SynCell2021
Virtual Poster Session
Welcome to the content page for SynCell2021's virtual poster session. Here you will find presenter's posters and pre-recorded presentations to view from May 11-20 2021.
Vote for your favourite presentation by clicking the star ⭐️. The crowd's-choice presenter will be featured during our live event in a lightning ⚡️ round alongside the winner's of the poster competition.
Conference participants can interact with the poster presenters during our live event on 18 May 2021 during 2, 1-hour long sessions and on 19 May 2021 during one session. Links for the poster sessions to come. You can register for the conference webinar on our main website.
More info: https://syncell2021.unm.edu
Filter displayed posters (74 keywords)
droplet-based microfluidics (2)
vesicle (2)
show more...
ATP synthesis (1)
Artificial membrane (1)
Artificial photosynthesis (1)
Atomic force Microscopy (1)
Bilayer tension (1)
Bioapplications (1)
Biomimicry (1)
Communication (1)
DNA (1)
Elastin Like Polypeptides (1)
Emulsion Phase Transfer (1)
Integrases (1)
Intrinsically Disordered Proteins (1)
JCVI-syn3A (1)
Label-free imaging (1)
Light-activated DNA (1)
Lipid Membranes (1)
Lipid Sponge Droplets (1)
Liquid-Liquid Phase Separation (1)
Mechanical Properties (1)
Mechanosensitive Channel (1)
Membraneless Organelles (1)
Microfludic (1)
MscL (1)
Multicompartment Vesicle (1)
Multifunctions (1)
Nucleic acid-protein interactions (1)
Quorum sensing (1)
RC (1)
Red blood cells (1)
Redox cofactors (1)
Regulatory Circuits (1)
SNARE-mediated fusion (1)
Stimuli-responsive (1)
Synthetic Organelles (1)
Synthetic cells (1)
artificial cell (1)
artificial membrane (1)
bc1 (1)
bilayer (1)
biomimetic (1)
bottom-up approach (1)
charge-mediated fusion (1)
chemical biology (1)
chemical oscillations (1)
chemoenzymatic synthesis (1)
dSTORM (1)
drop impact (1)
early Earth (1)
emergence (1)
extracellular matrix (ECM) (1)
high throughput screening (1)
hybrid protein coupling (1)
hybrid vesicles (1)
hydrodynamics (1)
hydrophobin (1)
light energy transduction (1)
membrane mixing (1)
membrane scaffold (1)
metabolic pathways (1)
natural products (1)
phospholipid (1)
photo-redox cycle (1)
protein capsules (1)
proteins (1)
protocells (1)
self-assembly (1)
sorting (1)
surface tension (1)
synthetic biology (1)
tetraspanins (1)
tissue engineering (1)
Show Posters:
Tracks
▼ 01. Syncells Back to top
Insights on the effect of cholesterol and sphingomyelin on tension and elasticity of plasma-like freestanding model membranes from natural lipids
Alessandra Griffo*, Carola Sparn, Friederike Nolle, Jean-Baptiste-Fleury, Ralf Seeman, Walter Nickel, Hendrik Hähl*
Abstract
The creation of model lipid membranes as free-standing platforms to study the processes occurring across the cellular membrane has been so far a field of remarkable interest. Such design in fact, allowed studies without drastically modify the physico-chemical properties and the dynamic nature of lipid bilayers. In the present study we develop unsupported membranes either for microfluidic technology than for atomic force microscopy and spectroscopy investigation and propose them as novel models for systematic studies. Our model membranes reflect the composition of the inner leaflet of plasma membrane and are enriched in sphingomyelin. Such composition in fact, revealed to be attractive to investigate the binding and insertion mechanisms of certain peripheral proteins as the Fibroblast Growth Factor 2 (FGF2), crucially involved in tumor angiogenesis processes. In addition, for a more realistic comparison with in vivo studies we chose lipids from extracts, that are more highly polydisperse. The described lipid bilayers are hence characterized mechanically and insights on the tension and the elasticity are explored.
Presented by
Alessandra Griffo
Institution
Saarland University, Saarbrucken, Germany
Other Affiliations
Heidelberg University Biochemistry Center, Heidelberg, Germany
Keywords
Lipid Membranes, Mechanical Properties, Bilayer tension, Microfludic, Atomic force Microscopy
Light-activated gene expression in synthetic cells
Jefferson M. Smith*, Denis Hartmann, Michael J. Booth
Abstract
Liposomes containing an in vitro transcription-translation system represent minimal synthetic cells that can perform specialised functions according to an encapsulated DNA template. Hence, regulation at the DNA-level enables control over synthetic cell activities. Protein expression inside the liposomes has previously been regulated using small-molecule sensitive transcription factors and riboswitches, however, these rely on the diffusion of small molecules across the lipid bilayer and are often leaky. Here, we utilise chemically modified, light activatable-DNA templates to provide tighter regulation over synthetic cell activities and activate these activities in a spatiotemporal manner using patterned light.
