DOCSLIB.ORG

High-Throughput Synthesis and Screening of Functional Coacervates Using Microfluidics Thomas Beneyton, Celina Love, Mathias Girault, T.-Y Tang, Jean-Christophe Baret

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
High-Throughput Synthesis and Screening of Functional Coacervates Using Microfluidics Thomas Beneyton, Celina Love, Mathias Girault, T.-Y Tang, Jean-Christophe Baret High-Throughput Synthesis and Screening of Functional Coacervates Using Microfluidics Thomas Beneyton, Celina Love, Mathias Girault, T.-Y Tang, Jean-Christophe Baret To cite this version: Thomas Beneyton, Celina Love, Mathias Girault, T.-Y Tang, Jean-Christophe Baret. High- Throughput Synthesis and Screening of Functional Coacervates Using Microfluidics. ChemSystem- sChem, Wiley, 2020, 10.1002/syst.202000022. hal-02870965 HAL Id: hal-02870965 https://hal.archives-ouvertes.fr/hal-02870965 Submitted on 17 Jun 2020 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Articles ChemSystemsChem doi.org/10.1002/syst.202000022 1 2 3 High-Throughput Synthesis and Screening of Functional 4 5 Coacervates Using Microfluidics 6 + [a] + [b, d] [a] [b, d] 7 Thomas Beneyton , Celina Love , Mathias Girault, T.-Y. Dora Tang,* and [a, c] 8 Jean-Christophe Baret* 9 10 11 To understand how membrane-free subcompartmentalization reaction rates of individual subcompartments and their sur- 12 can modulate biochemical reactions by coupled spatial enzyme rounding aqueous environment at the single coacervate level. 13 localization with substrate and product partitioning, we use Our results show that sub-compartmentalisation increases the 14 microfluidic strategies to synthesize, stabilize and characterize overall rates of reactions. This bottom-up synthetic strategy for 15 micron-sized functional coacervates in waterÀ oil emulsions. Our the production of synthetic organelles offers a physical model 16 methodologies have allowed for the first time to quantitatively for membrane-free compartmentalization in biology and pro- 17 characterize partition coefficients of a broad range of different vides insights into the role of sub-compartmentalisation in 18 molecules with different coacervate chemistries and to measure regulating out-of-equilibrium behaviours in biological systems. 19 20 1. Introduction tion is based on membrane bound compartments such as the 21 mitochondria, nucleus and Golgi apparatus or on membrane- 22 Compartmentalization is an essential feature of life, where free droplets such as stress granules or P-granules which are 23 hierarchical assembly of sub compartments provides a way to collectively known as condensates. The latter are believed to 24 spatially organize biology. Cellular micro and nano-compart- form via a liquid-liquid phase separation process of intrinsically 25 ments allows distinct chemical environments to coexist and disordered proteins (IDPs), RNA and structured proteins[10,11] and 26 support multi-step reactions. In addition, compartmentalization are hypothesized to regulate biological function by regulating 27 supports the generation and sustenance of chemical gradients the sequestration and therefore the local concentration of 28 essential to maintain out-of-equilibrium processes in cells.[1] molecules. Whilst these condensates have been hypothesized 29 Oparin hypothesized that compartmentalization would have to have potential implications in disease, gene expression 30 been crucial at the onset of life to enable the organization of regulation[12] and signaling,[13,14] how these membrane free 31 chemical and biochemical reactions by providing chemical droplets may alter out-of equilibrium behaviours is still not well 32 centers in which selected molecules were concentrated for understood. It can often be difficult to characterize the 33 prebiotic chemistry and/or biology.[2–9] In modern biology it is sequestration of different molecules into biological condensates 34 well known that compartmentalization occurs across length in vivo due to the molecular complexity and in vitro due to the 35 scales, from the sub-cellular level to the macroscopic assembly difficulty in expressing and purifying condensate forming 36 of compartments in multicellular organisms. Compartmentaliza- proteins. 