The artificial cell: biology-inspired compartmentalisation of chemical function

09:10-09:35 Navigating the endosome: the role of receptor ubiquitination and ubiquitin-binding adaptors

Professor Philip Woodman, University of Manchester, UK

Abstract

The endocytic pathway is the gateway between cells and their environment, and promotes diverse events such as nutrient uptake and cell migration. Membrane proteins (“cargoes”) enter endocytic vesicles and reach the endosome. From here they are recycled to the surface, or sorted to intralumenal vesicles (ILVs) to generate a multivesicular body (MVB) en route to the lysosome. The generation of transport intermediates at all points of the endocytic pathway involves the ordered recruitment of an array of membrane-sculpting adaptor proteins, which recognise cargo and coordinate the local induction of membrane curvature. The impairment of these adaptor proteins, or their defective regulation, gives rise to errors in compartmentalisation. One class of endocytic cargo that is transported to the MVB is exemplified by the epidermal growth factor receptor (EGFR). For these cargoes, the sorting signal is generated by receptor ubiquitination, a reversible post-translational modification. A broad range of ubiquitin-binding adaptor proteins recognise ubiquitinated cargoes and promote their endocytic sorting. These adaptors themselves are regulated by reversible ubiquitination, generating a network of ubiquitin homeostasis that governs endocytic trafficking. The key regulators of this network, ubiquitin ligases and deubiquitinating enzymes, must also coordinate their functions with membrane curvature.

09:45-10:10 Decision making in autophagy – How to loose specificity

Dr Thomas Wollert, Institut Pasteur, France

Abstract

Autophagy is a pivotal recycling pathway that operates in all eukaryotic cells. More than 40 AuTophaGy related (Atg) proteins are known in yeast and many of them are conserved in humans. In non-stressed, vegetative cells, autophagy mainly recycles unwanted or damaged cytoplasmic components by exclusively engulfing them into autophagic membrane sacks, termed phagophores. In stressed cells, however, autophagy loses its selectivity and degrades bulk cytoplasm. The regulation of this dramatic loss in selectivity is intimately intertwined with the onset of human diseases such as neurodegeneration or cancer. Its molecular bases remained, however, not well understood. Through a combination of classical biochemistry and cell biology with cutting edge in vitro reconstitutions, Dr Wollert and colleagues have been able to solve the long standing question how selectivity is regulated at a molecular level in yeast. They found that the decision is made at the earliest step in autophagy, i.e. nucleation of the phagophore.

10:20-10:50 Coffee

10:50-11:15 Compartmentalisation and crowding in artificial membranes

Professor Mark Wallace, King's College London, UK

Abstract

Diffusion of biomolecules in both the cytosol and in the plasma membrane are not simple random walks, instead the measured diffusivity depends on the timescale of observation. Our understanding of why and how this anomalous diffusion exists remains incomplete, and the presence of slower-moving obstacles, pinning sites, and compartmentalisation have all been suggested as potential contributors to anomalous diffusion. Ultimately, this confinement and compartmentalisation must affect the reaction rates of biomolecules, and understanding and exploiting these effects would be a useful tool in designing artificial cellular systems. Here Mark Wallace will present his group’s recent work to build artificial model membranes with defined nanoscale topographic and topological properties to control anomalous behaviour. Mark exploits a combination of single-molecule Total Internal Reflection Fluorescence and Interferometric Scattering microscopies to characterise diffusion using single-particle tracking that spans over four orders of magnitude in time.

11:25-11:50 Mimicking membrane scission in vitro

Professor Patricia Bassereau, Institut Curie, France

Abstract

Eukariotic cells are highly compartmentalized to achieve local special functions, but nevertheless have to exchange lipids and proteins between compartments and with the exterior of the cell. Thus, traffic intermediates (vesicles, tubules) have to be formed and detached from donor membranes to be transported and deliver their cargos and membrane to an acceptor compartment. Cell compartmentalization thus works hand-in-hand with membrane scission to allow for these exchanges. Reconstituted membrane systems have been instrumental to reveal mechanisms that drive membrane scission of bud neck in vivo, such as constriction by the ATPase dynamin involved in endocytosis or by line tension at the edge of membrane domains. Reconstitution approaches have similarly allowed recently to reveal a new scission mechanism, not based on constriction but on the friction between a protein scaffold and the membrane that produces a viscoelastic-like response and a scission when membrane tension exceeds lysis tension: Friction-Driven Scission (FDS). Professor Bassereau will show how this mechanism was shown using a combination of reconstituted systems and theoretical modelling.

