RESUMO
Genomically minimal cells, such as JCVI-syn3.0, offer a platform to clarify genes underlying core physiological processes. Although this minimal cell includes genes essential for population growth, the physiology of its single cells remained uncharacterized. To investigate striking morphological variation in JCVI-syn3.0 cells, we present an approach to characterize cell propagation and determine genes affecting cell morphology. Microfluidic chemostats allowed observation of intrinsic cell dynamics that result in irregular morphologies. A genome with 19 genes not retained in JCVI-syn3.0 generated JCVI-syn3A, which presents morphology similar to that of JCVI-syn1.0. We further identified seven of these 19 genes, including two known cell division genes, ftsZ and sepF, a hydrolase of unknown substrate, and four genes that encode membrane-associated proteins of unknown function, which are required together to restore a phenotype similar to that of JCVI-syn1.0. This result emphasizes the polygenic nature of cell division and morphology in a genomically minimal cell.
Assuntos
Proteínas de Bactérias/genética , Cromossomos Bacterianos/genética , DNA Bacteriano/genética , Genoma Bacteriano , Mycoplasma/genética , Biologia Sintética/métodos , Proteínas de Bactérias/antagonistas & inibidores , Sistemas CRISPR-Cas , Engenharia GenéticaRESUMO
The orientation of cell polarity depends on the position of the centrosome, the main microtubule-organizing center (MTOC). Microtubules (MTs) transmit pushing forces to the MTOC as they grow against the cell periphery. How the actin network regulates these forces remains unclear. Here, in a cell-free assay, we used purified proteins to reconstitute the interaction of a microtubule aster with actin networks of various architectures in cell-sized microwells. In the absence of actin filaments, MTOC positioning was highly sensitive to variations in microtubule length. The presence of a bulk actin network limited microtubule displacement, and MTOCs were held in place. In contrast, the assembly of a branched actin network along the well edges centered the MTOCs by maintaining an isotropic balance of pushing forces. An anisotropic peripheral actin network caused the MTOC to decenter by focusing the pushing forces. Overall, our results show that actin networks can limit the sensitivity of MTOC positioning to microtubule length and enforce robust MTOC centering or decentering depending on the isotropy of its architecture.
Assuntos
Actinas , Centrossomo , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Centrossomo/metabolismo , Centro Organizador dos Microtúbulos/metabolismo , Microtúbulos/metabolismoRESUMO
Artificial Intelligence (AI) is having a revolutionary impact on our societies. It is helping humans in facing the global challenges of this century. Traditionally, AI is developed in software or through neuromorphic engineering in hardware. More recently, a brand-new strategy has been proposed. It is the so-called Chemical AI (CAI), which exploits molecular, supramolecular, and systems chemistry in wetware to mimic human intelligence. In this work, two promising approaches for boosting CAI are described. One regards designing and implementing neural surrogates that can communicate through optical or chemical signals and give rise to networks for computational purposes and to develop micro/nanorobotics. The other approach concerns "bottom-up synthetic cells" that can be exploited for applications in various scenarios, including future nano-medicine. Both topics are presented at a basic level, mainly to inform the broader audience of non-specialists, and so favour the rise of interest in these frontier subjects.
Assuntos
Inteligência Artificial , Humanos , Células Artificiais/química , Redes Neurais de ComputaçãoRESUMO
Compartments are a fundamental feature of life, based variously on lipid membranes, protein shells, or biopolymer phase separation. Here, this combines self-assembling bacterial microcompartment (BMC) shell proteins and liquid-liquid phase separation (LLPS) to develop new forms of compartmentalization. It is found that BMC shell proteins assemble at the liquid-liquid interfaces between either 1) the dextran-rich droplets and PEG-rich continuous phase of a poly(ethyleneglycol)(PEG)/dextran aqueous two-phase system, or 2) the polypeptide-rich coacervate droplets and continuous dilute phase of a polylysine/polyaspartate complex coacervate system. Interfacial protein assemblies in the coacervate system are sensitive to the ratio of cationic to anionic polypeptides, consistent with electrostatically-driven assembly. In both systems, interfacial protein assembly competes with aggregation, with protein concentration and polycation availability impacting coating. These two LLPS systems are then combined to form a three-phase system wherein coacervate droplets are contained within dextran-rich phase droplets. Interfacial localization of BMC hexameric shell proteins is tunable in a three-phase system by changing the polyelectrolyte charge ratio. The tens-of-micron scale BMC shell protein-coated droplets introduced here can accommodate bioactive cargo such as enzymes or RNA and represent a new synthetic cell strategy for organizing biomimetic functionality.