Light-activated DNA is constructed by introducing 7 amino C6 thymine bases that are conjugated to photocleavable biotinylated linkers into the T7 promoter of a linear DNA template. Monovalent streptavidin binds at each biotin and sterically hinders transcription of the downstream gene by RNA polymerase in the absence of light. However, after UV irradiation, the photocleavable linker-streptavidin complex is liberated and transcription/translation can proceed. Using this approach, we have demonstrated that protein expression inside giant unilamellar vesicles can be activated using light as an external stimulus and using we can control this spatiotemporally using pattern illumination. Using this platform, we established a means to mediate synthetic cell-bacteria cell communication through the controlled in-situ synthesis of acyl homoserine lactones involved in quorum sensing.
Light-activated DNA is constructed by introducing 7 amino C6 thymine bases that are conjugated to photocleavable biotinylated linkers into the T7 promoter of a linear DNA template. Monovalent streptavidin binds at each biotin and sterically hinders transcription of the downstream gene by RNA polymerase in the absence of light. However, after UV irradiation, the photocleavable linker-streptavidin complex is liberated and transcription/translation can proceed. Using this approach, we have demonstrated that protein expression inside giant unilamellar vesicles can be activated using light as an external stimulus and using we can control this spatiotemporally using pattern illumination. Using this platform, we established a means to mediate synthetic cell-bacteria cell communication through the controlled in-situ synthesis of acyl homoserine lactones involved in quorum sensing.
Presented by
Jefferson Smith
Institution
University of Oxford, Department of Chemistry
Keywords
Light-activated DNA, Synthetic cells, Quorum sensing, Communication
Biomimetic rebuilding of multifunctional red blood cells
Jimin Guo
Abstract
The design and synthesis of artificial materials that mimic the structures, mechanical properties, and ultimately functionalities of biological cells remains a current holy grail of materials science and has the potential to transform a spectrum of biotechnologies. Here we report the design and construction of synthetic rebuilt red blood cells (RRBCs) that fully mimic the broad properties of native RBCs: size, biconcave shape, deformability, oxygen carrying capacity, long circulation time etc. Three successive processing steps: RBC bioreplication, layer-by-layer polymer deposition, and RBC ghost membrane fusion are employed for RRBC construction. A panel of physicochemical analyses including zeta potential measurement, fluorescence microscopy, and antibody-mediated agglutination assay, etc. proved the precise recapitulation of RBC shape, size, and membrane properties. Flow-based deformation studies carried out in a microfluidic blood capillary model confirmed the ability of rebuilt RBCs to deform and pass through small slits and reconstitute themselves in a manner comparable to native RBCs. Moreover, circulation studies of RRBCs conducted ex-ovo in a chick embryo and in vivo in a mouse model demonstrated the requirement of both deformability and a native cell membrane surface to achieve long-term circulation. To confer additional non-native functionalities to RRBCs, we developed modular procedures in which to load functional cargos such as hemoglobin, drugs, and magnetic nanoparticles within the RRBC interior to enable various functions, including oxygen delivery, MRI contrast imaging, therapeutic drug delivery, and magnetic manipulation. Taken together, RRBCs represent a new class of long circulating RBC-inspired artificial hybrid materials with a broad range of potential applications.
Presented by
Jimin Guo
Institution
Department of Chemical and Biological Engineering, the University of New Mexico
Other Affiliations
Center for Micro-Engineered Materials, the University of New Mexico
Keywords
Red blood cells, Biomimicry, Multifunctions, Bioapplications
A minimal pathway for the regeneration of redox cofactors
Michele Partipilo, Eleanor J. Ewins, Jacopo Frallicciardi, Tom Robinson, Bert Poolman & Dirk Jan Slotboom
Abstract
Metabolism is an intricate network of biochemical pathways that make a cell out-of-equilibrium, but only a limited group of fundamental molecules carry out this specific function. Among these are nicotinamide adenine dinucleotides NAD(H) and NADP(H), involved mostly in the transfer of reducing equivalents between reactants. An ambitious challenge of building a synthetic cell via bottom-up methodologies requires a metabolic pathway, thus ensuring the constant availability of nicotinamide cofactors. Here, we constructed a synthetic pathway confinable in the lumen of liposomes, in which the redox status of the cofactors NADH and NADPH is controlled by externally provided formate. The formate permeates the membrane where a luminal formate dehydrogenase uses NAD+ to form carbon dioxide and NADH. A soluble transhydrogenase subsequently utilizes NADH for reduction of NADP+ thereby making NAD+ available again for the first reaction. Consequently, this minimal system leads to production inside a cell-like system of the two central hub electron carriers NADH and NADPH, which are required for redox reactions leading to metabolic complexity. We further investigated our coupled redox system across different compartment scales, using large and giant unilamellar vesicles (LUVs and GUVs).
