of how compressive stress exerted by myosin motors on individual actin filaments may contribute to actin turnover [50] and how the degree of actin meshwork adhesion to the membrane may control the ratio between meshwork contraction and actin filament severing [51]. A first step to understand the physical basis for large-scale membrane shape transformations has been made by anchoring actin filaments inside and outside of synthetic liposomes (Figure 4a). Here, Current Opinion in Chemical Biology 2014, 22:18–26
myosin motor induced contraction of an outer membrane attached actin meshwork with a strong network connectivity showed occasional crushing events of liposomes (Figure 4a, right), while a weaker actin connectivity resulted in spontaneous rupture of the actin layer [49]. Biomimetic actin cortices inside liposomes were also used to investigate the contribution of the actin cytoskeleton to the effective membrane friction by performing nanotube extrusion experiments [50]. These experiments unveiled that the contribution of the actin cytoskeleton is additional but not dominant, and that the content of the membranes as well as the technique how the liposomes are prepared may play a major role. In summary, all these findings emphasize the power of bottom-up reconstitution approaches and may build a solid basis toward developing more complex contractile systems such as actomyosin ring structures that are www.sciencedirect.com
Cellular reconstruction in minimal membrane systems Rivas, Vogel and Schwille 23
Figure 4
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Reconstitution of actin-based assemblies on supported lipid membranes and giant vesicles. (a) Schematic representation of the assembly steps for a minimal actin cortex (MAC) (left). Small unilamellar vesicles are added to the support and result in the formation of a supported lipid bilayer. The bilayer contains functionalized lipids that with the aid of linker proteins can couple functionalized actin filaments to the lipid bilayer, eventually forming a MAC. TIRF images of a MAC before and after the addition of myosin motors (right); myosin motors (red) induce the contraction of the MAC and the formation of actomyosin clusters (right image). (b) Single confocal plane image and a reconstructed stack of confocal images of a GUV before and after coating with actin filaments (left). Adapted from [49]. Time-lapse images of an actin-coated liposome during myosin-induced contraction (right); the liposome crushed as a result of myosin contraction combined with strong actin network connectivity. (A), adapted from [50]. (B), adapted from [49]. All scale bars, 10 mm.
capable of driving force induced membrane shape changes to ultimately reconstitute a minimalistic version of eukaryotic cytokinesis.
Conclusions and outlook Can a simplified cytoskeleton or divisome of complex cellular organisms be reconstituted from purified elements, with the perspective of introducing and activating it for the division of protocells? The reconstitution approaches described here have been exploited to study the properties of selected cytoskeletal and cell division elements, the bacterial FtsZ and the eukaryotic actomyosin, which are both potential candidates for large-scale force-inducing systems. So far, the bacterial Z ring reconstitution, in spite of its successful assembly into ring-like structures, has not yet unambiguously revealed force induction [52]. In contrast, the actomyosin reconstitution has been shown to induce contractile large-scale forces, but so far not be brought into a ring structure that is coupled to a membrane [14,53]. These observations suggest that some elements are still missing from the current attempts to reconstitute www.sciencedirect.com
these molecular assemblies in proto-cellular systems, and further experimental and theoretical efforts are required to integrate biochemical and mechanical information on the systems within the complex architectures that have evolved to function within the cell [7,8,54,55,56]. Thus, despite of the remarkable progress made in the past years with regard to functional reconstitution of protein machineries, a minimal set of modules for controlled proto-cell division has yet to be identified. In order to achieve this ambitious goal, a deeper quantitative biochemical understanding, in terms of equilibrium and kinetic properties, of how these proteins work together as a system of multiple interacting elements, including the lipid membrane, is required [55,56–58]. The behavior of a membrane-confined system of soluble reactive proteins that combine reversibly with each other and with membrane components to produce a directed mechanical force influencing the shape of the confining enclosure will in all probability depend upon several factors: (1) the shape or local curvature of the enclosing membrane, (2) local Current Opinion in Chemical Biology 2014, 22:18–26
