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The evolution of land flora transformed the terrestrial environment. Land plants evolved from an ancestral charophycean alga from which they inherited developmental, biochemical, and cell biological attributes. Additional biochemical and physiological adaptations to land, and a life cycle with an alternation between multicellular haploid and diploid generations that facilitated efficient dispersal of desiccation tolerant spores, evolved in the ancestral land plant. We analyzed the genome of the liverwort Marchantia polymorpha, a member of a basal land plant lineage. Relative to charophycean algae, land plant genomes are characterized by genes encoding novel biochemical pathways, new phytohormone signaling pathways (notably auxin), expanded repertoires of signaling pathways, and increased diversity in some transcription factor families. Compared with other sequenced land plants, M. polymorpha exhibits low genetic redundancy in most regulatory pathways, with this portion of its genome resembling that predicted for the ancestral land plant. PAPERCLIP.
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Evolución Biológica , Embryophyta/genética , Genoma de Planta , Marchantia/genética , Adaptación Biológica , Embryophyta/fisiología , Regulación de la Expresión Génica de las Plantas , Marchantia/fisiología , Anotación de Secuencia Molecular , Transducción de Señal , Transcripción GenéticaRESUMEN
An alternative approach to acquire transmission travel time data is proposed, exploiting the geometry of devices commonly used in ultrasound computed tomography for medical imaging or non-destructive testing with ultrasonic waves. The intent is to (i) shorten acquisition time for devices with a large number of emitters, (ii) to eliminate the calibration step, and (iii) to suppress instrument noise. Inspired by seismic ambient field interferometry, the method rests on the active excitation of diffuse ultrasonic wavefields and the extraction of deterministic travel time information by inter-station correlation. To reduce stochastic errors and accelerate convergence, ensemble interferograms are obtained by phase-weighted stacking of observed and computed correlograms, generated with identical realizations of random sources. Mimicking an imaging setup, the accuracy of the travel time measurements as a function of the number of emitters and random realizations can be assessed both analytically and with spectral-element simulations for phantoms mimicking the model parameter distribution. The results warrant tomographic reconstructions with straight- or bent-ray approaches, where the effect of inherent stochastic fluctuations can be made significantly smaller than the effect of subjective choices on regularisation. This work constitutes a first conceptual study and a necessary prelude to future implementations.
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Tomografía Computarizada por Rayos X , Tomografía , Calibración , Fantasmas de Imagen , Tomografía/métodos , Tomografía Computarizada por Rayos X/métodos , Ultrasonografía/métodosRESUMEN
Computational models of acoustic wave propagation are frequently used in transcranial ultrasound therapy, for example, to calculate the intracranial pressure field or to calculate phase delays to correct for skull distortions. To allow intercomparison between the different modeling tools and techniques used by the community, an international working group was convened to formulate a set of numerical benchmarks. Here, these benchmarks are presented, along with intercomparison results. Nine different benchmarks of increasing geometric complexity are defined. These include a single-layer planar bone immersed in water, a multi-layer bone, and a whole skull. Two transducer configurations are considered (a focused bowl and a plane piston operating at 500 kHz), giving a total of 18 permutations of the benchmarks. Eleven different modeling tools are used to compute the benchmark results. The models span a wide range of numerical techniques, including the finite-difference time-domain method, angular spectrum method, pseudospectral method, boundary-element method, and spectral-element method. Good agreement is found between the models, particularly for the position, size, and magnitude of the acoustic focus within the skull. When comparing results for each model with every other model in a cross-comparison, the median values for each benchmark for the difference in focal pressure and position are less than 10% and 1 mm, respectively. The benchmark definitions, model results, and intercomparison codes are freely available to facilitate further comparisons.