Presented by
Michele Partipilo
Institution
University of Groningen, Groningen Institute of Biomolecular Sciences & Biotechnology, Membrane Enzymology Group
Other Affiliations
Max Planck Institute of Colloids and Interfaces of Potsdam, Department of Theory & Bio-Systems
Keywords
Redox cofactors, metabolic pathways, bottom-up approach
Single compartment approach for photo-autotrophic protocell preparation
Altamura E., Albanese P., Milano F., Giotta L., Trotta M., Ferretta A., Cocco T., Mavelli F.
Abstract
The construction from scratch of artificial cells by means of a “bottom up” approach is one of the most ambitious challenges in synthetic biology. Artificial cells capable of imitating the light phase of photosynthesis can be considered photoautotrophs. In bacterial photosynthesis, the first step in the light energy transduction process is the enzymatic photo-redox cycle catalysed by two membrane protein complexes, that is, the photosynthetic reaction centre (RC) and the ubiquinol oxidase (bc1B). In this work we studied this process in a micellar suspension of both proteins but coupling a bacterial RC with an ortholog bc1 extracted from mammalian mitochondria (bc1M). With this hybrid protein complex chain, the light transduction efficiency turns out to be enhanced up to 90% by tuning the enzymatic level ratio of the two protein complexes. These results pave the way towards the reconstitution of the entire photosynthetic machinery in artificial membranes for the realization of photoautotrophic artificial cells.
Presented by
Paola Albanese
Institution
University of Bari Aldo Moro, Chemistry Department
Keywords
Artificial photosynthesis, hybrid protein coupling, chemical oscillations, light energy transduction, photo-redox cycle, RC, bc1
Influence of Breast Cancer Lipid Changes on Membrane Oxygen Permeability
Qi Wang, Rachel J. Dotson, Gary Angles, Sally C. Pias
Abstract
Hypoxia is a typical condition for cancer cells. Breast cancer cells have been observed to have high levels of lipid tails that are generated through de novo biosynthesis. These lipid tails, lauroyl (L 12:0), myristoyl (M 14:0) and stearoyl (S 18:0), are four carbons and two carbons shorter, and two carbons longer, respectively, than the predominant palmitoyl (P 16:0) lipid tail. Another lipid tail, palmitoleoyl (Y 16:1), is two carbons shorter than the predominant oleoyl (O 18:1) lipid tail. The combination of P and O lipid tails with phosphatidylcholine (PC) headgroup is the most common phospholipid in normal breast cells. We hypothesized that lipid bilayers rich in de novo lipids would change oxygen permeability, due to the physical effects of the altered chain lengths. Atomic resolution molecular dynamics simulations were used to investigate this chain length effect. From the simulations, we have estimated the permeability of PYPC (16:0/16:1) to be 9.7 ± 0.5 cm/s at 37°C, compared with a higher permeability of 16.5 ± 0.6 cm/s for the “normal” phospholipid POPC (16:0/18:1). We have estimated the permeability of SOPC (18:0/18:1) also to be lower than POPC, at 10.8 ± 0.4 cm/s. However, SYPC (18:0/16:1) and LYPC (12:0/16:1) are much closer to POPC with permeabilities of 15.6 ± 0.9 cm/s and 17.4 ± 0.3 cm/s, respectively. The estimated permeabilities for MYPC (14:0/16:1), LOPC (12:0/18:1), and MOPC (14:0/18:1) are 18.3 ± 1.3 cm/s, 19.2 ± 0.8 cm/s, and 20.6 ± 2.4 cm/s and are greater than POPC. Lipid chain-length mismatch and lateral packing density may contribute to these observed changes in permeability. Investigation of chain-length effects in more complex membrane models will be required to understand the influence of de novo lipids on cellular oxygenation in pathological states.
Presented by
Qi Wang
Institution
New Mexico Institute of Mining and Technology
Keywords
Chemoselective generation of dynamic synthetic cells
Roberto J. Brea, Neal K. Devaraj
Abstract
The assembly of synthetic membranes provides a novel strategy to reconstruct life's functions and shed light on how life emerged on Earth. A significant challenge to the construction of artificial membranes is the development of simple methodologies to mimic native membrane generation. Here we describe chemoselective approaches that enable the precise fabrication of biomimetic membranes with dynamic properties, which could be used for building synthetic cells with lifelike properties, including the ability to undergo evolution, growing, or division. Additionally, the biocompatibility of such methodologies makes them a powerful tool for the efficient encapsulation of relevant biomolecules. These studies offer a deeper understanding of the nature of living systems, bring new insights into the origin of cellular life, and afford novel synthetic chassis for advancing synthetic biology.