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variations in the plasticity of the membrane arising from an inhomogeneous distribution of membrane components (lipids, proteins), (3) the distribution of sites on the membrane to which the reactive proteins and their complexes may be anchored, (4) the stoichiometry of each of the reactive species, and (5) the composition of other confined macromolecular and small molecule solutes which, even though not binding to the reactive species, may modulate the behavior of these species through nonspecific interaction. The complexity of the system as a whole requires that each of these factors be studied individually and in combination. As these systems have co-evolved together with the particular compartment shapes of their hosts, it is essential to be able to subject them to environments that display essential spatial features of these organisms [1,59]. Thus, flexible vesicle membranes without any mimicry of shapes induced by peptidoglycan walls or protein cytoskeleton will likely not be appropriate. If the assembly of these structures prove too complex to be functionally reconstituted, cutting-edge nano-fabrication and microfabrication procedures, in which volumes or surfaces can be precisely and reproducibly shaped, will have to be employed. As many of these minimal cellular modules, such as FtsZ, will be potentially derived from microorganisms and operate on extremely small dimensions, we will have to investigate them by advanced biophysical methods, particularly super resolution microscopy. The use of cell-free expression systems [60,61] or cells in which certain elements have been removed [62] will help us in capturing functional modules that are hard to be purified, as many membranetargeted proteins. Finally, after having understood the principles of a fundamental set of modules, we may even be able to construct minimal functional elements from non-protein-based macromolecules, for example, DNA nanostructures, known as DNA origami [63], and to engineer assemblies with novel functionalities in ways that cannot be easily achieved in the cell.
Acknowledgements We thank Allen Minton (NIH) for useful comments. This work was supported in part by Human Frontier Science Program through Grant RGP0050/2010-C012 (to PS and GR), by the DFG Leibniz Prize (to PS), and by the European Commission through Contract HEALTH-F3-2009223431 and by the Spanish Government through Grant BIO2011-28941C03-03 (both to GR).
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest 1.
Vahey MD, Fletcher DA: The biology of boundary conditions: cellular reconstitution in one, two, and three dimensions. Curr Opin Cell Biol 2014, 26:60-68. This review states the importance of performing bottom-up reconstitution studies in environments that faithfully reproduce the natural spatial constraints.
Current Opinion in Chemical Biology 2014, 22:18–26
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Vale RD: Preface: the role of reconstitution in cytoskeleton research. Methods Enzymol 2014, 540:xix-xxiii.
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Balasubramanian MK, Srinivasan R, Huang Y, Ng KH: Comparing contractile apparatus-driven cytokinesis mechanisms across kingdoms. Cytoskeleton (Hoboken) 2012, 69:942-956.
4.
Loose M, Schwille P: Biomimetic membrane systems to study cellular organization. J Struct Biol 2009, 168:143-151.
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Sezgin E, Schwille P: Model membrane platforms to study protein–membrane interactions. Mol Membr Biol 2012, 29:144154.
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Liu AP, Fletcher DA: Biology under construction: in vitro reconstitution of cellular function. Nat Mol Cell Biol Rev 2009, 10:644-650.
7.
Martos A, Jime´nez M, Rivas G, Schwille P: Towards a bottom-up reconstitution of bacterial cell division. Trends Cell Biol 2012, 22:634-643.
8.
Mullins RD, Hansen SD: In vitro studies on actin filament and network dynamics. Curr Opin Cell Biol 2013, 25:6-13.
9.
Derganc J, Antonny B, Copic A: Membrane bending: the power of protein imbalance. Trends Biochem Sci 2013, 38:576-584.