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Benchmarking , Transductores , Simulación por Computador , Cráneo/diagnóstico por imagen , Ultrasonografía/métodosRESUMEN
Building on recombinant DNA technology, leaps in synthesis, assembly, and analysis of DNA have revolutionized genetics and molecular biology over the past two decades (Kosuri and Church, 2014). These technological advances have accelerated the emergence of synthetic biology as a new discipline (Cameron et al., 2014). Synthetic biology is characterized by efforts targeted at the modification of existing and the design of novel biological systems based on principles adopted from information technology and engineering (Andrianantoandro et al., 2006; Khalil and Collins, 2010). As in more traditional engineering disciplines such as mechanical, electrical and civil engineering, synthetic biologists utilize abstraction, decoupling and standardization to make the design of biological systems more efficient and scalable. To facilitate the management of complexity, synthetic biology relies on an abstraction hierarchy composed of multiple levels (Endy, 2005): DNA as genetic material, "parts" as elements of DNA encoding basic biological functions (e.g. promoter, ribosome-binding site, terminator sequence), "devices" as any combination of parts implementing a human-defined function, and "systems" as any combination of devices fulfilling a predefined purpose. Parts are designated to perform predictable and modular functions in the context of higher-level devices or systems, which are successively refined through a cycle of designing, building, and testing.
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Plastidios/genética , Biología Sintética/tendencias , Ingeniería Genética , Genoma del Cloroplasto , Plastidios/metabolismoRESUMEN
Ultrasound computed tomography (USCT) is an emerging modality to image the acoustic properties of the breast tissue for cancer diagnosis. With the need of improving the diagnostic accuracy of USCT, while maintaining the cost low, recent research is mainly focused on improving (1) the reconstruction methods and (2) the acquisition systems. D-optimal sequential experimental design (D-SOED) offers a method to integrate these aspects into a common systematic framework. The transducer configuration is optimized to minimize the uncertainties in the estimated model parameters, and to reduce the time to solution by identifying redundancies in the data. This work presents a formulation to jointly optimize the experiment for transmission and reflection data and, in particular, to estimate the speed of sound and reflectivity of the tissue using either ray-based or wave-based imaging methods. Uncertainties in the parameters can be quantified by extracting properties of the posterior covariance operator, which is analytically computed by linearizing the forward problem with respect to the prior knowledge about parameters. D-SOED is first introduced by an illustrative toy example, and then applied to real data. This shows that the time to solution can be substantially reduced, without altering the final image, by selecting the most informative measurements.
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Recently, the liverwort Marchantia polymorpha has received increasing attention as a basal plant model for multicellular studies. Its ease of handling, well-characterized plastome and proven protocols for biolistic plastid transformation qualify M. polymorpha as an attractive platform to study the evolution of chloroplasts during the transition from water to land. In addition, chloroplasts of M. polymorpha provide a convenient test-bed for the characterization of genetic elements involved in plastid gene expression due to the absence of mechanisms for RNA editing. While reporter genes have proven valuable to the qualitative and quantitative study of gene expression in chloroplasts, expression of green fluorescent protein (GFP) in chloroplasts of M. polymorpha has proven problematic. We report the design of a codon-optimized gfp varian, mturq2cp, which allowed successful expression of a cyan fluorescent protein under control of the tobacco psbA promoter from the chloroplast genome of M. polymorpha. We demonstrate the utility of mturq2cp in (i) early screening for transplastomic events following biolistic transformation of M. polymorpha spores; (ii) visualization of stromules as elements of plastid structure in Marchantia; and (iii) quantitative microscopy for the analysis of promoter activity.
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Genoma del Cloroplasto , Proteínas Fluorescentes Verdes/metabolismo , Marchantia/genética , Secuencia de Aminoácidos , Secuencia de Bases , Fluorescencia , Proteínas Fluorescentes Verdes/química , Proteínas Fluorescentes Verdes/genética , Datos de Secuencia Molecular , Plantas Modificadas Genéticamente , Transformación GenéticaRESUMEN
In vitro recombination methods have enabled one-step construction of large DNA sequences from multiple parts. Although synthetic biological circuits can in principle be assembled in the same fashion, they typically contain repeated sequence elements such as standard promoters and terminators that interfere with homologous recombination. Here we use a computational approach to design synthetic, biologically inactive unique nucleotide sequences (UNSes) that facilitate accurate ordered assembly. Importantly, our designed UNSes make it possible to assemble parts with repeated terminator and insulator sequences, and thereby create insulated functional genetic circuits in bacteria and mammalian cells. Using UNS-guided assembly to construct repeating promoter-gene-terminator parts, we systematically varied gene expression to optimize production of a deoxychromoviridans biosynthetic pathway in Escherichia coli. We then used this system to construct complex eukaryotic AND-logic gates for genomic integration into embryonic stem cells. Construction was performed by using a standardized series of UNS-bearing BioBrick-compatible vectors, which enable modular assembly and facilitate reuse of individual parts. UNS-guided isothermal assembly is broadly applicable to the construction and optimization of genetic circuits and particularly those requiring tight insulation, such as complex biosynthetic pathways, sensors, counters and logic gates.