Presented by
Roberto J. Brea
<rbreafernandez@ucsd.edu>
Institution
University of California, San Diego
Keywords
artificial cell, biomimetic, phospholipid, self-assembly, vesicle
▼ 02. Synthetic Genomes Back to top
Genetic circuits based on serine-integrases as regulatory networks in the minimal cell Mycoplasma mycoides JCVI-Syn3A
Marco A. de OLIVEIRA¹,², Daniela BITTENCOURT², Yo SUZUKI³, John I. GLASS³, Elibio L. RECH²
Abstract
Gene expression regulation often relies on multi-element, complex circuits, so a simplified system able to control clusters of genes simultaneously will be an important component in a synthetic cell. We propose that serine-integrases acting on defined groups of genes can work as such systems. We evaluated the use of two serine-integrases (INT9 and INT13) as genetic switches capable of turning the expression of a reporter gene on by flipping its DNA sequence upon induction in the synthetic minimal cell Mycoplasma mycoides JCVI-Syn3A. Cells were transformed with a plasmid containing one integrase gene under control of a Tetracycline inducible promoter as well as the mCherry gene in a reverse orientation relative to its promoter, therefore silenced, flanked by the att sites of that integrase. All the plasmids also had a Cre/loxP system to allow insertion on a landing pad present in the Syn-3 genome and a selection marker. After incubation with tetracycline we measured mCherry signal intensity in a microplate reader. Results show that INT9 activation led to a significant increase in fluorescence intensity and the expression of the flipped reporter gene continued after tetracycline removal. INT13 induction did not result in apparent expression of mCherry. A decrease observed on the levels of mCherry signal in the positive controls also indicates that the integrases are better used when transiently expressed. The next step will be the insertion of att sites flanking multiple endogenous genes of Syn-3A. The results presented give us directions for the assembly of a synthetic regulatory network.
Presented by
Marco de Oliveira
<mrc.toni@gmail.com>
Institution
1University of Brasília, Department of Cell Biology, Brasilia, DF, Brazil
Other Affiliations
2Laboratory of Synthetic Biology, EMBRAPA Genetic Resources and Biotechnology, Brasilia, DF, Brazil; ³Synthetic Biology and Bioenergy Group, J. Craig Venter Institute, La Jolla, CA, United States.
Keywords
Integrases, JCVI-syn3A, Regulatory Circuits
▼ 03. Synthetic Organelles Back to top
Lipid Sponge Droplets as Programmable Synthetic Organelles
Ahanjit Bhattacharya, Neal K. Devaraj
Abstract
Cells and organelles spatially and temporally orchestrate biochemical reactions to a degree of precision that is still unattainable in synthetic reaction systems. Additionally, organelles such as the endoplasmic -
reticulum (ER) contain highly interconnected and dense membrane networks that provide large reaction spaces for both transmembrane and soluble enzymes. Here we present lipid sponge droplets to emulate functions of organelles such as the ER. We demonstrate that lipid sponge droplets can be programmed to internally concentrate specific proteins, to host and accelerate biochemical transformations, and to rapidly and reversibly sequester and release proteins to control enzymatic reactions. The self-assembled and programmable nature of lipid sponge droplets will facilitate the integration of complex functions for bottom-up synthetic biology.
Presented by
Ahanjit Bhattacharya
Institution
University of California San Diego
Other Affiliations
Stanford University
Keywords
Synthetic Organelles, Lipid Sponge Droplets
Chemoenzymatic Generation of Phospholipid Membranes Mediated by Type I Fatty Acid Synthase
Satyam Khanal, Michael D. Burkart, Neal K. Devaraj
Abstract
The de novo formation of lipid membranes from minimal reactive precursors is a major goal in synthetic cell research. In nature, the synthesis of membrane phospholipids is orchestrated by numerous enzymes, including fatty acid synthases and membrane-bound acyltransferases. However, these enzymatic pathways are difficult to fully reproduce in vitro. Here, we describe a chemoenzymatic strategy for lipid membrane generation that utilizes a soluble bacterial fatty acid synthase (cgFAS I) to synthesize palmitoyl-CoA in situ from acetyl-CoA and malonyl-CoA. The fatty acid derivative spontaneously reacts with a cysteine-modified lysophospholipid by native chemical ligation (NCL), affording a non-canonical amidophospholipid that self-assembles into micron-sized membrane-bound vesicles. Our results demonstrate that combining the specificity and efficiency of a type I fatty acid synthase with a highly selective bioconjugation reaction provides a biomimetic route for the de novo formation of membrane-bound vesicles.