10. Kozlov MM, Felix Campelo F, Liska N, Chernomordik LV, Marrink SJ, McMahon HT: Mechanisms shaping cell membranes. Curr Opin Cell Biol 2014, 29:53-60. 11. Andes-Koback M, Keating CD: Complete budding and asymmetric division of primitive model cells to produce daughter vesicles with different interior and membrane compositions. J Am Chem Soc 2011, 133:9545-9555. 12. Bries Y, Walde P, Schuppler M, Loessner MJ: How did bacterial ancestors reproduce? Lessons from L-form cells and giant lipid vesicles. Bioessays 2012, 34:1078-1084. 13. Mendes Pinto I, Rubinstein B, Li R: Force to divide: structural and mechanical requirements for actomyosin ring contraction. Biophys J 2013, 105:547-554. 14. Pollard TD: Mechanics of cytokinesis in eukaryotes. Curr Opin Cell Biol 2010, 22:50-56. 15. Vogel SK, Schwille P: Minimal systems to study membrane– cytoskeleton interactions. Curr Opin Biotechnol 2012, 23:758765. 16. Garcı´a-Sa´ez AJ, Carrer DC, Schwille P: Fluorescence correlation spectroscopy for the study of membrane dynamics and organization in giant unilamellar vesicles. Methods Mol Biol 2010, 606:493-508. 17. Walde P, Cosentino K, Engel H, Stano P: Giant vesicles: preparations and applications. Chembiochem 2010, 11:848865. 18. Jime´nez M, Martos A, Cabre´ EJ, Raso A, Rivas G: Giant vesicles: a powerful tool to reconstruct bacterial division assemblies in cell-like compartments. Environ Microbiol 2013, 15:3158-3168. 19. Takiguchi K, Negishi M, Tanaka-Takiguchi Y, Homma M, Yoshikawa K: Transformation of actoHMM assembly confined in cell-sized liposome. Langmuir 2011, 27:11528-11535. 20. Richmond DL, Schmid EM, Martens S, Stachowiak JC, Liska N, Fletcher DA: Forming giant vesicles with controlled membrane composition, asymmetry, and contents. Proc Natl Acad Sci U S A 2011, 108:9431-9436. 21. Abkarian M, Loiseau E, Massiera G: Continuous droplet interface crossing encapsulation (cDICE) for high throughput monodisperse vesicle design. Soft Matter 2011, 7:4610-4614. 22. Mellouli S, Monterroso B, Vutukuri HR, te Brinke E, Chokkalingam V, Rivas G, Huck WTS: Self-organization of the bacterial cell-division protein FtsZ in confined environments. Soft Matter 2013, 9:10493-10500. 23. Abu Shah E, Keren K: Symmetry breaking in reconstituted actin cortices. Elife 2014, 3:e01433. 24. Mim C, Unger VM: Membrane curvature and its generation by BAR proteins. Trends Biochem Sci 2012, 37:526-533. www.sciencedirect.com
Cellular reconstruction in minimal membrane systems Rivas, Vogel and Schwille 25
25. Zieske K, Schwille P: Reconstitution of pole-to-pole oscillations of min proteins in microengineered polydimethylsiloxane compartments. Angew Chem Int Ed Engl 2013, 52:459-462. 26. Zieske K, Schweizer J, Schwille P: Surface topology assisted alignment of Min protein waves. FEBS Lett 2014 http:// dx.doi.org/10.1016/j.febslet.2014.06.026. 27. Arumugam S, Chwastek G, Fischer-Friedrich E, Ehrig C, Mo¨nch I, Schwille P: Surface topology engineering of membranes for the mechanical investigation of the tubulin homologue FtsZ. Angew Chem Int Ed Engl 2012, 51:11858-11862. This paper describes the behavior of polymers of a membrane-targeted FtsZ variant on micro-structured substrates of different curvature supporting lipid bilayers. FtsZ polymers preferentially orient along curvatures close to the diameter of E. coli cells, suggesting that additional factors than FtsZ alone are required to facilitate cell constriction. 28. Herrig A, Janke M, Austermann J, Gerke V, Janshoff A, Steinem C: Cooperative adsorption of erzin on PIP2-containing membranes. Biochemistry 2006, 45:13025-13034. 29. Groves JT, Parthasarathy R, Forstner MB: Fluorescence imaging of membrane dynamics. Annu Rev Biomed Eng 2008, 10:311338. 30. Castellana ET, Cremer PS: Solid supported lipid bilayers: from biophysical studies to sensor design. Surf Sci Rep 2006, 61:429-444. 