Assuntos
Proteínas de Bactérias , Dextranos , Proteínas de Bactérias/metabolismoRESUMO
Executing gene circuits by cell-free transcription-translation into cell-sized compartments, such as liposomes, is one of the major bottom-up approaches to building minimal cells. The dynamic synthesis and proper self-assembly of macromolecular structures inside liposomes, the cytoskeleton in particular, stands as a central limitation to the development of cell analogs genetically programmed. In this work, we express the Escherichia coli gene mreB inside vesicles with bilayers made of lipid-polyethylene glycol (PEG). We demonstrate that two-dimensional molecular crowding, emulated by the PEG molecules at the lipid bilayer, is enough to promote the polymerization of the protein MreB at the inner membrane into a sturdy cytoskeleton capable of transforming spherical liposomes into elongated shapes, such as rod-like compartments. We quantitatively describe this mechanism with respect to the size of liposomes, lipid composition of the membrane, crowding at the membrane, and strength of MreB synthesis. So far unexplored, molecular crowding at the surface of synthetic cells emerges as an additional development with potential broad applications. The symmetry breaking observed could be an important step toward compartment self-reproduction.
Assuntos
Células Artificiais/metabolismo , Membrana Celular/metabolismo , Forma Celular , Citoesqueleto/metabolismo , Proteínas de Escherichia coli/química , Escherichia coli/metabolismo , Lipossomos/metabolismo , Membrana Celular/química , Citoesqueleto/química , Escherichia coli/citologia , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Lipossomos/química , Polimerização , Biossíntese de Proteínas , Conformação ProteicaRESUMO
In eukaryotic cells, the membraneless organelles (MLOs) formed via liquid-liquid phase separation (LLPS) are found to interact intimately with membranous organelles (MOs). One major mode is the clustering of MOs by MLOs, such as the formation of clusters of synaptic vesicles at nerve terminals mediated by the synapsin-rich MLOs. Aqueous droplets, including complex coacervates and aqueous two-phase systems, have been plausible MLO-mimics to emulate or elucidate biological processes. However, neither of them can cluster lipid vesicles (LVs) like MLOs. In this work, we develop a synthetic droplet assembled from a combination of two different interactions underlying the formation of these two droplets, namely, associative and segregative interactions, which we call segregative-associative (SA) droplets. The SA droplets cluster and disperse LVs recapitulating the key functional features of synapsin condensates, which can be attributed to the weak electrostatic interaction environment provided by SA droplets. This work suggests LLPS with combined segregative and associative interactions as a possible route for synaptic clustering of lipid vesicles and highlights SA droplets as plausible MLO-mimics and models for studying and mimicking related cellular dynamics.
Assuntos
Organelas , Sinapsinas , Células Eucarióticas , LipídeosRESUMO
A minimal synthetic cell should contain a substrate for information storage and have the capability to divide. Notable efforts were made to assemble functional synthetic cells from the bottom up, however often lacking the capability to reproduce. Here, we develop a mechanism to fully control reversible cargo loading and division of DNA-containing giant unilamellar vesicles (GUVs) with light. We make use of the photosensitizer Chlorin e6 (Ce6) which self-assembles into lipid bilayers and leads to local lipid peroxidation upon illumination. On the time scale of minutes, illumination induces the formation of transient pores, which we exploit for cargo encapsulation or controlled release. In combination with osmosis, complete division of two daughter GUVs can be triggered within seconds of illumination due to a spontaneous curvature increase. We ultimately demonstrate the division of a selected DNA-containing GUV with full spatiotemporal control-proving the relevance of the division mechanism for bottom-up synthetic biology.