Presented by
Satyam Khanal
Institution
University of California, San Diego
Keywords
Artificial membrane, chemical biology, chemoenzymatic synthesis, synthetic biology
Recombinant Intrinsically Disordered Proteins for Triggered Sequestration of Nucleic Acids via Liquid-Liquid Phase Separation
Telmo Díez Pérez, Jacqueline A. De Lora, Adam D. Quintana, Andrew P. Shreve, Gabriel P. López, Nick J. Carroll
Abstract
The mechanisms underlying the formation of condensed phase cellular bodies that contribute to the regulation of information flow within the cellular cytoplasm, such as membrane-less organelles (MLO) with different nucleic acid compositions, are gaining attention from experimentalists and theorists alike. To better understand the dynamics fundamental parameters that drive of liquid-liquid phase separation (LLPS) that govern and cause the formation of such condensed phase cellular bodies such as ribonucleoprotein (RNP) membraneless organelles (MLO), we investigate a model intrinsically disordered polypeptide (IDP), and its electrostatic binding to single-stranded DNA (ssDNA). We demonstrate controllable DNA capture within elastin-like polypeptide (ELP) coacervate bodiescondensates and as well as characterize the influence of DNA and salt on ELP LLPS behavior. Using charge shielding NaCl counterions, we validate the mechanism driving E3-DNA complexation to be electrostatic interaction and show that a modified Flory-Huggins (FH) model theoretical treatment can describe the totality of our experimental observations. In particular, we modulate ELP coacervate-DNA binding behavior in bulk and within droplet microenvironments to characterize ssDNA capture by E3 condensates as a function of NaCl., and We then introduce a modified FH theory to construct phase diagrams using the modified FH theory that describe molecular component partitioning, specifically the (e.g. degree of DNA capture,) within discrete coacervate protein and water rich phases. Finally, we present the first application of this type class of minimal synthetic model systems by demonstrating a two-step DNA purification assay. Taken together, this simple ELP/DNA solution platform represents an important contribution to synthetic biology and DNA technologies and even haswith further implications into origin of life discussions.
Presented by
Telmo Diez Perez
Institution
University of New Mexico
Other Affiliations
Center for Biomedical Engineering, University of New Mexico, Albuquerque, NM 87131, USA, / Center for Micro-Engineered Materials, University of New Mexico, Albuquerque, NM 87131, USA, / Department of Chemical and Biological Engineering, University of New Mexico, Albuquerque, NM 87131, USA, and / Max Planck Institute for Medical Research, Department of Cellular Biophysics, 70569 Stuttgart, Germany
Keywords
Membraneless Organelles, Elastin Like Polypeptides, Intrinsically Disordered Proteins, Liquid-Liquid Phase Separation, Nucleic acid-protein interactions, DNA
▼ 04. Compartmentalization Back to top
Pure protein bilayers and vesicles made from fungal hydrophobins: an alternative platform for synthetic cells
Hendrik Hähl, Friederike Nolle, Alessandra Griffo, Kirstin Kochems, Päivi Laaksonen, Michael Lienemann, Ralf Seemann, Jean-Baptiste Fleury, and Karin Jacobs
Abstract
Hydrophobins are small proteins whose unique feature is a pronounced amphiphilicity rendering them naturally occurring Janus particles. This feature allows them to cover rapidly any type of interface. One class of hydrophobins produced by filamentous fungi, as e.g. the protein HFBI from Trichoderma reesei, builds interfacial monolayers that exhibit a highly ordered two-dimensional structure as well as very high lateral cohesion. Contacting two of these hydrophobin monolayers, e.g. as droplet-interface-bilayers or in a microfluidic setup, results in a stable pure-protein bilayer [1]. Compared to usual lipid bilayers, the hydrophobin bilayers feature a similar thickness but can sustain much higher lateral tension and exhibit a water permeability, which is nearly two orders of magnitudes lower. Moreover, the rather simple ion channel gramicidin A can be inserted into these bilayers and functions there as in a lipid environment [2]. The monolayer can, however, also be contacted with the hydrophilic sides and thus form a membrane between oily compartments. From both types of bilayers, i.e. between either aqueous or oily phases, vesicles can be formed, e.g. by the microfluidic jetting technique. These lipid-free pure protein vesicles may serve as an alternative platform for synthetic cells offering a different matrix for the study of membrane proteins.
[1] Hähl, H. et al., Langmuir 34, 8542 (2018). [2] Hähl, H. et al., Adv Mater 29, 1602888 (2017).
[1] Hähl, H. et al., Langmuir 34, 8542 (2018). [2] Hähl, H. et al., Adv Mater 29, 1602888 (2017).