14:10-14:35 Adaptive compartments with life-like behaviour

Professor Jan Van Hest, Technische Universiteit Eindhoven, The Netherlands

Abstract

Compartmentalization is generally regarded as one of the key prerequisites for life. In living cells, not only the cell itself is a compartment, with its properties controlled by the semipermeable cell membrane, but also the organelles play a crucial role in protecting and controlling biological processes. To better understand the role of compartmentalization, there is clear need for model systems that can be adapted in a highly controlled fashion, and in which life-like properties can be installed. Polymer-based compartments are robust and chemically versatile, and as such are a useful platform for the development of life-like compartments. In this contribution both an artificial organelle and cell system will be discussed. The artificial organelles are composed of biodegradable amphiphilic block copolymers that self-assemble into vesicular structures. These so-called polymersomes are loaded with enzymes and are semi-permeable for small molecule substrates. Upon introduction in living cells, they affect metabolic pathways as artificial organelles. A different type of polymersome is created via a shape change process in which a bowl-shaped structure is obtained. Within the cavity of the bowl enzymes are loaded which provide the nanostructure with motility upon conversion of chemical energy into kinetic energy. The synthetic cell platform is composed of a complex polymer coacervate, stabilized by a biodegradable block copolymer. The specific feature of the polymer membrane is its semipermeable character. Enzymes inside the protocell can therefore still be reached by their substrates, and small molecule products can be excreted. This allows protocell communication with this robust synthetic platform.

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14:45-15:10 Biomimetic synthesis of artificial magnetosomes

Dr Sarah Staniland, University of Sheffield, UK

Abstract

Magnetic nanoparticles (MNPs) have many applications, particularly over a range of emerging biomedicine (diagnostic and therapeutic). For such applications the MNPs are required to be biocompatible and have a mono-disperse size and shape distribution to ensure their magnetic behaviour is consistent, which can require chemical synthesis under harsh and toxic conditions, which may compromise biocompatibility. Magnet bacteria synthesis nanoparticles of magnetite within lipid vesicles (known as magnetosomes) within their cells. Magnetosomes show great potential for such biomedical applications, however, magnetosomes themselves may not be ideal with respect to economy of production and adaptability for different specifications. Sarah Staniland’s group have therefore taken inspiration from magnetic bacteria to create artificial magnetosome-like theranostic magnetic nanovesicles in situ under mild reaction conditions. Here Sarah describes how her group mimics the magnetosomes with iron ions imported into base filled vesicles to form a range of magnetovesicles, from magnetic nanoparticles embedded in the membrane to magnetic nanoparticle core liposome similar to a native magnetosome. Sarah will show how different magnetovesicles vary in their hyperthermic and MRI response and demonstrate how the group can control and tune the particle size and number to tailor them for different applications.

15:20-15:50 Tea

15:50-16:15 Model systems for membraneless subcellular organelles: Compartmentalization of biomolecules and reactions by liquid-liquid phase coexistence

Professor Christine Keating, Penn State University, USA

Abstract

Biological cells are highly organized with numerous subcellular compartments, many of which lack membranous boundaries such as those that surround organelles. Christine Keating’s group are developing simple experimental models for these membraneless organelles based on liquid-liquid phase separation, which is a common phenomenon in aqueous solutions of macromolecules. Solutes such as ions, small molecules, and biopolymers can become compartmentalized by partitioning into one of the phases. The group is studying mechanisms for, and consequences of, this type of compartmentalization using a variety of simple model systems composed of phase-separating aqueous polymer solutions. Through these types of studies, Christine hopes to uncover underlying physiochemical mechanisms in cellular organization and to identify new avenues for biomimetic systems for applications in biotechnology and materials science. For example, compartmentalization of catalysts and/or reactants into polymer-rich droplets can lead to control over the sites and rates of reactions. This approach has led to increased ribozyme reaction rates and development of liposome-stabilized water-in-water emulsions that act as artificial mineralizing vesicles.