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Vías Biosintéticas/genética , Regulación de la Expresión Génica , Redes Reguladoras de Genes , Ingeniería Genética/métodos , Secuencia de Bases , Células Madre Embrionarias/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Elementos Aisladores , Nucleótidos/química , Biología Sintética/métodos , Regiones Terminadoras GenéticasRESUMEN
The chloroplast genomes of most plants and algae contain a large inverted repeat (IR) region that separates two single-copy regions and harbours the ribosomal RNA operon. We have addressed the functional importance of the IR region by removing an entire copy of the 25.3-kb IR from the tobacco plastid genome. Using plastid transformation and subsequent selectable marker gene elimination, we precisely excised the IR, thus generating plants with a substantially reduced plastid genome size. We show that the lack of the IR results in a mildly reduced plastid ribosome number, suggesting a gene dosage benefit from the duplicated presence of the ribosomal RNA operon. Moreover, the IR deletion plants contain an increased number of plastid genomes, suggesting that genome copy number is regulated by measuring total plastid DNA content rather than by counting genomes. Together, our findings (1) demonstrate that the IR can enhance the translation capacity of the plastid, (2) reveal the relationship between genome size and genome copy number, and (3) provide a simplified plastid genome structure that will facilitate future synthetic biology applications.
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Dosificación de Gen , Genoma de Plastidios , Secuencias Invertidas Repetidas , Nicotiana , Nicotiana/genética , Secuencias Invertidas Repetidas/genética , Plastidios/genética , Tamaño del Genoma , Variaciones en el Número de Copia de ADN , Genoma de PlantaRESUMEN
The application of tissue-engineered heart valves in the high-pressure circulatory system is still challenging. One possible solution is the development of biohybrid scaffolds with textile reinforcement to achieve improved mechanical properties. In this article, we present a manufacturing process of bio-inspired fiber reinforcement for an aortic valve scaffold. The reinforcement structure consists of polyvinylidene difluoride monofilament fibers that are biomimetically arranged by a novel winding process. The fibers were embedded and fixated into electrospun polycarbonate urethane on a cylindrical collector. The scaffold was characterized by biaxial tensile strength, bending stiffness, burst pressure and hemodynamically in a mock circulation system. The produced fiber-reinforced scaffold showed adequate acute mechanical and hemodynamic properties. The transvalvular pressure gradient was 3.02 ± 0.26 mmHg with an effective orifice area of 2.12 ± 0.22 cm2. The valves sustained aortic conditions, fulfilling the ISO-5840 standards. The fiber-reinforced scaffold failed in a circumferential direction at a stress of 461.64 ± 58.87 N/m and a strain of 49.43 ± 7.53%. These values were above the levels of tested native heart valve tissue. Overall, we demonstrated a novel manufacturing approach to develop a fiber-reinforced biomimetic scaffold for aortic heart valve tissue engineering. The characterization showed that this approach is promising for an in situ valve replacement.
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STUDY DESIGN: Retrospective evaluation of prospectively collected data. OBJECTIVE: Analyzing time course and stages of interbody fusion of a uniformly operated cohort, defining a grading system and establishing diagnosis-dependent periods of bone healing. METHODS: Sequential lateral radiographs of 238 patients (313 levels) with interbody fusion operated thoracoscopically were analyzed. RESULTS: Evaluation of 1696 radiographs with a mean follow-up of 65.19 months and average numbers of 5.42 (2-18) images per level was performed. Diagnoses were Pyogenic Spondylitis (74), Fracture (96), Ankylosing Spondylitis (38) and Degenerative Disease (105). No case with Grade 2 deteriorated to Grade 5. On average, Grade 4 persisted for 113 days, Grade 3 for 197 days, Grade 2 for 286 days and Grade 1 for 316 days. The first 95% of levels ("Green Zone", ≤ Grade 2) fused at 1 year, the remaining 4% levels fused between 12 and 17 months ("Yellow Zone") and the last 1% ("Red Zone") fused after 510 days. CONCLUSION: Sequential lateral radiographs permit evaluation of interbody fusion. Grade 2 is the threshold point for fusion; once accomplished, failure is unlikely. If fusion (Grade 2,1 or 0) is not reached within 510 days, it should be regarded as failed. The 510-day-threshold could reduce the necessity of CT scanning for assessing fusion.
