20 research outputs found
Large-area biomolecule nanopatterns on diblock copolymer surfaces for cell adhesion studies
Cell membrane receptors bind to extracellular ligands, triggering intracellular signal transduction pathways that result in specific cell function. Some receptors require to be associated forming clusters for effective signaling. Increasing evidences suggest that receptor clustering is subjected to spatially controlled ligand distribution at the nanoscale. Herein we present a method to produce in an easy, straightforward process, nanopatterns of biomolecular ligands to study ligand鈥搑eceptor processes involving multivalent interactions. We based our platform in self-assembled diblock copolymers composed of poly(styrene) (PS) and poly(methyl methacrylate) (PMMA) that form PMMA nanodomains in a closed-packed hexagonal arrangement. Upon PMMA selective functionalization, biomolecular nanopatterns over large areas are produced. Nanopattern size and spacing can be controlled by the composition of the block-copolymer selected. Nanopatterns of cell adhesive peptides of different size and spacing were produced, and their impact in integrin receptor clustering and the formation of cell focal adhesions was studied. Cells on ligand nanopatterns showed an increased number of focal contacts, which were, in turn, more matured than those found in cells cultured on randomly presenting ligands. These findings suggest that our methodology is a suitable, versatile tool to study and control receptor clustering signaling and downstream cell behavior through a surface-based ligand patterning technique
Cells as active particles in asymmetric potentials: Motility under external gradients
Cell migration is a crucial event during development and in disease. Mechanical constraints and chemical gradients can contribute to the establishment of cell direction, but their respective roles remain poorly understood. Using a microfabricated topographical ratchet, we show that the nucleus dictates the direction of cell movement through mechanical guidance by its environment. We demonstrate that this direction can be tuned by combining the topographical ratchet with a biochemical gradient of fibronectin adhesion. We report competition and cooperation between the two external cues. We also quantitatively compare the measurements associated with the trajectory of a model that treats cells as fluctuating particles trapped in a periodic asymmetric potential. We show that the cell nucleus contributes to the strength of the trap, whereas cell protrusions guided by the adhesive gradients add a constant tunable bias to the direction of cell motion
Nanopatterns of surface-bound ephrinB1 produce multivalent ligand-receptor interactions that tune EphB2 receptor clustering
Here we present a nanostructured surface able to produce multivalent interactions between surface-bound ephrinB1 ligands and membrane EphB2 receptors. We created ephrinB1 nanopatterns of regular size (<30 nm in diameter) by using self-assembled diblock copolymers. Next, we used a statistically enhanced version of the Number and Brightness technique, which can discriminate with molecular sensitivity the oligomeric states of diffusive species to quantitatively track the EphB2 receptor oligomerization process in real time. The results indicate that a stimulation using randomly distributed surface-bound ligands was not sufficient to fully induce receptor aggregation. Conversely, when nanopatterned onto our substrates, the ligands effectively induced a strong receptor oligomerization. This presentation of ligands improved the clustering efficiency of conventional ligand delivery systems, as it required a 9-fold lower ligand surface coverage and included faster receptor clustering kinetics compared to traditional cross-linked ligands. In conclusion, nanostructured diblock copolymers constitute a novel strategy to induce multivalent ligand-receptor interactions leading to a stronger, faster, and more efficient receptor activation, thus providing a useful strategy to precisely tune and potentiate receptor responses. The efficiency of these materials at inducing cell responses can benefit applications such as the design of new bioactive materials and drug-delivery systems
Eph-ephrin signaling modulated by polymerization and condensation of receptors
Eph receptor signaling plays key roles in vertebrate tissue boundary formation, axonal pathfinding, and stem cell regeneration by steering cells to positions defined by its ligand ephrin. Some of the key events in Eph-ephrin signaling are understood: ephrin binding triggers the clustering of the Eph receptor, fostering transphosphorylation and signal transduction into the cell. However, a quantitative and mechanistic understanding of how the signal is processed by the recipient cell into precise and proportional responses is largely lacking. Studying Eph activation kinetics requires spatiotemporal data on the number and distribution of receptor oligomers, which is beyond the quantitative power offered by prevalent imaging methods. Here we describe an enhanced fluorescence fluctuation imaging analysis, which employs statistical resampling to measure the Eph receptor aggregation distribution within each pixel of an image. By performing this analysis over time courses extending tens of minutes, the information-rich 4D space (x, y, oligomerization, time) results were coupled to straightforward biophysical models of protein aggregation. This analysis reveals that Eph clustering can be explained by the combined contribution of polymerization of receptors into clusters, followed by their condensation into far larger aggregates. The modeling reveals that these two competing oligomerization mechanisms play distinct roles: polymerization mediates the activation of the receptor by assembling monomers into 6- to 8-mer oligomers; condensation of the preassembled oligomers into large clusters containing hundreds of monomers dampens the signaling. We propose that the polymerization鈥揷ondensation dynamics creates mechanistic explanation for how cells properly respond to variable ligand concentrations and gradients
Using enhanced number and brightness to measure protein oligomerization dynamics in live cells