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16:25-16:50 Protein design in the cell: towards a synthetic proteome

Professor Dek Woolfson, University of Bristol, UK

Abstract

Protein design, that is, the construction of entirely new protein sequences that fold into prescribed structures, has come of age. It is now possible to design proteins de novo using simple rules of thumb or computational design methods. The designs can be made rapidly via peptide synthesis or the expression of synthetic genes; and the resulting proteins can usually be characterised all the way through to high-resolution X-ray crystal structures. Contemporary questions in the protein-design field include: What do we do with these new-found skills? What protein structures and functions do we target? How far can we move past the confines of natural protein structures and functions? And, how can we take protein design from an exercise largely done in silico and in vitro into a truly synthetic biology and take it in vivo? This talk will address these questions with reference to simple through to complex and functional protein designs that we have explored over the past 5–10 years. These use a straightforward protein structure, called the alpha-helical coiled coil, which are bundles of 2 or more alpha helices found in many protein-protein interactions. As such it provides an excellent basis for building proteins from the bottom up. The vast majority of coiled-coil designs have been based on simple rules of thumb learnt from natural proteins or derived empirically through experiment. These rules relate sequence to structure to guide the specification of coiled-coil oligomerization state, strand orientation, partner selection, and, to some extent, stability. This has been extremely informative and productive, and design and engineering is probably more advanced for coiled coils than for any other protein structure. However, to move past the low-hanging fruit of coiled-coil design, and into the so-called dark matter of protein structures, we will all have to learn new tricks. To address this Professor Woolfson has begun to tackle coiled-coil design parametrically using computational methods. Professor Woolfson has developed easy-to-use computational modelling tools and a more-sophisticated suite of programs called ISAMBARD that allow the rapid generation and optimisation of protein designs in silico. The first part of the talk will describe how a serendipitous discovery of a 6-stranded alpha-helical barrel led his group to develop these computational methods, and how they have used these to deliver entirely new non-natural protein structures predictably. It will show the utility of this approach to make water-soluble protein-like barrels, which the group has engineered to form materials, bind small molecules, and catalyse simple reactions. Secondly, Professor Woolfson will demonstrate how we might take protein design in vivo with recent work with the Warren lab (Kent) and the Verkade lab (Bristol). He has engineered hybrids of a de novo heterodimer and a natural component of bacterial microcompartments. When expressed in E. coli, the hybrid assemblies to form a cytoscaffold that permeates the cells, and can act as a support for the co-localisation of functional enzymes.

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09:00-09:25 Regenerating the nuclear envelope during exit from mitosis

Dr Jeremy Carlton, KCL and Francis Crick Institute, UK

Abstract

During cell division, as well as separating their duplicated genomes, cells must also deconstruct, separate and then reconstruct many of their cytoplasmic organelles.  Mammalian nuclei are surrounded by a double-membraned organelle called the nuclear envelope that is continuous with the endoplasmic reticulum. During mitotic exit, sheets of ER envelope the forming daughter nuclei and become fused together to generate a sealed nuclear envelope. In addition to its role in membrane abscission during cytokinesis, viral budding, endosomal sorting and plasma membrane repair, the endosomal sorting complex required for transport-III (ESCRT-III) machinery has recently been shown to participate in nuclear envelope sealing during mitotic exit. Nuclear envelope localisation of ESCRT-III is dependent upon the ESCRT-III component CHMP7 and the inner nuclear membrane protein LEM2. However, it is unclear how ESCRT-III actually engages nuclear membranes. Here, Dr Carlton shows that the N-terminus of CHMP7 acts as a novel membrane-binding module. This membrane-binding ability allows CHMP7 to bind to the endoplasmic reticulum (ER), an organelle continuous with the NE, and provides a platform to direct NE-recruitment of ESCRT-III during mitotic exit. Dr Carlton also identifies novel activities in the C-terminus of CHMP7 that restrict its activity to the inner-nuclear membrane and help us understand how this complex can help regenerate the nucleus. Dr Carlton finds that mutations that compromise CHMP7 function also prevent assembly of downstream ESCRT-III components at the reforming NE and proper establishment of post-mitotic nucleo-cytoplasmic compartmentalisation. These data identify a novel membrane-binding activity within an ESCRT-III subunit that is essential for post-mitotic nuclear regeneration.