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Ultrasound computed tomography (USCT) has great potential for 3-D quantitative imaging of acoustic breast tissue properties. Typical devices include high-frequency transducers, which makes tomography techniques based on numerical wave propagation simulations computationally challenging, especially in 3-D. Therefore, despite the finite-frequency nature of ultrasonic waves, ray-theoretical approaches to transmission tomography are still widely used. This article introduces a finite-frequency traveltime tomography to medical ultrasound. In addition to being computationally tractable for 3-D imaging at high frequencies, the method has two main advantages: 1) it correctly accounts for the frequency dependence and volumetric sensitivity of traveltime measurements, which are related to off-ray-path scattering and diffraction. 2) It naturally enables out-of-plane imaging and the construction of 3-D images from 2-D slice-by-slice acquisition systems. Our method rests on the availability of calibration data in water, used to linearize the forward problem and to provide analytical expressions of cross correlation traveltime sensitivity. As a consequence of the finite-frequency content, sensitivity is distributed in multiple Fresnel volumes, thereby providing out-of-plane sensitivity. To improve computational efficiency, we develop a memory-efficient implementation by encoding the Jacobian operator with a 1-D parameterization, which allows us to extend the method to large-scale domains. We validate our tomographic approach using laboratory measurements collected with a 2-D setup of transducers and using a cylindrically symmetric phantom. We then demonstrate its applicability for 3-D reconstructions by simulating a slice-by-slice acquisition system using the same data set.
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Imagenología Tridimensional/métodos , Tomografía/métodos , Ultrasonografía/métodos , Fantasmas de ImagenRESUMEN
Horizon scanning is intended to identify the opportunities and threats associated with technological, regulatory and social change. In 2017 some of the present authors conducted a horizon scan for bioengineering (Wintle et al., 2017). Here we report the results of a new horizon scan that is based on inputs from a larger and more international group of 38 participants. The final list of 20 issues includes topics spanning from the political (the regulation of genomic data, increased philanthropic funding and malicious uses of neurochemicals) to the environmental (crops for changing climates and agricultural gene drives). The early identification of such issues is relevant to researchers, policy-makers and the wider public.
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Bioingeniería , Cambio Climático , Predicción , Agricultura , Biotecnología , Femenino , Ingeniería Genética , Humanos , Internacionalidad , Masculino , Plantas Modificadas Genéticamente , PolíticaRESUMEN
A number of significant muscle diseases, such as cachexia, sarcopenia, systemic chronic inflammation, along with inflammatory myopathies share TNF-α-dominated inflammation in their pathogenesis. In addition, inflammatory episodes may increase susceptibility to drug toxicity. To assess the effect of TNF-α-induced inflammation on drug responses, we engineered 3D, human skeletal myobundles, chronically exposed them to TNF-α during maturation, and measured the combined response of TNF-α and the chemotherapeutic doxorubicin on muscle function. First, the myobundle inflammatory environment was characterized by assessing the effects of TNF-α on 2D human skeletal muscle cultures and 3D human myobundles. High doses of TNF-α inhibited maturation in human 2D cultures and maturation and function in 3D myobundles. Then, a tetanus force dose-response curve was constructed to characterize doxorubicin's effects on function alone. The combination of TNF-α and 10 nM doxorubicin exhibited a synergistic effect on both twitch and tetanus force production. Overall, the results demonstrated that inflammation of a 3D, human skeletal muscle inflammatory system alters the response to doxorubicin.