Assuntos
Células Artificiais , Lipossomas Unilamelares , DNA , Bicamadas Lipídicas , Biologia SintéticaRESUMO
The ultimate goal of bottom-up synthetic biology is recreating life in its simplest form. However, in its quest to find the minimal functional units of life, this field contributes more than its main aim by also offering a range of tools for asking, and experimentally approaching, biological questions. This Review focusses on how bottom-up reconstitution has furthered our understanding of cell biology. Studying cell biological processes in vitro has a long tradition, but only recent technological advances have enabled researchers to reconstitute increasingly complex biomolecular systems by controlling their multi-component composition and their spatiotemporal arrangements. We illustrate this progress using the example of cytoskeletal processes. Our understanding of these has been greatly enhanced by reconstitution experiments, from the first in vitro experiments 70â years ago to recent work on minimal cytoskeleton systems (including this Special Issue of Journal of Cell Science). Importantly, reconstitution approaches are not limited to the cytoskeleton field. Thus, we also discuss progress in other areas, such as the shaping of biomembranes and cellular signalling, and prompt the reader to add their subfield of cell biology to this list in the future.
Assuntos
Células Artificiais/ultraestrutura , Citoesqueleto/ultraestrutura , Transdução de Sinais , Biologia Sintética/métodos , Lipossomas Unilamelares/química , Actinas/metabolismo , Actinas/ultraestrutura , Células Artificiais/metabolismo , Compartimento Celular , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Citoesqueleto/metabolismo , Cinética , Microfluídica/métodos , Microtecnologia/métodos , Modelos Biológicos , Miosinas/metabolismo , Miosinas/ultraestrutura , Biologia Sintética/instrumentação , Termodinâmica , Lipossomas Unilamelares/metabolismoRESUMO
The cell-free molecular synthesis of biochemical systems is a rapidly growing field of research. Advances in the Human Genome Project, DNA synthesis, and other technologies have allowed the in vitro construction of biochemical systems, termed cell-free biology, to emerge as an exciting domain of bioengineering. Cell-free biology ranges from the molecular to the cell-population scales, using an ever-expanding variety of experimental platforms and toolboxes. In this review, we discuss the ongoing efforts undertaken in the three major classes of cell-free biology methodologies, namely protein-based, nucleic acids-based, and cell-free transcription-translation systems, and provide our perspectives on the current challenges as well as the major goals in each of the subfields.
Assuntos
Bioengenharia/tendências , Sistema Livre de Células , Biologia Sintética/tendências , Actinas/metabolismo , Relógios Circadianos , Citoesqueleto/metabolismo , Perfilação da Expressão Gênica , Redes Reguladoras de Genes , Humanos , Microtúbulos/metabolismo , Nanoestruturas , Ácidos Nucleicos/química , Biossíntese de Proteínas , Proteínas/química , Transcrição GênicaRESUMO
This paper studies a system of Ordinary Differential Equations modeling a chemical reaction network and derives from it a simulation tool mimicking Loss of Function and Gain of Function mutations found in cancer cells. More specifically, from a theoretical perspective, our approach focuses on the determination of moiety conservation laws for the system and their relation with the corresponding stoichiometric surfaces. Then we show that Loss of Function mutations can be implemented in the model via modification of the initial conditions in the system, while Gain of Function mutations can be implemented by eliminating specific reactions. Finally, the model is utilized to examine in detail the G1-S phase of a colorectal cancer cell.
Assuntos
Neoplasias Colorretais , Mutação com Perda de Função , Modelos Biológicos , Neoplasias Colorretais/patologia , Simulação por Computador , Humanos , CinéticaRESUMO
The effect of the two-dimensional glycan display on glycan-lectin recognition remains poorly understood despite the importance of these interactions in a plethora of cellular processes, in (patho)physiology, as well as its potential for advanced therapeutics. Faced with this challenge we utilized glycodendrimersomes, a type of synthetic vesicles whose membrane mimics the surface of a cell and offers a means to probe the carbohydrate biological activity. These single-component vesicles were formed by the self-assembly of sequence-defined mannose-Janus dendrimers, which serve as surrogates for glycolipids. Using atomic force microscopy and molecular modeling we demonstrated that even mannose, a monosaccharide, was capable of organizing the sugar moieties into periodic nanoarrays without the need of the formation of liquid-ordered phases as assumed necessary for rafts. Kinetics studies of Concanavalin A binding revealed that those nanoarrays resulted in a new effective ligand yielding a ten-fold increase in the kinetic and thermodynamic constant of association.