Presented by
Hendrik Hähl
Institution
Department of Experimental Physics and Center for Biophysics, Saarland University, 66123 Saarbrücken, Germany
Other Affiliations
VTT Technical Research Centre of Finland Ltd., Espoo 02150, Finland; HAMK Tech, Häme University of Applied Sciences, Hämeenlinna 13101, Finland
Keywords
artificial membrane, hydrophobin, proteins, bilayer, vesicle
Charge vs. SNARE-mediated fusion of biomimetic polymer/lipid hybrid compartments: Which one is more efficient?
Nika Marušič1, Lado Otrin1, Ziliang Zhao2, Fotis L. Kyrilis3, Farzad Hamdi3, Panagiotis L. Kastritis3, Rumiana Dimova2, Tanja Vidaković-Koch1, Ivan Ivanov1, Kai Sundmacher1
Abstract
Mechanical characteristics (e.g. membrane rigidity, tightness) of cellular compartments are alterable by partial or complete replacement of natural building blocks with synthetic alternatives, but often at the expense of other properties (e.g. fluidity, thickness). Since the scope of bottom-up synthetic biology aims to reconstitute essential life processes such as selective transport and energy transduction, we seek to retain properties of the interfaces, necessary for interaction with membrane proteins. PDMS-g-PEO polymer appears to accommodate these demands. Furthermore, the said polymer increased functional and chemical stability of our artificial organelles.
Fusion plays a crucial role in various cell functions, such as exo- and endocytosis, membrane remodeling, signal transduction, intracellular trafficking, and others, which we also aim to mimic in the context of bottom-up synthetic biology. Here, we studied protein-free and protein-mediated fusion of PDMS-g-PEO/lipid hybrid compartments. For protein-free fusion we prepared hybrid vesicles, which contained either anionic or cationic lipids, and fused them based on the opposite membrane charge. Meanwhile, to achieve protein-mediated fusion, we developed reconstitution protocols for the insertion of SNARE fusogenic peptides into nano and microcompartments. In both cases, fusion efficiency was studied via membrane mixing (FRET) and content mixing, whereby the latter was monitored through coupling between two membrane proteins – proton pump bo3 oxidase and F1FO-ATPase. Remarkably, fusion efficiency of protein-free oppositely charged hybrids surpassed the one determined in liposomes (80 vs. 20 % membrane mixing, respectively), while the fusion efficiency of SNARE-functionalized hybrids matched the one measured in liposomes (~ 20 % in 60 min). Moreover, ATP synthesis upon fusion of neutral and cationic hybrids exceeded the one seen in oppositely charged liposomes, while the SNAREs-induced content mixing was similar across all systems. To obtain further insights into fusion mechanism we studied biophysical properties of fusogenic compartments, in particular vesicles surface charge, membrane disorder, bending rigidity and morphology. In this context, hybrid-membrane specific intermediates of SNARE-mediated fusion were revealed with cryo-EM. In future works, SNARE-mediated fusion will be explored to achieve growth of artificial cell via multiple sequential fusion events, and for the integration of various functional modules.
Presented by
Nika Marušič
Institution
1Max Planck Institute for Dynamics of Complex Technical Systems, Magdeburg, 2Max Planck Institute of Colloids and Interfaces, Potsdam, 3Martin Luther University Halle-Wittenberg, Halle/Saale
Keywords
hybrid vesicles, charge-mediated fusion, SNARE-mediated fusion, membrane mixing, ATP synthesis
Generation of Extracellular Matrix Protein-based Microcapsules for Investigating Single Cells
Sadaf Pashapour, Ilia Platzmann, Friedrich Frischknecht and Joachim P. Spatz
Abstract
Interactions of cells with the extracellular matrix (ECM) are involved in nearly every cellular response in vivo, and consequently activate multiple signaling pathways that initiate, drive and regulate nearly all motions of a cell. Therefore, cell-ECM interactions also underscore their central physiological roles, as well as their involvement in a wide variety of disease states. Consequently, engineering 3D ECM niche systems for controlled manipulation of cells in vitro has become an important strategy, particularly in medical applications, and can contribute to the understanding of the mechanisms underlying the ability of cells to perform their tasks as a response to environmental information. In this research, we have established a novel droplet-based microfluidic approach for the controlled assembly of cell-laden ECM-based protein microcapsules. Towards this end, water-in-oil emulsion droplets consisting of charged blockcopolymer surfactants are used as a template for the charge-mediated formation of a laminin- or laminin/collagen-based continuous layer on the inner droplet periphery. A double inlet microfluidic device is implemented to encapsulate cells, proteins and the appropriate ionic conditions for the controlled polymerization of the protein layer. Sequential release of the assembled cell-laden ECM based microcapsules from the polymer-stabilized droplets into a physiological environment allows for analysis of cell-ECM interactions on a single-cell level. By tailoring the biochemical properties of our system, we are able to produce a wide variety of ECM-based microcapsules that are tunable in terms of protein composition and cellular encapsulation. Ultimately, this technology can be used for a variety of applications. One possibility is the investigation of the communication of cells through a protein barrier or the usage of those cell-laden protein microcapsules for fabricating 3D tissue structures for biomedical and biophysical applications.