37 Coacervate microdroplets formed through liquid-liquid 38 + phase separation between oppositely charged polyelectrolytes 39 [a] Dr. T. Beneyton, Dr. M. Girault, Prof. J.-C. Baret Univ. Bordeaux, CNRS and small molecules offer an alternative synthetic model to 40 CRPP, UMR 5031 biological condensates as studies have shown that the mecha- 41 115 avenue Albert Schweitzer nism of biological condensate formation is partly driven by 42 33600 Pessac (France) E-mail: [email protected] coacervation.[15,16] In addition, these chemically enriched mem- 43 + [b] C. Love, Dr. T.-Y. D. Tang brane-free droplets with a highly charged and crowded 44 Max Planck Institute for Molecular Cell Biology and Genetics interior[17,18] passively upconcentrate molecules and have been 45 Pfotenhauerstrasse 108 01307 Dresden (Germany) shown to support enzymatic reactions where alterations in 46 E-mail: [email protected] diffusion length scales or in secondary structure of enzymes 47 [c] Prof. J.-C. Baret affects the rates of reactions[19–24] thereby modulating biochem- 48 Institut Universitaire de France 75005 Paris France [d] C. Love,+ Dr. T.-Y. D. Tang ical processes. Artificial systems formed by coacervation will 49 Cluster of Excellence Physics of Life therefore be interesting models to test hypothesis on the 50 TU Dresden, 01062 Dresden (Germany) dynamic properties of membrane-free organelles and offer new 51 [+] These authors contributed equally to this work routes to generate controlled mimics of those. 52 Supporting information for this article is available on the WWW under Moreover, coacervates represent interesting materials[25] and 53 https://doi.org/10.1002/syst.202000022 © 2020 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA. This are promising tools in bottom-up synthetic biology, where one 54 is an open access article under the terms of the Creative Commons Attri- of its aims is to construct artificial living systems from 55 bution Non-Commercial License, which permits use, distribution and re- elementary ingredients and modules.[26] Despite their advan- 56 production in any medium, provided the original work is properly cited and is not used for commercial purposes. tages for a range of applications the assembly of functional 57 ChemSystemsChem 2020, 2, e2000022 (1 of 9) © 2020 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA Wiley VCH Dienstag, 26.05.2020 2099 / 164441 [S. 1/9] 1 Articles ChemSystemsChem doi.org/10.1002/syst.202000022 coacervate-based compartments is hindered by the intrinsic underlying mechanism of the transport process. We provide 1 transient nature of the structures and the difficulty in handling quantitative measurements with statistics over large popula- 2 and characterizing known concentrations of molecules within tions of coacervates. Our quantitative approach demonstrates 3 the coacervates at the micron scale. Recently, the control of for the first time that transport in coacervates at the micron 4 coacervate formation was achieved in micron-sized compart- scale is driven by equilibrium processes, without energy barriers 5 ments using microfluidics,[27,28] with the demonstration that the to partitioning. 6 coacervation process can be controlled by a chemical reaction 7 acting on the molecule that makes up the coacervate. Whilst 8 such a synthetic biology approach represents progress in 2. Results and Discussion 9 building structural representations of compartmentalization 10 there still remains key challenges for the synthesis and To achieve stable individuated coacervate microdroplets we 11 characterisation of functional model systems that can be used microfluidics to spontaneously assemble coacervates with- 12 produced reliably and robustly in a high throughput manner. in water-in-oil (w/o) droplets. Traditionally, coacervate micro- 13 New microfluidic methods for the high-throughput produc- droplets are formed by mixing stock aqueous solutions, by 14 tion, functionalisation and analysis of coacervates would hand, containing either a polycation (Polylysine (Plys) or poly 15 provide unprecedented quantitative approaches of molecular (diallyldimethylammounium chloride) (PDDA)) with a polyanion 16 dynamic processes on the single droplets level at high (carboxymethylÀ dextran (CMÀ Dex) or adenosine triphosphate 17 throughput and provide rational models for enzymatically (ATP)) at fixed molar ratios (Figure S1). When prepared by hand, 18 active subcompartments as found in biological systems.