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Antibióticos Antineoplásicos/toxicidad , Doxorrubicina/toxicidad , Músculo Esquelético/efectos de los fármacos , Factor de Necrosis Tumoral alfa/farmacología , Animales , Línea Celular , Humanos , Ratones , Modelos Biológicos , Músculo Esquelético/fisiología , Mioblastos Esqueléticos/efectos de los fármacos , Mioblastos Esqueléticos/fisiología , Ingeniería de TejidosRESUMEN
Modern genetic tools allow the dissection and emulation of fundamental mechanisms shaping morphogenesis in multicellular organisms. Several synthetic genetic circuits for control of multicellular patterning have been reported to date. However, hierarchical induction of gene expression domains has received little attention from synthetic biologists, despite its importance in biological self-organization. Here we report a synthetic genetic system implementing population-based AND-logic for programmed autonomous induction of bacterial gene expression domains. We develop a ratiometric assay for bacteriophage T7 RNA polymerase activity and use it to systematically characterize different intact and split enzyme variants. We then utilize the best-performing variant to build a three-color patterning system responsive to two different homoserine lactones. We validate the AND gate-like behavior of this system both in cell suspension and in surface culture. Finally, we use the synthetic circuit in a membrane-based spatial assay to demonstrate programmed hierarchical patterning of gene expression across bacterial populations.
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Bacterias/genética , Bacterias/metabolismo , ARN Polimerasas Dirigidas por ADN/metabolismo , Genes Sintéticos , Ingeniería Genética , Regiones Promotoras Genéticas , Biología Sintética/instrumentación , Biología Sintética/métodos , Proteínas Virales/metabolismoRESUMEN
Droplet-based microfluidics has been used to facilitate high-throughput analysis of individual prokaryote and mammalian cells. However, there is a scarcity of similar workflows applicable to rapid phenotyping of plant systems where phenotyping analyses typically are time-consuming and low-throughput. We report on-chip encapsulation and analysis of protoplasts isolated from the emergent plant model Marchantia polymorpha at processing rates of >100,000 cells per hour. We use our microfluidic system to quantify the stochastic properties of a heat-inducible promoter across a population of transgenic protoplasts to demonstrate its potential for assessing gene expression activity in response to environmental conditions. We further demonstrate on-chip sorting of droplets containing YFP-expressing protoplasts from wild type cells using dielectrophoresis force. This work opens the door to droplet-based microfluidic analysis of plant cells for applications ranging from high-throughput characterisation of DNA parts to single-cell genomics to selection of rare plant phenotypes.
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Ensayos Analíticos de Alto Rendimiento/métodos , Marchantia/citología , Técnicas Analíticas Microfluídicas/métodos , Protoplastos/química , Análisis de la Célula Individual/métodos , Agrobacterium tumefaciens/genética , Proteínas Bacterianas/análisis , Proteínas Bacterianas/genética , Separación Celular/instrumentación , Separación Celular/métodos , Composición de Medicamentos , Diseño de Equipo , Regulación de la Expresión Génica de las Plantas , Genes Reporteros , Genómica/métodos , Ensayos Analíticos de Alto Rendimiento/instrumentación , Calor , Dispositivos Laboratorio en un Chip , Proteínas Luminiscentes/análisis , Proteínas Luminiscentes/genética , Marchantia/química , Marchantia/genética , Microscopía Fluorescente , Plantas Modificadas Genéticamente , Regiones Promotoras Genéticas , Análisis de la Célula Individual/instrumentación , Procesos Estocásticos , Transformación GenéticaRESUMEN
Plants are attractive platforms for synthetic biology and metabolic engineering. Plants' modular and plastic body plans, capacity for photosynthesis, extensive secondary metabolism, and agronomic systems for large-scale production make them ideal targets for genetic reprogramming. However, efforts in this area have been constrained by slow growth, long life cycles, the requirement for specialized facilities, a paucity of efficient tools for genetic manipulation, and the complexity of multicellularity. There is a need for better experimental and theoretical frameworks to understand the way genetic networks, cellular populations, and tissue-wide physical processes interact at different scales. We highlight new approaches to the DNA-based manipulation of plants and the use of advanced quantitative imaging techniques in simple plant models such as Marchantia polymorpha. These offer the prospects of improved understanding of plant dynamics and new approaches to rational engineering of plant traits.