Presented by
Sadaf Pashapour
<sadaf.pashapour@mr.mpg.de>
Institution
Max-Planck Institute for Medical Research, Heidelberg, Germany
Other Affiliations
Heidelberg University, Germany
Keywords
extracellular matrix (ECM), protein capsules, tissue engineering, droplet-based microfluidics
Tetraspanin Scaffold Regulation of Epidermal Growth Factor Receptor Biology on the Plasma Membrane
Sebastian Restrepo Cruz, Sandeep Pallikkuth, Keith A. Lidke, Diane S. Lidke, and Jennifer M. Gillette
Abstract
Molecular interactions on the plasma membrane can significantly influence the outcomes of signaling events crucial to cell function. Tetraspanins play a vital role as scaffolding proteins on the membrane, interacting with various different proteins as well as other tetraspanins to form structural platforms termed Tetraspanin-Enriched Microdomains (TEMs). By facilitating compartmentalization, tetraspanins have been shown to regulate numerous cellular processes, including signaling and adhesion. Here, we assess the role of tetraspanin CD82 in the regulation of Epidermal Growth Factor Receptor (EGFR) biology. Genetic and biochemical studies have previously shown that different tetraspanins can directly and indirectly influence EGFR signaling and internalization. However, the mechanism by which tetraspanins modulate the spatiotemporal properties of EGFR remains unclear. Using direct stochastic optical reconstruction microscopy (dSTORM) to achieve sub-diffraction limit resolution, we mapped the relative organization of EGFR with tetraspanins CD82 on the plasma membrane of HeLa and HEK293 cells. Analysis of the distribution of these molecules reveals a positive association between EGFR and CD82, in addition to a re-organization of CD82 following EGF treatment. To further analyze the impact of CD82 on the function of EGFR, we generated cell lines overexpressing either CD82 or a CD82 palmitoylation mutant and measured EGFR phosphorylation following EGF stimulation. We find that while CD82 overexpression increases basal EGFR activation, ligand-dependent activation is significantly attenuated. Collectively, these studies suggest that the CD82 scaffold directly interacts with EGFR to regulate receptor organization and signaling. Future studies will address the contribution of tetraspanin- tetraspanin and tetraspanin-cholesterol interactions to EGFR signaling and dynamics.
Presented by
Sebastian Restrepo Cruz
Institution
University of New Mexico, Department of Pathology
Keywords
tetraspanins, membrane scaffold, dSTORM
Monitor, categorize and manipulate label-free water-in-oil droplets in microfluidic systems
Tobias Neckernuss, Christoph Frey, Jonas Pfeil, Daniel Geiger, Patricia Schwilling, Ilia Platzman, Joachim Spatz and Othmar Marti
Abstract
A key point of droplet based microfluidics is the availability of powerful but easy-to-implement methods for high throughput real-time analysis and automated manipulation of the droplets. We developed a novel optical device, consisting of a fast camera with integrated data processing for smart and fast algorithms enabling label-free real-time monitoring and active manipulation of passing droplets. The device continuously analyzes up to 3000 particles per second in real-time with respect to bright-field image parameters like size, brightness, granularity, circumference, speed and many more. According to these parameters and combinations thereof, the passing droplets can be sorted. We measure different droplet production parameters and demonstrate label-free detection of cells encapsulated in droplets. Furthermore, we performed label-free sorting of cell laden droplets from empty droplets. The peripheral sorting electronics are controlled by our device. Decision making is based on predefined parameter ranges that are compared to the measurement results of the droplets right before the sorting gate. Similarly, in another experiment we demonstrate efficient sorting of droplets depending on size.