31. Baciu CL, Becker J, Janshoff A, So¨nnichsen C: Protein– membrane interaction probed by single plasmonic nanoparticles. Nano Lett 2008, 8:1724-1728. 32. Eisele NB, Frey S, Piehler J, Go¨rlich D, Richter RP: Ultrathin nucleoporin phenylalanine–glycine repeat films and their interaction with nuclear transport receptors. EMBO Rep 2010, 11:366-372. 33. Schu¨ler MA, Denisov IG, Sligar SG: Nanodiscs as a new tool to examine lipid-protein interactions. Methods Mol Biol 2013, 974:415-433. 34. Rico AI, Krupka M, Vicente M: In the beginning, Escherichia coli assembled the proto-ring: an initial phase of division. J Biol Chem 2013, 288:20830-20836. 35. Osawa M, Anderson DE, Erickson HP: Reconstitution of contractile FtsZ rings in liposomes. Science 2008, 320:792-794. 36. Osawa M, Erickson HP: Liposome division by a simple bacterial division machinery. Proc Natl Acad Sci U S A 2013, 110:1100011004. In this study, the authors tether native FtsZ to liposome membranes by the action of a gain-of-function FtsA* variant. FtsZ–FtsA* ring-like structures are formed inside vesicles and, in few cases, are able to constrict vesicles. 37. Cabre´ EJ, Sa´nchez-Gorostiaga A, Carrara P, Ropero N, Casanova M, Palacios P, Stano P, Jime´nez M, Rivas G, Vicente M: Bacterial division proteins FtsZ and ZipA induce vesicle shrinkage and cell membrane invagination. J Biol Chem 2012, 288:26625-26634. A minimal division machinery assembly was reconstructed in permeable giant vesicles containing FtsZ and ZipA. These vesicles shrank upon FtsZ polymerization, partly mimicking membrane constriction. 38. Herna´ndez-Rocamora VM, Reija B, Garcı´a C, Natale P, Alfonso C, Minton AP, Zorrilla S, Rivas G, Vicente M: Dynamic interaction of the Escherichia coli cell division ZipA and FtsZ proteins evidenced in nanodiscs. J Biol Chem 2012, 287:30097-30104. 39. Lo´pez-Montero I, Lo´pez-Navajas P, Mingorance J, Ve´lez M, Vicente M, Monroy F: Membrane reconstitution of FtsZ–ZipA complex inside giant spherical vesicles made of E. coli lipids: large membrane dilation and analysis of membrane plasticity. Biochim Biophys Acta 2013, 1828:687-698. 40. Mateos-Gil P, Ma´rquez I, Lo´pez-Navajas P, Jime´nez M, Vicente M, Mingorance J, Rivas G, Ve´lez M: FtsZ polymers bound to lipid bilayers through ZipA form dynamic two-dimensional networks. Biochim Biophys Acta 2012, 1818:806-813. This atomic force microscopy study shows that FtsZ polymers bound to lipid bilayers through ZipA form dynamic two-dimensional networks that retain their capacity to dynamically restructure, to fragment, to anneal and to condense laterally. www.sciencedirect.com
41. Encinar M, Kralicek AV, Martos A, Krupka M, Cid S, Alonso A, Rico AI, Jime´nez M, Ve´lez M: Polymorphism of FtsZ filaments on lipid surfaces: role of monomer orientation. Langmuir 2013, 29:9436-9446. 42. Arumugam S, Petrasˇek Z, Schwille P: MinCDE exploits the dynamic nature of FtsZ filaments for its spatial regulation. Proc Natl Acad Sci U S A 2014, 111:E1192-E1200. This paper proposes a new mechanism of spatial positioning of FtsZ filaments by the Min protein system, building on a constant dynamic turnover of FtsZ monomers all along the filaments that it is enhanced in the presence of MinC. 43. Loose M, Mitchison TJ: The bacterial cell division proteins FtsA and FtsZ self-organize into dynamic cytoskeletal patterns. Nat Cell Biol 2014, 16:38-46. This paper describes the co-reconstitution of FtsZ and FtsA in supported bilayers, showing that FtsA (in the presence of ATP) not only recruits FtsZ polymers to the membrane but also the formation of FtsA–FtsZ polymer networks that undergo rapid chiral treadmilling. 44. Carlier MF, Laurent V, Santolini J, Melki R, Didry D, Xia GX, Hong Y, Chua NH, Pantaloni D: Actin depolymerizing factor (ADF/cofilin) enhances the rate of filament turnover: implication in actinbased motility. J Cell Biol 1997, 136:1307-1322. 