Protein dimerization and oligomerization are essential to most cellular functions, yet measurement of the size of these oligomers in live cells, especially when their size changes over time and space, remains a challenge. A commonly used approach for studying protein aggregates in cells is number and brightness (N&B), a fluorescence microscopy method that is capable of measuring the apparent average number of molecules and their oligomerization (brightness) in each pixel from a series of fluorescence microscopy images. We have recently expanded this approach in order to allow resampling of the raw data to resolve the statistical weighting of coexisting species within each pixel. This feature makes enhanced N&B (eN&B) optimal for capturing the temporal aspects of protein oligomerization when a distribution of oligomers shifts toward a larger central size over time. In this protocol, we demonstrate the application of eN&B by quantifying receptor clustering dynamics using electron-multiplying charge-coupled device (EMCCD)-based total internal reflection microscopy (TIRF) imaging. TIRF provides a superior signal-to-noise ratio, but we also provide guidelines for implementing eN&B in confocal microscopes. For each time point, eN&B requires the acquisition of 200 frames, and it takes a few seconds up to 2 min to complete a single time point. We provide an eN&B (and standard N&B) MATLAB software package amenable to any standard confocal or TIRF microscope. The software requires a high-RAM computer (64 Gb) to run and includes a photobleaching detrending algorithm, which allows extension of the live imaging for more than an hour
Study of cell response over nanopatterned ligands on diblock copolymer surfaces
[eng] Cells in tissues are exposed to extracellular signals that integrate and appropriately translate into specific responses. Receptors at the cell membrane recognize a variety of soluble ligands, extracellular matrix proteins and molecules presented by the neighboring cells. Ligand-receptor recognition event triggers intracellular signal transduction pathways modulating the resulting cell function. Some receptors do not function individually as signaling units but require interactions and associations with other receptors in multimolecular complexes. This process is known as receptor clustering and is an evolutionarily preserved mechanism responsible for the integration of highly complex signals. Increasing evidences suggest that this exceptional integration is subjected to spatially controlled ligand distribution at the nanoscale. Recent developments in highly sophisticated nanofabrication approaches have allowed to experimentally address this detailed spatial regulation on cell signaling. However, it is still unclear how the nanoscale distribution of ligands can impact on the dynamics of receptor activation and signaling processes.
Herein we present a nanostructured platform to create patterns of ligands in regular nanosized (< 30 nm) clusters. We based our platform in self-assembled diblock copolymers composed of poly(styrene) (PS) and poly(methyl methacrylate) (PMMA) that tend to segregate into nanodomains. The hexagonal arrangement of the PMMA domains acts as template to be replicated by the ligand distribution. Thanks to the versatile functionalization strategy developed, any amine-bearing molecule can be covalently immobilized. The spatial distribution of ligand was analyzed by Atomic force microscopy (AFM) and stochastic reconstruction microscopy (STORM), unveiling the high level of fidelity between the nanopatterned ligands and the underlying polymeric template.
To validate these substrates as platforms for systematic study of receptor clustering processes, an adhesive peptide which promotes focal adhesion formation, was immobilized on the nanopatterned surfaces. While the overall ligand surface density was maintained constant, the spatial distribution of ligands showed a remarkable impact on focal adhesion formation. Cells on nanopatterns showed an increased number of focal contacts, which were, in turn, more matured than those found in cells cultured on randomly presenting ligands. These findings suggest that ligand presentation in a clustered format might promote multivalent ligand-receptor interactions which can help to shed light on receptor oligomerization processes.
In addition, the nanopatterned substrates developed were used to investigate the dynamics of the process of Eph receptor assembly into oligomeric clusters upon stimulation with ephrin ligands. It is known that Eph receptor oligomer composition is crucial in the fine-tuning of receptor signaling, as it will trigger intracellular signals feedback which will modulate cell response. Oligomerization processes, which imply resolving the temporal evolution of nanometric size objects in diffusive environments such as cell membranes are beyond the reach of live-imaging tools. We in here resolve the oligomerization kinetics of the Eph receptor in live cells with the required spatial and temporal resolution by using an enhanced version of the Number and Brightness (eN&B) technique, which can discriminate with molecular sensitivity the oligomeric species. The results demonstrated that stimulation through surface-bound ligands with a random distribution was not sufficient to activate the receptor signaling. Conversely, when nanopatterned on our substrates, ligands effectively induced receptor oligomerization. In addition, surface-induced ligand clustering by our nanopatterning approach accelerated the dynamics of receptor oligomerization process when compared to antibody-induced ligand clustering. Such an efficiency was induced even when ligand surface coverage was 9-fold lower in the nanopatterned presentation. Therefore, our ligand presenting platform is thought to induce multivalent ligand-receptor interactions, and might be a useful strategy to precisely tune and potentiate receptor responses. It has promising applications in biotechnology and biomedicine, such as cell culture systems to provide insight into relevant receptor clustering processes and design of new bioactive materials and drug-delivery systems.[spa] En los tejidos, las c茅lulas reciben m煤ltiples se帽ales tanto de naturaleza f铆sica como qu铆mica del entorno que las rodea. Inmersas en un entorno tridimensional, las c茅lulas interact煤an entre s铆 y con la matriz proteica que las envuelve. Adem谩s, hasta ellas difunden diversos factores solubles que transmiten se帽ales qu铆micas revelantes implicadas en el correcto funcionamiento celular. Ante tan complejo entorno, las c茅lulas son capaces de reconocer de manera diferencial los est铆mulos que reciben y responder a todos ellos a trav茅s de complejos mecanismos intracelulares de se帽alizaci贸n.