Presented by
Tobias Neckernuss
<tobias.neckernuss@uni-ulm.de>
Institution
Institute for Experimental Physics, Ulm University, Max-Planck-Institute for Medical Research, Heidelberg, Sensific GmbH
Other Affiliations
Institute for Experimental Physics, Ulm University, Max-Planck-Institute for Medical Research, Heidelberg, Sensific GmbH
Keywords
Label-free imaging, sorting, droplet-based microfluidics
▼ 05. Astrobiology Back to top
Hydrodynamically-active oily ocean surface as a cradle for the emergence of life
Franky Djutanta, Rachael Kha, Bernard Yurke, and Rizal F. Hariadi
Abstract
Geological settings that provide the energy sources for the synthesis of building blocks of life, concentrating information-carrying molecules, and driving a simple form of self-replication and Darwinian evolution, are candidate locals where life may have arisen. Hydrothermal vents, closed basins, and tide pools have been found to support the synthesis of organic molecules and polymers, but these products are subjected to loss either by burial through sedimentation or by dilution through hydrodynamic transport. These processes hinder organic compound accumulation to densities sufficient for the initiation of life. Here, we argue for and provide experimental support for the hypothesis that a hydrodynamically-active prebiotic oily ocean surface may have been the setting for the origin of life by fostering the birth, growth, self-replication, and death of oil droplets. The oil is hypothesized to consist of organic compounds produced from the transformation of buried micrometeorite kerogen into prebiotic petroleum through the maturation process beneath the seafloor, much like the formation of petroleum of biological origins today. Due to their low density and fluidity, these hydrocarbons seep upwards through rock pores and fractures to form oil slicks on the ocean surface. Ocean gyres provide confinement for the accumulation of this oil. Hydrodynamic forces, generated by falling rain or breaking waves, acting on these slicks, generate oil droplets whose surface can act as a substrate to which the components of a protobiont adhere to avoid loss by dilution in the ocean. By suppressing fusion through surfactant coatings and by growth via the scavenging of organic compounds from seawater, such droplets could have served as a platform on which molecular machinery evolved, driven by natural selection acting on the droplet composition. A surprising finding is that raindrop impact can induce the encapsulation of seawater within an oil droplet, suggesting a mechanism for the production of organelle-like structures. A key feature of the hypothesis is that hydrodynamic forces provide a mechanism to drive a primitive form of replication by fragmenting a parent oil droplet into daughter oil droplets. By taking a global geophysical perspective on where organic compounds would have accumulated to the greatest concentration in the prebiotic world, we have identified an active environment, the ocean surface, where life may have arisen. We have offered an origin of life hypothesis for which key elements are demonstrable in the laboratory or for which modern geophysical analogs exist.
Presented by
Franky Djutanta
<fdjutant@asu.edu>
Institution
Arizona State University & Boise State University
Keywords
emergence, early Earth, protocells, hydrodynamics, surface tension, drop impact
▼ 06. Synthetic Signalling Back to top
Building mechanosensitive signalling pathways in synthetic cells using membrane engineering
James W. Hindley, Yuval Elani, Robert V. Law, Oscar Ces
Abstract
The mechanosensitive channel of large conductance (MscL) is a large, non-specific protein channel (MWCO ~ 10 kDa) that can be gated by multiple stimuli, including osmotic stress, small molecule binding and membrane compositional changes. This work utilises different membrane engineering methods to produce single- and multi-compartment vesicles which utilise MscL in different contexts. This enables a) the construction of a synthetic signalling pathway through controlled activation of phospholipase (sPLA2) - Membrane - MscL interactions in a vesicle-based synthetic cell and b) the construction of mechanosensitive giant vesicles that can be gated by the binding of small molecules to the channel lumen, enabling triggered activation of the vesicle. Such environment-responsive functionalities will be critical in the future translation of synthetic cells from the lab bench to the clinic in biomedical applications such as diagnostics and drug delivery.
Presented by
James Hindley
<j.hindley14@imperial.ac.uk>
Institution
Imperial College London
Keywords
Mechanosensitive Channel, MscL, Multicompartment Vesicle, Emulsion Phase Transfer, Stimuli-responsive
▼ 07. Synthetic Proteins Back to top
Engineering High Throughput Biosynthesis of Natural Product-Like Cyclic Peptides
Jillian Stafford and Mark C. Walker
Abstract
Chemicals produced by living systems, or natural products, are an indispensable tools in a wide variety of fields ranging from medicine to materials science. Although tens of thousands of natural products have been characterized to date, genome sequencing efforts over the past two decades have revealed that the known set of natural products are produced by a small fraction of the biosynthetic pathways that exist in nature. This observation highlights that much of natural product “chemical space” remains to be explored for new compounds. This project will focus on leveraging the biosynthetic machinery bacteria use to produce the ribosomally synthesized and post-translationally modified peptide (RiPP) class of cyclic peptide natural products to develop a yeast surface display platform for the production and screening of millions of natural product-like compounds. We are developing an assay for characterizing the substrate tolerance of these enzymes will be developed. With this method, enzymes with the capacity to cyclize many diverse peptides will be identified and design principles for cyclic peptide libraries revealed.
Presented by
Mark Walker
Institution
University of New Mexico, Department of Chemistry and Chemical Biology
Keywords
natural products, high throughput screening