The artificial cell: biology-inspired compartmentalisation of chemical function

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.

Professor Philip Woodman, University of Manchester, UK

Professor Philip Woodman, University of Manchester, UK

Philip Woodman is Professor of Cell Biology at the University of Manchester UK. He obtained a PhD from Cambridge University in 1986, and after a post-doc with Graham Warren in Dundee and at the CRUK London research institute moved to Manchester in 1991 as an MRC Senior Research Fellow. He works on the sorting of signalling receptors within the endosome, with a particular interest in the mechanisms of epidermal growth factor receptor down-regulation.

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.

Dr Thomas Wollert, Institut Pasteur, France

Dr Thomas Wollert, Institut Pasteur, France

Thomas Wollert studied Biochemistry and Biophysical Chemistry in Potsdam and Hannover (Germany) and obtained his Masters degree in 2003. He carried out his PhD studies at the Helmholtz Centre for Infection Biology, Braunschweig and obtained a PhD in structural biology from the University of Braunschweig (Germany). Thomas did his Postdoc studies in James Hurley’s Laboratory at the National Institut of Health, USA were he investigated the molecular mechanisms of membrane trafficking complexes. He became independent Max-Planck Research Group leader at the MPI of Biochemistry in Martinsried (Germany) in 2010 where he started his research on autophagy using a combination of biophysical, biochemical and cell biology approaches. In 2017, Thomas joined the Institut Pasteur Paris (France) as Directeur de Recherche continuing his research on autophagy.

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.

Professor Mark Wallace, King's College London, UK

Professor Mark Wallace, King's College London, UK

Mark studied Chemical Physics as an undergraduate at the University of Bristol, followed by a PhD in Chemistry at the University of Cambridge under the supervision of Professor David Klenerman. He was awarded the 2002 Gregorio Weber International Prize in Biological Fluorescence for this work. Mark then spent 2 years as a postdoctoral fellow at Stanford University, working with Professor Richard Zare. He returned to the UK in 2002 to undertake a second postdoctoral position at the National Institute for Medical Research with Dr Justin Molloy, before moving to Oxford in 2005 as a Royal Society University Research Fellow. He was subsequently appointed as a University lecturer and fellow of Wadham College in 2006. He joined King’s in 2016 as part of its expansion of Chemistry. In 2009 Mark was appointed to the steering committee of the British Biophysical Society. Patents arising from his work have also been licensed in the UK Mark has also been active in raising awareness of his group’s research beyond the lab, including video podcasting and participation in the 2014 “I’m a scientist get me out of here” competition. He was awarded the RSC Norman Heatley Award in 2015 in recognition of his work. He was made a Fellow of the RSC in the same year.

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.

Professor Patricia Bassereau, Institut Curie, France

Professor Patricia Bassereau, Institut Curie, France

Patricia Bassereau is currently CNRS Directrice de Recherche (equivalent to professor) at the Institut Curie in Paris where she is the leader of the group 'Membranes and cellular functions'. She started her carrier in Soft Matter in Montpellier (GDPC)  and spent one year as a visiting scientist at the IBM Almaden Center (San Jose, USA). She moved to the Institut Curie in 1993 to work on questions related to 'Physics of the cell'. She develops a multidisciplinary approach, largely based on synthetic biology and on the development and study of biomimetic systems, as well as quantitative mechanical and microscopy methods to understand the role of biological membranes in cellular functions. Additionnally, she studies the mechanics of single filopodia in living cells, and in vitro the generation of cell protrusions.

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.

Professor Jan Van Hest, Technische Universiteit Eindhoven, The Netherlands

Professor Jan Van Hest, Technische Universiteit Eindhoven, The Netherlands

Jan van Hest obtained his PhD from Eindhoven University of Technology in 1996 with prof. E.W. Meijer. He subsequently worked as a postdoc with prof D.A. Tirrell at the University of Massachusetts. In 1997 he joined the chemical company DSM in the Netherlands. In 2000 he was appointed full professor in Bio-organic chemistry at Radboud University Nijmegen. As of September 2016 he holds the chair of Bio-organic Chemistry at Eindhoven University of Technology. He has won a number of awards, amongst which a Dutch Science Foundation VICI grant (2010) and an ERC Advanced grant (2016).  In 2012 he was one of the main grantees of the 27 M€ Gravitation Program on functional molecular systems (FMS). He currently coordinates the Horizon 2020 ITN Network Nanomed, dedicated to developing smart delivery carriers in medicine. The group’s focus is to develop well-defined compartments for nanomedicine and artificial cell research.