[29,30] coacervates were highly polydisperse as observed by optical 19 To address these issues, we have designed and synthesised microscopy (Figure S2), where diameters typically span one 20 an active membrane-free subcompartment with coupled fea- order of magnitude (and therefore volumes vary over three 21 tures of localized enzyme reactions and passive molecular orders of magnitude). We observe that the droplets coarsen 22 sequestration. Structurally, this comprises a coacervate micro- with a continuous change of coacervate size distribution over 23 droplet surrounded by a finite volume of aqueous media timescales ranging from hours to days. Eventually, all of the 24 provided by a surfactant stabilized water-in oil
Load more

Recommended publications
  • Light-Powered CO 2 Fixation in a Chloroplast Mimic with Natural And
    Light-powered CO 2 fixation in a chloroplast mimic with natural and synthetic parts Tarryn Miller, Thomas Beneyton, Thomas Schwander, Christoph Diehl, Mathias Girault, Richard Mclean, Tanguy Chotel, Peter Claus, Niña Socorro Cortina, Jean-Christophe Baret, et al. To cite this version: Tarryn Miller, Thomas Beneyton, Thomas Schwander, Christoph Diehl, Mathias Girault, et al.. Light- powered CO 2 fixation in a chloroplast mimic with natural and synthetic parts. Science, American Association for the Advancement of Science, 2020, 368 (6491), pp.649-654. 10.1126/science.aaz6802. hal-02870971 HAL Id: hal-02870971 https://hal.archives-ouvertes.fr/hal-02870971 Submitted on 24 Feb 2021 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Light-powered CO2 fixation in a chloroplast mimic with natural and synthetic parts Tarryn E. Miller1, Thomas Beneyton3, Thomas Schwander1, Christoph Diehl1, Mathias Girault3, Richard McLean1, Tanguy Chotel3, Peter Claus1, Niña Socorro Cortina1, Jean- Christophe Baret3,4*, Tobias J. Erb1,2* 1Max Planck Institute for terrestrial Microbiology, Department of Biochemistry and Synthetic Metabolism 2Center for Synthetic Microbiology, Karl-von-Frisch-Str. 10, D-35043 Marburg, Germany. 3Univ. Bordeaux, CNRS, CRPP UMR 5031, Pessac, France.
    [Show full text]
  • Cell Paintballing Using Optically Targeted Coacervate Microdroplets† Cite This: Chem
    Chemical Science View Article Online EDGE ARTICLE View Journal | View Issue Cell paintballing using optically targeted coacervate microdroplets† Cite this: Chem. Sci.,2015,6,6106 James P. K. Armstrong,‡*abc Sam N. Olof,‡§*abd Monika D. Jakimowicz,abcd Anthony P. Hollander,{c Stephen Mann,b Sean A. Davis,b Mervyn J. Miles,d Avinash J. Patil*b and Adam W. Perriman*bc We present a new approach for the directed delivery of biomolecular payloads to individual cells with high spatial precision. This was accomplished via active sequestration of proteins, oligonucleotides or molecular dyes into coacervate microdroplets, which were then delivered to specific regions of stem cell membranes using a dynamic holographic assembler, resulting in spontaneous coacervate microdroplet–membrane Received 23rd June 2015 fusion. The facile preparation, high sequestration efficiency and inherent membrane affinity of the Accepted 20th July 2015 microdroplets make this novel “cell paintballing” technology a highly advantageous option for spatially- DOI: 10.1039/c5sc02266e directed cell functionalization, with potential applications in single cell stimulation, transfection and www.rsc.org/chemicalscience differentiation. Creative Commons Attribution 3.0 Unported Licence. Introduction approaches have been used to visualize sub-cellular structure,7 provide nutrients during tissue engineering,8 improve in vivo The emergence of crosscutting techniques such as cellular cell targeting,9 facilitate the external manipulation of cells,10 levitation,1 bio-microuidics2 and live-cell microscopy,3 as well and initiate signalling cascades for cancer therapies.11 Such as advances in single-cell proteomics,4 genomics5 and spec- biotechnologies are limited to some extent by their indiscrim- troscopy,6 has created a demand for new approaches to inter- inate nature, with widespread membrane labelling of the entire rogate cells on an individual basis.