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Botánica/métodos , Biología Sintética/métodos , Cloroplastos/genética , Ingeniería Genética/métodos , Plantas/genética , Plantas Modificadas Genéticamente/metabolismo , Transformación GenéticaRESUMEN
Advances in biological engineering are likely to have substantial impacts on global society. To explore these potential impacts we ran a horizon scanning exercise to capture a range of perspectives on the opportunities and risks presented by biological engineering. We first identified 70 potential issues, and then used an iterative process to prioritise 20 issues that we considered to be emerging, to have potential global impact, and to be relatively unknown outside the field of biological engineering. The issues identified may be of interest to researchers, businesses and policy makers in sectors such as health, energy, agriculture and the environment.
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Bioingeniería/tendencias , Investigación/tendencias , Cambio Climático , Conservación de los Recursos Naturales , HumanosRESUMEN
Recombination-based DNA construction methods, such as Gibson assembly, have made it possible to easily and simultaneously assemble multiple DNA parts, and they hold promise for the development and optimization of metabolic pathways and functional genetic circuits. Over time, however, these pathways and circuits have become more complex, and the increasing need for standardization and insulation of genetic parts has resulted in sequence redundancies--for example, repeated terminator and insulator sequences--that complicate recombination-based assembly. We and others have recently developed DNA assembly methods, which we refer to collectively as unique nucleotide sequence (UNS)-guided assembly, in which individual DNA parts are flanked with UNSs to facilitate the ordered, recombination-based assembly of repetitive sequences. Here we present a detailed protocol for UNS-guided assembly that enables researchers to convert multiple DNA parts into sequenced, correctly assembled constructs, or into high-quality combinatorial libraries in only 2-3 d. If the DNA parts must be generated from scratch, an additional 2-5 d are necessary. This protocol requires no specialized equipment and can easily be implemented by a student with experience in basic cloning techniques.
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Secuencia de Bases/genética , Ingeniería Genética/métodos , Secuencias Repetitivas de Ácidos Nucleicos/genética , Biología Sintética/métodos , Clonación Molecular/métodos , Biblioteca de GenesRESUMEN
As a reference platform for in vitro synthetic biology, we have developed a prototype flow microreactor for enzymatic biosynthesis. We report the design, implementation, and computer-aided optimisation of a three-step model pathway within a microfluidic reactor. A packed bed format was shown to be optimal for enzyme compartmentalisation after experimental evaluation of several approaches. The specific substrate conversion efficiency could significantly be improved by an optimised parameter set obtained by computational modelling. Our microreactor design provides a platform to explore new in vitro synthetic biology solutions for industrial biosynthesis.
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Técnicas Analíticas Microfluídicas/instrumentación , Biología Sintética/instrumentación , Simulación por Computador , Diseño Asistido por Computadora , Enzimas Inmovilizadas/química , Enzimas Inmovilizadas/metabolismo , Diseño de EquipoRESUMEN
The collective migration of cells is fundamental to epithelial biology. One of the hallmarks of collective behavior in migrating cohesive epithelial cell sheets is the emergence of so called leader cells. These cells exhibit a distinct morphology with a large and highly active lamellipodium. Although it is generally accepted that they play a crucial part in collective migration, the biophysical factors that regulate their formation remain unknown.Here we show that a geometry-based cue like local variation of curvature of the collective's perimeter is capable of triggering leader cell formation and promoting enhanced motility at defined positions. Remarkably, the extent of this effect scales with the magnitude of the curvature.Cytoskeletal tension was found to be important for geometry induced leader cell formation, as cells treated with tension reducing agents appeared less sensitive to local curvature variation. Accordingly, traction force microscopy revealed an increased level of shear stress at highly curved positions even before the cell migration had actually started, indicating the presence of a collective polarization induced by the geometry of the confinement.Together our findings suggest that high curvature leads to locally increased stress accumulation, mediated via cell-substrate interaction as well as via cytoskeleton tension. The stress accumulation in turn enhances the probability of leader cell formation as well as cell motility. This work defines the importance of geometric cue such as local curvature in the collective migration dynamics of epithelial cells and thus shows implications for the biophysical regulation of epithelium during wound healing, embryonic development, and oncogenesis.