Assuntos
Dendrímeros/química , Manose/química , Sítios de Ligação , Concanavalina A/química , Cinética , Microscopia de Força Atômica , Modelos Moleculares , Estrutura Molecular , TermodinâmicaRESUMO
We are aiming for a blue print for synthesizing (moderately complex) subcellular systems from molecular components and ultimately for constructing life. However, without comprehensive instructions and design principles, we rely on simple reaction routes to operate the essential functions of life. The first forms of synthetic life will not make every building block for polymers de novo according to complex pathways, rather they will be fed with amino acids, fatty acids and nucleotides. Controlled energy supply is crucial for any synthetic cell, no matter how complex. Herein, we describe the simplest pathways for the efficient generation of ATP and electrochemical ion gradients. We have estimated the demand for ATP by polymer synthesis and maintenance processes in small cell-like systems, and we describe circuits to control the need for ATP. We also present fluorescence-based sensors for pH, ionic strength, excluded volume, ATP/ADP, and viscosity, which allow the major physicochemical conditions inside cells to be monitored and tuned.
Assuntos
Trifosfato de Adenosina/metabolismo , Células Artificiais/metabolismo , Metabolismo Energético , Células Artificiais/citologia , Compartimento Celular , Redes e Vias Metabólicas , Biologia SintéticaRESUMO
One of the primary challenges facing synthetic biology is reconstituting a living system from its component parts. A particularly difficult landmark is reconstituting a self-organizing system that can undergo autonomous chromosome compaction, segregation, and cell division. Here, we discuss how the syn3.0 minimal genome can inform us of the core self-organizing principles of a living cell and how these self-organizing processes can be built from the bottom up. The review underscores the importance of fundamental biology in rebuilding life from its molecular constituents.
Assuntos
Células Artificiais/citologia , Divisão Celular , Cromossomos , Biologia SintéticaRESUMO
Building automated cancer screening systems based on image analysis is currently a hot topic in computer vision and medical imaging community. One of the biggest challenges of such systems, especially those using state-of-the-art deep learning techniques, is that they usually require a large amount of training data to be accurate. However, in the medical field, the confidentiality of the data and the need for medical expertise to label them significantly reduce the amount of training data available. A common practice to overcome this problem is to apply data set augmentation techniques to artificially increase the size of the training data set. Classical data set augmentation methods such as geometrical or color transformations are efficient but still produce a limited amount of new data. Hence, there has been interest in data set augmentation methods using generative models able to synthesize a wider variety of new data. VitaDX is actually developing an automated bladder cancer screening system based on the analysis of cell images contained in urinary cytology digital slides. Currently, the number of available labeled cell images is limited and therefore exploitation of the full potential of deep learning techniques is not possible. In an attempt to increase the number of labeled cell images, a new generic generator for 2D cell images has been developed and is described in this article. This framework combines previous works on cell image generation and a recent style transfer method referred to as doodle-style transfer in this article. To the best of our knowledge, we are the first to use a doodle-style transfer method for synthetic cell image generation. This framework is quite modular and could be applied to other cell image generation problems. A statistical evaluation has shown that features of real and synthetic cell images followed roughly the same distribution. Finally, the realism of the synthetic cell images has been assessed through a visual evaluation performed with the help of medical experts. © 2019 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.