    [Show full text]
  • Phase Transition of RNA−Protein Complexes Into Ordered Hollow Condensates
    Phase transition of RNA−protein complexes into ordered hollow condensates Ibraheem Alshareedaha,1, Mahdi Muhammad Moosaa,1, Muralikrishna Rajub, Davit A. Potoyanb,2, and Priya R. Banerjeea,2 aDepartment of Physics, University at Buffalo, The State University of New York, Buffalo, NY 14260; and bDepartment of Chemistry, Iowa State University, Ames, IA 50011 Edited by David A. Weitz, Harvard University, Cambridge, MA, and approved May 27, 2020 (received for review December 20, 2019) Liquid−liquid phase separation of multivalent intrinsically disor- morphologies (14–16).Inanothersystem,itwasobservedthat dered protein−RNA complexes is ubiquitous in both natural and simple overexpression of TDP-43, a stress granule protein, biomimetic systems. So far, isotropic liquid droplets are the most can give rise to multilayered compartments with vacuolated commonly observed topology of RNA−protein condensates in ex- nucleoplasm-filled internal space (17). However, the physical periments and simulations. Here, by systematically studying the driving forces behind these hollow morphologies remain phase behavior of RNA−protein complexes across varied mixture poorly understood. compositions, we report a hollow vesicle-like condensate phase of Recently, we demonstrated that RNA can mediate a reentrant nucleoprotein assemblies that is distinct from RNA−protein drop- phase transition of ribonucleoproteins (RNPs) containing arginine- lets. We show that these vesicular condensates are stable at specific rich low-complexity domains (LCDs) through multivalent heterotypic mixture compositions and concentration regimes within the phase interactions (18, 19). At the substoichiometric regime, RNA triggers diagram and are formed through the phase separation of anisotropic RNP phase separation, whereas, at superstoichiometric ratios, excess protein−RNA complexes. Similar to membranes composed of am- RNA leads to droplet dissolution due to charge inversion on the phiphilic lipids, these nucleoprotein−RNA vesicular membranes ex- surface of RNP−RNA complexes (18).
    [Show full text]
  • Multiphase Complex Coacervate Droplets Tiemei Lu and Evan Spruijt*
    This is an open access article published under a Creative Commons Non-Commercial No Derivative Works (CC-BY-NC-ND) Attribution License, which permits copying and redistribution of the article, and creation of adaptations, all for non-commercial purposes. pubs.acs.org/JACS Article Multiphase Complex Coacervate Droplets Tiemei Lu and Evan Spruijt* Cite This: https://dx.doi.org/10.1021/jacs.9b11468 Read Online ACCESS Metrics & More Article Recommendations *sı Supporting Information ABSTRACT: Liquid−liquid phase separation plays an important role in cellular organization. Many subcellular condensed bodies are hierarchically organized into multiple coexisting domains or layers. However, our molecular understanding of the assembly and internal organization of these multicomponent droplets is still incomplete, and rules for the coexistence of condensed phases are lacking. Here, we show that the formation of hierarchically organized multiphase droplets with up to three coexisting layers is a generic phenomenon in mixtures of complex coacervates, which serve as models of charge- driven liquid−liquid phase separated systems. We present simple theoretical guidelines to explain both the hierarchical arrangement and the demixing transition in multiphase droplets using the interfacial tensions and critical salt concentration as inputs. Multiple coacervates can coexist if they differ sufficiently in macromolecular density, and we show that the associated differences in critical salt concentration can be used to predict multiphase droplet formation. We also show that the coexisting coacervates present distinct chemical environments that can concentrate guest molecules to different extents. Our findings suggest that condensate immiscibility may be a very general feature in biological systems, which could be exploited to design self-organized synthetic compartments to control biomolecular processes.
    [Show full text]
  • Systemic Dissemination of Viral Vectors During Intratumoral Injection
    Molecular Cancer Therapeutics 1233 Systemic dissemination of viral vectors during intratumoral injection Yong Wang,1 Jim Kang Hu,2 Ava Krol,1 therapy (1, 2). However, the efficacy of gene therapy is Yong-Ping Li,2 Chuan-Yuan Li,2 and Fan Yuan1 still limited by the delivery of therapeutic genes into 1 2 target cells. Systemic delivery of viral vectors is inade- Departments of Biomedical Engineering and Radiation quate, primarily due to the poor interstitial penetration in Oncology, Duke University, Durham, NC solid tumors (3, 4) and normal tissue toxicity caused by viral vectors and/or gene products (5–15). Different approaches have been developed for reducing Abstract the toxicity in normal tissues. One is to switch to non-viral Intratumoral injection is a routine method for local viral vectors, such as cationic liposomes or polymers (16, 17). gene delivery that may improve interstitial transport of viral Non-viral vectors are less toxic and may have similar vectors in tumor tissues and reduce systemic toxicity. transfection efficiency in vitro as viral vectors. However, However, the concentration of transgene products in non-viral vectors are in general less efficient in vivo. The normal organs, such as in the liver, may still exceed normal second approach is to use tissue targeting viral vectors tissue tolerance if the products are highly toxic. The (18–23), which can be achieved through at least two elevated concentration in normal tissues is likely to be mechanisms. One is to incorporate specific molecular caused by the dissemination of viral vectors from the structures on the vector surface that can bind to unique tumor.