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.

Dr Sarah Staniland, University of Sheffield, UK

Dr Sarah Staniland, University of Sheffield, UK

Sarah S Staniland obtained her undergraduate degree from the University of Edinburgh (MChem 2001) and stayed in chemistry there to complete a PhD in magnetic materials under the supervision of Profs. Andrew Harrison and Neil Robertson (2005). After her PhD she won a prestigious independent EPSRC life science interface fellowship (2005-2008) at the University of Edinburgh where she initiated the research she is currently active in. This helped her transition from the chemical material sciences to interdisciplinary work at the interface with biology. She took this opportunity to live and work in various places globally, from Cape Town to Tokyo, forming lasting collaboration with key leaders in their field. She moved to the University of Leeds to take up a Lectureship in Bionanoscience in the School of Physics and Astronomy where she was promoted to Associate Professor (2008-2013). She has now moved to Sheffield as a Senior Lecturer in Bionanoscience recently promoted to Reader (2016).

Her research interests centres around the biomimetic synthesis of nanomaterials, particularly nanomagnetic particles and how they are produced within magnetic bacteria. 

From a basis of material chemistry and PhD in magnetic materials, Sarah has moved into a multidiscipline approach of using biology to control material synthesis. Key to this is mimicing the biomineralisaiton in magnetosomes to form precise mangetic nanoparticles within vesicles.

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.

Professor Christine Keating, Penn State University, USA

Professor Christine Keating, Penn State University, USA

Christine D Keating received her BS in Biology and Chemistry from St Francis College (Loretto, PA) in 1991 and her PhD in Chemistry from Pennsylvania State University in 1997.  She is currently a Professor of Chemistry at Pennsylvania State University and is affiliated with the Materials Research Institute and the Huck Institute for Life Sciences. She has been honored as a Sloan Research Fellow, Beckman Young Investigator, Camille Dreyfus Teacher-Scholar, and recipient of an NSF CAREER award. Dr Keating was named the Unilever Outstanding Young Investigator in Colloid and Surfactant Science (2004), the Bodil Schmidt Nielsen Fellow & Bioengineer in Residence at Mount Desert Island Biological Laboratory in 2010, and a Fellow of the American Association for the Advancement of Science in 2014. Dr Keating is author or coauthor of over 100 scientific articles. Her research interests combine materials science, colloid chemistry, and cell biology.

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.

Professor Dek Woolfson, University of Bristol, UK

Professor Dek Woolfson, University of Bristol, UK

Professor Dek Woolfson took his first degree in Chemistry at the University of Oxford, UK.  He then did a PhD at the University of Cambridge followed by post-doctoral research at University College London and the University of California, Berkeley. After 10 years as Lecturer through to Professor of Biochemistry at the University of Sussex, he moved to the University of Bristol in 2005 to take up a joint chair in Chemistry and Biochemistry.

Dek’s research has always been at the interface between chemistry and biology, applying chemical methods and principles to understand biological phenomena.  Specifically, his group is interested in the challenge of rational protein design, and how this can be applied in synthetic biology and biotechnology. His particular emphasis is on making completely new protein structures and biomaterials for applications in cell biology and medicine.

In 2011, Dek became the first recipient of the Medimmune Protein and Peptide Science Award of the Royal Society of Chemistry; and in 2014 he received a Royal Society Wolfson Research Merit Award. Dek is Director of the Bristol BioDesign Institute and Co-Director of BrisSynBio, a £13.6M BBSRC/EPSRC-funded Synthetic Biology Research Centre.