Assuntos
Aprendizado Profundo , Detecção Precoce de Câncer/métodos , Processamento de Imagem Assistida por Computador/métodos , Neoplasias da Bexiga Urinária/diagnóstico por imagem , Técnicas Citológicas , Detecção Precoce de Câncer/instrumentação , Humanos , Processamento de Imagem Assistida por Computador/instrumentação , Neoplasias da Bexiga Urinária/diagnóstico , Urina/citologia , Urotélio/citologiaRESUMO
Cell motility is an important but complex process; as cells move, new adhesions form at the front and adhesions disassemble at the back. To replicate this dynamic and spatiotemporally controlled asymmetry of adhesions and achieve motility in a minimal synthetic cell, we controlled the adhesion of a model giant unilamellar vesicle (GUV) to the substrate with light. For this purpose, we immobilized the proteins iLID and Micro, which interact under blue light and dissociate from each other in the dark, on a substrate and a GUV, respectively. Under blue light, the protein interaction leads to adhesion of the vesicle to the substrate, which is reversible in the dark. The high spatiotemporal control provided by light, allowed partly illuminating the GUV and generating an asymmetry in adhesions. Consequently, the GUV moves into the illuminated area, a process that can be repeated over multiple cycles. Thus, our system reproduces the dynamic spatiotemporal distribution of adhesions and establishes mimetic motility of a synthetic cell.
RESUMO
Cell-free cytoplasmic extracts prepared from Xenopus eggs and embryos have for decades provided a biochemical system with which to interrogate complex cell biological processes in vitro. Recently, the application of microfabrication and microfluidic strategies in biology has narrowed the gap between in vitro and in vivo studies by enabling formation of cell-size compartments containing functional cytoplasm. These approaches provide numerous advantages over traditional biochemical experiments performed in a test tube. Most notably, the cell-free cytoplasm is confined using a two- or three-dimensional boundary, which mimics the natural configuration of a cell. This strategy enables characterization of the spatial organization of a cell, and the role that boundaries play in regulating intracellular assembly and function. In this review, we describe the marriage of Xenopus cell-free cytoplasm and confinement technologies to generate synthetic cell-like systems, the recent biological insights they have enabled, and the promise they hold for future scientific discovery.
Assuntos
Extratos Celulares/genética , Sistema Livre de Células , Citoplasma/genética , Xenopus laevis/genética , Actinas/genética , Actinas/metabolismo , Animais , Citoplasma/metabolismo , Microfluídica , Óvulo/crescimento & desenvolvimento , Óvulo/metabolismoRESUMO
DNA assembly is the key technology of the emerging interdisciplinary field of synthetic biology. While the assembly of smaller DNA fragments is usually performed in vitro, high molecular weight DNA molecules are assembled in vivo via homologous recombination in the host cell. Escherichia coli, Bacillus subtilis and Saccharomyces cerevisiae are the main hosts used for DNA assembly in vivo. Progress in DNA assembly over the last few years has paved the way for the construction of whole genomes. This review provides an update on recent synthetic biology advances with particular emphasis on high molecular weight DNA assembly in vivo in E. coli, B. subtilis and S. cerevisiae. Special attention is paid to the assembly of whole genomes, such as those of the first synthetic cell, synthetic yeast and minimal genomes.
Assuntos
DNA Bacteriano/química , DNA Bacteriano/genética , Edição de Genes/métodos , Engenharia Genética/métodos , Recombinação Genética/genética , Biologia Sintética/métodos , Genoma Bacteriano/genética , Peso MolecularRESUMO
Microalgae are currently being considered as a clean, sustainable and renewable energy source. Enzymes that catalyse the metabolic pathways for biofuel production are specific and require strict regulation and co-ordination. Thorough knowledge of these key enzymes along with their regulatory molecules is essential to enable rational metabolic engineering, to drive the metabolic flux towards the desired metabolites of importance. This paper reviews two key enzymes that play their role in production of bio-oil: DGAT (acyl-CoA:diacylglycerol acyltransferase) and PDAT (phospholipid:diacylglycerol acyltransferase). It also deals with the transcription factors that control the enzymes while cell undergoes a metabolic shift under stress. The paper also discusses the association of other enzymes and pathways that provide substrates and precursors for oil accumulation. Finally a futuristic solution has been proposed about a synthetic algal cell platform that would be committed towards biofuel synthesis.