    [Show full text]
  • Active Coacervate Droplets Are Protocells That Grow and Resist Ostwald Ripening
    1 Active coacervate droplets are protocells that grow and resist Ostwald ripening 2 3 Karina K. Nakashima1, Merlijn H. I. van Haren1, Alain A. M. André1, Irina Robu1 and 4 Evan Spruijt1* 5 6 1 Institute for Molecules and Materials, Radboud University, Heyendaalseweg 135, 7 6525 AJ Nijmegen, the Netherlands. 8 * 9 Correspondence: [email protected] 10 11 Keywords: protocells, complex coacervates, artificial growth model, active droplets 12 13 Abstract 14 Active coacervate droplets are liquid condensates coupled to a chemical reaction that turns over 15 their components, keeping the droplets out of equilibrium. This turnover can be used to drive active 16 processes such as growth, and provide an insight into the chemical requirements underlying 17 (proto)cellular behaviour. Moreover, controlled growth is a key requirement to achieve population 18 fitness and survival. Here we present a minimal, nucleotide-based coacervate model for active 19 droplets, and report three key findings that make these droplets into evolvable protocells. First, we 20 show that coacervate droplets form and grow by the fuel-driven synthesis of new coacervate 21 material. Second, we find that these droplets do not undergo Ostwald ripening, which we attribute 22 to the attractive electrostatic interactions within complex coacervates, active or passive. Finally, 23 we show that the droplet growth rate reflects experimental conditions such as substrate, enzyme 24 and protein concentration, and that a different droplet composition (addition of RNA) leads to 25 altered growth rates and droplet fitness. These findings together make active coacervate droplets a 26 powerful platform to mimic cellular growth at a single-droplet level, and to study fitness at a 27 population level.
    [Show full text]
  • Appendix 1*) for Essential Information Very Well Illustrated in Google and Wikipedia
    Appendix 1*) For Essential Information Very Well Illustrated in Google and Wikipedia *) in Support of the Text with Literature Citations. Referrals to illustrations in Appendix 2. Cancer in the Plant. The insertion of the Agrobacterium tumefaciens circular plasmid T (transferred) DNA into the genome of its new host, the plant (Gelvin BS. Microbiol Molecular Biol Rev 2003;67:16–37). The plant cancer “crown gall” (agrocallus; Agrobacterial crown gall) consists of malignantly transformed cells replicating the agrobacterial T DNA plasmid (reviewed in postscript Table XXXV). For reference: Koncz C Mayerhofer R Koncz-Kálmán Zs et al EMBO J 1990;9:1337–1346. Transfer of potentially oncogenic bacterial genes and proteins to patients: Septicemic Bacteroides enterotoxigenic (Sinkovics J G & Smith JP Cancer 1970;25:663–671; Viljoen KS et al PLoS One 2015;10(3):e0119462); Bartonella bacilliformis etc (Guy L et al PLoS Genet 2013;9(3):e1003393; Harms A & Dehio C Clin Microbiol Rev 2012;25:42–78; Llosa M et al Trends Microbiol 2012;20:355–9; Minnick MF et al PLoS Negl Trop Dis 2014;6(7):e2919); Helicobacter pylori (Bonsor DA et al J Biol Chem 2015;pii:jbc.M115.641829; Su YL et al J Immunol 2015;194:3997–4007; Vaziri F et al Pathog Dis 2015;73(3). pii.ftu021); Porphyromonas gingivalis (Katz J et al Int J Oral Sci 2011;3:209–215); Tuberculous infections with A. tumefaciens in patients (Ramirez FC et al Clin Infect Dis 1992;15:938–940). DNA-binding Antibodies. DNA- (or RNA-) binding proteins use zink finger motifs, leucine zippers and winged (beta-sheet loops) helix-turn helix motifs (HTH, two helices separated by the loop, RNA/DNA-binding domains) in recognition of RNA/DNA receptors for attachment.