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09:35-10:00 Synthetic biology of minimal cellular systems

Professor Petra Schwille, Max Planck Institute of Biochemistry, Germany

Abstract

In recent years, biophysics has accumulated an impressive selection of cutting-edge techniques to analyse biological systems with ultimate sensitivity and precision, down to the single molecule level. However, a strictly quantitative application of most of these techniques in living cells or organisms has been extremely challenging, because of the enormous complexity and redundancy of cellular modules and elements. The more physiological a system under study, the harder it is to define a manageable number of relevant control parameters. This renders it necessary to accumulate ever more sophisticated techniques and assays in order to master a single biological problem, and thus, often extends experiments and publications in the life sciences to hardly manageable sizes. An alternative approach is to limit the methodological toolbox in a biological study without sacrificing the biophysical standards of quantitation. Instead, the biological phenomenon will have to be reduced to its fundamental features by reconstituting it in a bottom-up approach. The strive for identifying such minimal biological systems, particularly of subcellular structures or modules, has in the past years been very successful, and crucial in vitro experiments with reduced complexity can nowadays be performed, e.g., on reconstituted cytoskeleton and membrane systems. As a particularly exciting example for the power of minimal systems, we recently demonstrated the self-organization of MinCDE, essential proteins of the bacterial cell division machinery, leading to a protein-based pacemaker and spatiotemporal cue for downstream events, such as the positioning of divisome proteins. In her talk, Petra Schwille will discuss some recent results of her group’s work on membrane-based systems, using single molecule optics and biological reconstitution assays. Petra will further discuss the perspective of assembling a minimal system to reconstitute cell division.

10:40-11:05 Buckling of an epithelium growing under spherical confinement

Dr Aurelien Roux, University of Geneva, Switzerland

Abstract

Many organs, such as the gut or the spine are formed through folding of an epithelium. While genetic regulations of cell fates leading to epithelium folding have been investigated, mechanisms by which forces sufficient to deform the epithelium are generated are less studied. Here, Aurelien Roux shows that cells forming an epithelium on to the inner surface of spherical elastic shells protrude inward while growing. By measuring the pressure and local forces applied onto the elastic shell, Aurelien shows that this folding is induced by compressive stresses arising within the epithelial layer: while growing under spherical confinement, epithelial cells are subjected to lateral compression, which induces epithelium buckling. While several fold initiations can be observed within one capsule, final shapes often show a single fold. These findings are recapitulated by an analytical model of the epithelium buckling from which the Roux group can estimate local compressive forces and rigidity. As proposed for gastrulation or neurulation, this study shows that forces arising from epithelium proliferation are sufficient to drive epithelium folding.

11:15-11:40 Signalling reactions on membrane surfaces: the roles of space, force, and time

Professor Jay Groves, University of California, Berkeley, USA

Abstract

Most intracellular signal transduction reactions take place on the membrane. The membrane provides much more than just a surface environment on which signalling molecules are concentrated. There is a growing realization that multiple physical and chemical mechanisms allow the membrane to actively participate in the signalling reactions. Using a combination of single molecule imaging and spectroscopic techniques, Professor Jay Groves’ research seeks to directly resolve the actual mechanics of signalling reactions on membrane surfaces both in reconstituted systems and in living cells. These observations are revealing new insights into cellular signalling processes as well as some unexpected functional behaviours of proteins on the membrane surface. The Groves’ lab has recently discovered a type of signalling reaction phenomenon that enables geometrical features of the membrane surface to couple directly to the outcome of a signalling process.