Assuntos
Chlamydomonas/metabolismo , Microalgas/metabolismo , Nitrogênio/metabolismo , Diacilglicerol O-Aciltransferase/metabolismo , Redes e Vias Metabólicas/fisiologiaRESUMO
Calpain (intracellular Ca(2+)-dependent protease) and calpastatin (calpain specific endogenous inhibitor) are widely distributed in biological systems, and have been implicated in many cellular physiological and pathological processes. Calpastatin level is of central importance to the control of calpain activity. We demonstrated for the first time that calpastatin is overexpressed in mycoplasma-contaminated cultured cells (SH-SY5Y cells that are infected by a strain of Mycoplasma hyorhinis (NDMh)). We have found that the calpastatin-upregulating activity resides in the mycoplasmal membrane lipoproteins, and is associated with NF-κB activation. Calpain-promoted proteolysis is attenuated in the NDMh lipoprotein-treated cells. Here we show that the NDMh lipoproteins promoted an increase in calpastatin in SH-SY5Y cells via the TLR2/TAK1/NF-κB pathway. The synthetic mycoplasmal lipopeptide MALP-2 and the bacterial lipopeptide PAM3CSK4 (TLR2 agonists) also promoted calpastatin upregulation. LPS (TLR4 agonist) activated NF-κB without calpastatin increase in the cell. In contrast, lipoteichoic acid (TLR2 agonist) upregulated calpastatin not via NF-κB activation, but via the MEK1/ELK1 pathway. Zymosan and peptidoglycan, TLR2 agonists that lack lipids, did not induce calpastatin upregulation. Cell treatment with a calpastatin-upregulating agonist (lipoteichoic acid) led to the attenuation of Ca(2+)-promoted calpain activity, whereas agonists that do not upregulate calpastatin (LPS, Zymosan) were ineffective. Overall, the results indicate that in these non-immune cells, calpastatin is upregulated by TLR2-agonists containing lipids, with more than one downstream pathway involved. Such agonists may be useful for studying mechanisms and factors involved in calpastatin regulation. In addition, suitable TLR2 agonists may be of interest in devising treatments for pathological processes involving excessive calpain activation.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Lipopeptídeos/farmacologia , Mycoplasma hyorhinis/metabolismo , Neuroblastoma/metabolismo , Receptor 2 Toll-Like/metabolismo , Calpaína/metabolismo , Humanos , Immunoblotting , Lipopolissacarídeos/farmacologia , Lipoproteínas/metabolismo , MAP Quinase Quinase 1/metabolismo , NF-kappa B/metabolismo , Neuroblastoma/patologia , Proteólise , Ácidos Teicoicos/farmacologia , Células Tumorais Cultivadas , Regulação para Cima , Proteínas Elk-1 do Domínio ets/metabolismoRESUMO
Artificial cells, synthetic cells, or minimal cells are microengineered cell-like structures that mimic the biological functions of cells. Artificial cells are typically biological or polymeric membranes where biologically active components, including proteins, genes, and enzymes, are encapsulated. The goal of engineering artificial cells is to build a living cell with the least amount of parts and complexity. Artificial cells hold great potential for several applications, including membrane protein interactions, gene expression, biomaterials, and drug development. It is critical to generate robust, stable artificial cells using high throughput, easy-to-control, and flexible techniques. Recently, droplet-based microfluidic techniques have shown great potential for the synthesis of vesicles and artificial cells. Here, we summarized the recent advances in droplet-based microfluidic techniques for the fabrication of vesicles and artificial cells. We first reviewed the different types of droplet-based microfluidic devices, including flow-focusing, T-junction, and coflowing. Next, we discussed the formation of multi-compartmental vesicles and artificial cells based on droplet-based microfluidics. The applications of artificial cells for studying gene expression dynamics, artificial cell-cell communications, and mechanobiology are highlighted and discussed. Finally, the current challenges and future outlook of droplet-based microfluidic methods for engineering artificial cells are discussed. This review will provide insights into scientific research in synthetic biology, microfluidic devices, membrane interactions, and mechanobiology.