    [Show full text]
  • Controlled Delivery of Bioactive Factors for Tissue Regeneration
    CONTROLLED DELIVERY OF BIOACTIVE FACTORS FOR TISSUE REGENERATION by Noah Ray Johnson Bachelor of Science, The Pennsylvania State University, 2010 Submitted to the Graduate Faculty of Swanson School of Engineering in partial fulfillment of the requirements for the degree of Ph.D. in Bioengineering University of Pittsburgh 2014 UNIVERSITY OF PITTSBURGH SWANSON SCHOOL OF ENGINEERING This dissertation was presented by Noah Ray Johnson It was defended on November 25, 2014 and approved by William J. Federspiel, Ph.D., Professor, Department of Bioengineering Patricia A. Hebda, Ph.D., Professor, Departments of Otolaryngology and Pathology Alan Wells, M.D., DMSc, Professor, Department of Pathology Dissertation Director: Yadong Wang, Ph.D., Professor, Department of Bioengineering ii Copyright © by Noah Ray Johnson 2014 iii CONTROLLED DELIVERY OF BIOACTIVE FACTORS FOR TISSUE REGENERATION Noah Ray Johnson, Ph.D. University of Pittsburgh, 2014 Growth factors have enormous clinical potential as they orchestrate all repair and regenerative processes in the body. However, their half-lives in vivo when applied alone are very short, on the order of minutes to hours. Therefore, large doses and multiple applications are necessary which is expensive and raises safety concerns considering their potency. To address this issue we developed a coacervate delivery system which protects growth factors from degradation and sustains and localizes their release at the site of injection. By imitating the native signaling environment involving ligands, proteoglycans, and cell receptors our delivery vehicle also enhances growth factor bioactivity, enabling the use of minute and clinically-safe dosages. This dissertation describes the translational potential of this growth factor therapy to address three significant clinical needs: 1) Diabetic wound healing, 2) Cardiac repair following myocardial infarction, 3) Bone regeneration for congenital defects and trauma.
    [Show full text]
  • Compartmentalised RNA Catalysis in Membrane-Free Coacervate Protocells
    ARTICLE DOI: 10.1038/s41467-018-06072-w OPEN Compartmentalised RNA catalysis in membrane-free coacervate protocells Björn Drobot1, Juan M. Iglesias-Artola 1, Kristian Le Vay 2, Viktoria Mayr2, Mrityunjoy Kar1, Moritz Kreysing 1, Hannes Mutschler 2 & T-Y Dora Tang 1 Phase separation of mixtures of oppositely charged polymers provides a simple and direct route to compartmentalisation via complex coacervation, which may have been important for 1234567890():,; driving primitive reactions as part of the RNA world hypothesis. However, to date, RNA catalysis has not been reconciled with coacervation. Here we demonstrate that RNA catalysis is viable within coacervate microdroplets and further show that these membrane-free dro- plets can selectively retain longer length RNAs while permitting transfer of lower molecular weight oligonucleotides. 1 Max-Planck Institute for Molecular Cell Biology and Genetics, Pfotenhauerstraße 108, 01307 Dresden, Germany. 2 Max-Planck Institute for Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany. These authors contributed equally: Juan M. Iglesias-Artola, Kristian Le Vay. Correspondence and requests for materials should be addressed to M.K. (email: [email protected]) or to H.M. (email: [email protected]) or to T-Y.D.T. (email: [email protected]) NATURE COMMUNICATIONS | (2018) 9:3643 | DOI: 10.1038/s41467-018-06072-w | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-06072-w ompartmentalisation driven by spontaneous self-assembly ab processes is crucial for spatial localisation and con- 5′ C G centration of reactants in modern biology and may have G G BHQ1 been important during the origin of life.