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11:50-12:15 Harnessing Nature's ability to create membrane compartmentalisation

Dr Paul Beales, University of Leeds, UK
Dr Barbara Ciani, University of Sheffield, UK

Abstract

A biological cell can be thought of as a complex chemical reactor where vast numbers of interactions are simultaneously taking place. To prevent unwanted cross-talk and interference within the ‘noise’ of all these concurrent chemical pathways, a cell compartmentalises these processes localizing different functions within individual membrane-bound structures (organelles). Confinement of chemical processes also allows a cell to maintain incompatible environments that are optimal for each organelle's function, which would not be possible within a single ‘pot’. If we are to mimic this complexity within synthetic ‘nanoreactors’, we need to develop ways of mimicking cellular compartmentalisation within synthetic structures. Here, Dr Barbara Ciani and Dr Paul Beales will show that it is possible to create multi-compartment architectures, in vitro, using a purified membrane remodelling protein complex. Barbara and Paul will also show how this in vitro system allows us to learn what controls the membrane shaping action of these proteins and therefore regulate the encapsulation of cargo.

13:45-14:10 A cellular transition to evolutionary dynamics

Professor Lee Cronin, University of Glasgow, UK

Abstract

Complex chemical systems contain not only diverse chemistry, but also couple molecular processes to the environment via dynamical interactions. Whereas the formation of dissipative chemical fluxes in polymer systems, soft materials, or inorganic crystal gardens are controlled by macroscopic dynamics that can be fine-grained, the exploration of the development of complexity at the molecular level has not been widely explored.  In this talk Professor Cronin will present his group’s research in this area that aims to drive the development of complexity in molecular systems by coupling them to non-equilibrium dynamics associated with proto-cell systems. The ultimate aim is to explore how evolutionary dynamics and complex molecular machinery might emerge giving a new avenue for the design of molecular machines and autonomous life-like systems. To explore this Professor Cronin has developed a series of fully autonomous chemical robots aim to look at flows, proto-cells, and self-replicating chemistry.

14:20-14:45 Microfluidic technologies for the bottom-up construction of artificial cells

Professor Oscar Ces, Imperial College London, UK

Abstract

This talk will outline microfluidic strategies for bottom-up synthetic biology that are being used to construct multi-compartment artificial cells where the contents and connectivity of each compartment can be controlled. These compartments are separated by biologically functional membranes that can facilitate transport between the compartments themselves and between the compartments and external environment. These technologies have enabled us to engineer multi-step enzymatic signalling cascades into the cells leading to in-situ chemical synthesis and systems that are capable of sensing and responding to their environment. In addition, we have developed hybrid systems fusing living and non-living components to generate higher order systems. Finally, this talk will provide an overview of microfluidic single cell analysis tools for counting the protein copy number in single cells and artificial cells.

14:55-15:25 Tea

15:25-15:50 Collective behaviour in synthetic protocell communities

Professor Stephen Mann FRS, University of Bristol, UK

Abstract

Recent progress in the chemical construction of micro-compartmentalized colloidal objects comprising integrated biomimetic functions is paving the way towards rudimentary forms of artificial cell-like entities (protocells) for modelling complex biological systems, exploring the origin of life, and advancing future proto-living technologies. Although several new types of protocells are currently available, the design of synthetic protocell communities and investigation of their collective properties has received little attention. In this talk, Professor Mann reviews some recent experiments undertaken in his laboratory that demonstrate simple forms of higher-order dynamic behaviour in synthetic protocells. Professor Mann will discuss four new areas of investigation: (i) enzyme-powered motility and collective migration in buoyant organoclay/DNA protocells; (ii) artificial predatory behaviour in mixed populations of proteinosomes and coacervate micro-droplets; and (iii) artificial phagocytosis behaviour in a binary population of magnetic and silica colloidosomes. He will use these new model systems to discuss pathways towards chemical cognition, modulated reactivity, basic signalling pathways and non-equilibrium activation in compartmentalized artificial micro-ensembles.

16:00-16:30 Final discussion/Overview