45. Clark AG, Wartlick O, Salbreux G, Paluch EK: Stresses at the cell surface during animal cell morphogenesis. Curr Biol 2014, 24:R484-R494. Elegant review about the actomyosin cortex and how myosin generated stresses on the molecular level control actin cortex and membrane tension. 46. Vogel SK, Heinemann F, Chwastek G, Schwille P: The design of MACs (minimal actin cortices). Cytoskeleton (Hoboken) 2013, 70:706-717. 47. Heinemann F, Vogel SK, Schwille P: Lateral membrane diffusion modulated by a minimal actin cortex. Biophys J 2013, 104:14651475. 48. Campillo C, Sens P, Ko¨ster D, Pontani LL, Levy D, Bassereau P, Nassoy P, Sykes C: Unexpected membrane dynamics unveiled by membrane nanotube extrusion. Biophys J 2013, 104:12481256. 49. Carvalho K, Tsai FC, Lees E, Voituriez R, Koenderink GH, Sykes C: Cell-sized liposomes reveal how actomyosin cortical tension drives shape change. Proc Natl Acad Sci U S A 2013, 110:1645616461. In this study, the authors present actin coated lipid vesicles that upon myosin contraction either burst in the case of strong actin connectivity or display rupture and peeling away of the actin coat when the actin connectivity is weak. 50. Vogel SK, Petrasek Z, Heinemann F, Schwille P: Myosin motors fragment and compact membrane-bound actin filaments. Elife 2013, 2:e00116. This article gives the first direct evidence that myosin motors can break and compact membrane coupled actin filaments in a reconstituted actomyosin cortex. Based on the experimental data and a theoretical description the authors provide a mechanism for actin breakage by myosin motors that could potentially contribute to actin turnover in vivo. 51. Murrell MP, Gardel ML: F-actin buckling coordinates contractility and severing in a biomimetic actomyosin cortex. Proc Natl Acad Sci U S A 2012, 109:20820-20825. 52. Meier EL, Goley ED: Form and function of the bacterial cytokinetic ring. Curr Opin Cell Biol 2014, 26:19-27. 53. Mishra M, Huang J, Balasubramanian MK: The yeast actin cytoskeleton. FEMS Microbiol Rev 2014, 38:213-227. 54. Vicente M: Will test tubes ever undergo division? In Curtis T, Daran JM, Pronk JT, Frey J, Jansson JK, Robbins-Pianka A, et al.: Crystal ball – 2013. Microb Biotechnol 2013, 6:3-16. Commentary on the state of the art of bacterial division, with focus on central issues that are still unresolved. 55. Dogterom M, Surrey T: Microtubule organization in vitro. Curr Opin Cell Biol 2013, 25:23-29. Comprehensive review on bottom-up reconstitution approaches to study microtubule cytoskeleton self-organization and function. Current Opinion in Chemical Biology 2014, 22:18–26
26 Synthetic biology
56. Hyman A: Whither systems biology. Philos Trans R Soc B 2011, 366:3635-3637. 57. Pollard TD: No questions about exciting questions in cell biology. PLoS Biol 2013, 11:e1001734. 58. Rivas G, Alfonso C, Jime´nez M, Monterroso B, Zorrila S: Macromolecular interactions of the bacterial division FtsZ protein: from quantitative biochemistry and crowding to reconstructing minimal divisomes in the test tube. Biophys Rev 2013, 5:63-77. 59. Stachowiak MR, Laplante C, Chin HF, Guirao B, Karatekin E, Pollard TD, O’Shaughnessy B: Mechanism of cytokinetic contractile ring constriction in fission yeast. Dev Cell 2014, 29:547-561.
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60. Maeda YT, Nakadai T, Shin J, Uryu K, Noireaux V, Libchaber A: Assembly of MreB filaments on liposome membranes: a synthetic biology approach. ACS Synth Biol 2012, 1:53-59. 61. Nourina Z, Scott A, Danelon C: Toward the assembly of a minimal divisome. Syst Synth Biol 2014 http://dx.doi.org/ 10.1007/s11693-014-9150-x. 62. Pazos M, Casanova M, Palacios P, Margolin W, Natale P, Vicente M: FtsZ placement in nucleoid-free bacteria. PLOS ONE 2014, 9:e91984. 63. Langecker M, Arnaut V, Martin TG, List J, Renner S, Mayer M, Dietz H, Simmel FC: Synthetic lipid membrane channels formed by designed DNA nanostructures. Science 2012, 338:932-936.
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