    [Show full text]
  • Phospholipid Membrane Formation Templated by Coacervate Droplets
    bioRxiv preprint doi: https://doi.org/10.1101/2021.02.17.431720; this version posted February 18, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Phospholipid membrane formation templated by coacervate droplets Fatma Pir Cakmak,†‡ Allyson M. Marianelli,† Christine D. Keating* Department of Chemistry, Pennsylvania State University, University Park, Pennsylvania 16802, USA. † These authors contributed equally * Corresponding author; E-mail: [email protected] Abstract: We report formation of coacervate-supported phospholipid membranes by hydrating a dried lipid film in the presence of coacervate droplets. In contrast to traditional giant lipid vesicles formed by gentle hydration in the absence of coacervates, the coacervate-templated membrane vesicles are more uniform in size, shape, and apparent lamellarity. Due to their fully-coacervate model cytoplasm, these simple artificial cells are macromolecularly crowded and can be easily pre-loaded with high concentrations of proteins or nucleic acids. Coacervate-supported membranes were characterized by fluorescence imaging, polarization, fluorescence recovery after photobleaching of labeled lipids, lipid quenching experiments, and solute uptake experiments. Our findings are consistent with the presence of lipid membranes around the coacervates, with many droplets fully coated with what appear to be continuous lipid bilayers. Within the same population, other coacervate droplets are coated with membranes having defects or pores that permit solute entry, and still others are coated with multilayered membranes. These membranes surrounding protein- based coacervate droplets provided protection from a protease added to the external solution.
    [Show full text]
  • S41467-020-20124-0.Pdf
    ARTICLE https://doi.org/10.1038/s41467-020-20124-0 OPEN Programmed spatial organization of biomacromolecules into discrete, coacervate-based protocells Wiggert J. Altenburg 1,2, N. Amy Yewdall1,2, Daan F. M. Vervoort1,2, Marleen H. M. E. van Stevendaal2,3, ✉ ✉ Alexander F. Mason2,3 & Jan C. M. van Hest 1,2,3 1234567890():,; The cell cytosol is crowded with high concentrations of many different biomacromolecules, which is difficult to mimic in bottom-up synthetic cell research and limits the functionality of existing protocellular platforms. There is thus a clear need for a general, biocompatible, and accessible tool to more accurately emulate this environment. Herein, we describe the development of a discrete, membrane-bound coacervate-based protocellular platform that utilizes the well-known binding motif between Ni2+-nitrilotriacetic acid and His-tagged proteins to exercise a high level of control over the loading of biologically relevant macro- molecules. This platform can accrete proteins in a controlled, efficient, and benign manner, culminating in the enhancement of an encapsulated two-enzyme cascade and protease- mediated cargo secretion, highlighting the potency of this methodology. This versatile approach for programmed spatial organization of biologically relevant proteins expands the protocellular toolbox, and paves the way for the development of the next generation of complex yet well-regulated synthetic cells. 1 Department of Biomedical Engineering, Eindhoven University of Technology, PO Box 513, 5600 MB Eindhoven, The Netherlands. 2 Institute for Complex Molecular Systems, Eindhoven University of Technology, PO Box 513, 5600 MB Eindhoven, The Netherlands. 3 Department of Chemical Engineering and ✉ Chemistry, Eindhoven University of Technology, PO Box 513, 5600 MB Eindhoven, The Netherlands.
    [Show full text]
  • Origin of Species Before Origin of Life: the Role of Speciation in Chemical Evolution
    life Review Origin of Species before Origin of Life: The Role of Speciation in Chemical Evolution Tony Z. Jia 1,2,* , Melina Caudan 1 and Irena Mamajanov 1,* 1 Earth-Life Science Institute, Tokyo Institute of Technology, 2-12-1-IE-1 Ookayama, Meguro-ku, Tokyo 152-8550, Japan; [email protected] 2 Blue Marble Space Institute of Science, 1001 4th Ave., Suite 3201, Seattle, WA 98154, USA * Correspondence: [email protected] (T.Z.J.); [email protected] (I.M.) Abstract: Speciation, an evolutionary process by which new species form, is ultimately responsible for the incredible biodiversity that we observe on Earth every day. Such biodiversity is one of the critical features which contributes to the survivability of biospheres and modern life. While speciation and biodiversity have been amply studied in organismic evolution and modern life, it has not yet been applied to a great extent to understanding the evolutionary dynamics of primitive life. In particular, one unanswered question is at what point in the history of life did speciation as a phenomenon emerge in the first place. Here, we discuss the mechanisms by which speciation could have occurred before the origins of life in the context of chemical evolution. Specifically, we discuss that primitive compartments formed before the emergence of the last universal common ancestor (LUCA) could have provided a mechanism by which primitive chemical systems underwent speciation. In particular, we introduce a variety of primitive compartment structures, and associated functions, that may have plausibly been present on early Earth, followed by examples of both discriminate and indiscriminate speciation affected by primitive modes of compartmentalization.
    [Show full text]