Recientemente, se han desarrollado herramientas altamente sofisticadas que permiten estudiar el comportamiento celular ante una presentaci贸n definida de ligandos. Se ha demostrado que fen贸menos tan relevantes como la adhesi贸n, la proliferaci贸n o la diferenciaci贸n celular son sensibles a la distribuci贸n espacial nanom茅trica de ligandos en superficie. M煤ltiples receptores celulares, cuando son estimulados por sus correspondientes ligandos, necesitan agruparse y formar cl煤sters que modulan la transmisi贸n de la se帽al. Desafortunadamente, todav铆a se desconocen los pormenores de la activaci贸n y la din谩mica de agregaci贸n de los mismos ante las m煤ltiples combinaciones espaciales de ligandos.
Por este motivo, este trabajo tiene como objetivo el desarrollo de superficies que permitan la presentaci贸n controlada de ligandos en grupos nanom茅tricos para analizar el efecto de los mismos en los procesos de se帽alizaci贸n intracelular. Para abordar este ambicioso objetivo, se desarroll贸 una plataforma a partir de copol铆meros en bloque cuya principal particularidad es que se autoensamblan, generando estructuras nanom茅tricas. El copol铆mero en bloque m谩s utilizado en este 谩mbito es el compuesto por poliestireno y poli(metil metacrilato) (PS-b-PMMA). En este estudio se utilizaron dos copol铆meros en bloque con distinta fracci贸n volum茅trica de cada uno de los componentes, de manera que se autoensamblan generando cilindros nanom茅tricos de PMMA inmersos en una matriz de PS. Cuando se depositan en una capa fina sobre un sustrato de silicio o de vidrio, y se controla tanto el grosor de la capa como la energ铆a superficial del sustrato, se puede conseguir que los cilindros se posicionen de forma perpendicular y ordenada sobre la superficie.
Para ello, en primer lugar se modific贸 la energ铆a superficial del sustrato mediante el anclaje de pol铆meros con una disposici贸n de mon贸meros aleatoria. Por otro lado, el grosor de la capa fina se control贸 mediante la concentraci贸n de la soluci贸n empleada y esta capa
fina se someti贸 a un tratamiento t茅rmico a 220掳C en vac铆o que permite equilibrar las tensiones superficiales del PS y del PMMA. De este modo, se fabricaron dos plataformas nanoestructuradas con patrones circulares compuestos de cilindros de PMMA (21 y 28 nm de di谩metro) separados por una matriz de PS.
Una vez obtenidas las plataformas nanostructuradas, se dise帽贸 un proceso de funcionalizaci贸n que permitiera la localizaci贸n de peque帽os grupos de ligandos sobre los dominios nanom茅tricos de PMMA. Para ello, se realiz贸 una hidr贸lisis superficial de los grupos metilos del PMMA, generando as铆 grupos 谩cidos m谩s reactivos que posibilitan la uni贸n covalente de cualquier mol茅cula con un grupo amino terminal. En este tipo de mol茅culas se incluyen todas las prote铆nas y peque帽os p茅ptidos, lo cual pone de manifiesto la gran versatilidad de la estrategia de funcionalizaci贸n.
La caracterizaci贸n de la disposici贸n espacial de los ligandos se realiz贸 mediante microscop铆a de fuerzas at贸micas, y se corrobor贸 utilizando una novedosa t茅cnica de alta resoluci贸n denominada microscop铆a de reconstrucci贸n 贸ptica estoc谩stica, que permite confirmar el estado de agregaci贸n de los ligandos biol贸gicamente activos.
Para validar la utilidad de estas superficies nanoestructuradas, primeramente se inmoviliz贸 un conocido ligando de adhesi贸n celular y se monitoriz贸 la respuesta celular, en concreto evaluando la formaci贸n de contactos focales. Los resultados demostraron que sobre estas superficies, los fibroblastos se expand铆an de tal manera que el 谩rea ocupada por las c茅lulas era equivalente en todos los sustratos. En cambio, cuando se analiz贸 en detalle las estructuras macromoleculares que forman los receptores en la membrana celular tras la activaci贸n por parte del ligando, se observaron diferencias significativas. El n煤mero de contactos focales formados en la superficie donde los grupos de ligandos estaban m谩s separados, era menor que en aquellos cuya distancia entre ligandos era menor. Por otro lado, aquellas superficies donde los ligandos se presentaban en grupos fomentaban la maduraci贸n de los contactos focales, revelando de este modo que este proceso puede manipularse utilizando estrategias de presentaci贸n de ligandos como la desarrollada en esta tesis.
Tras verificar el potencial de nuestras plataformas, se indag贸 en el proceso de agregaci贸n del receptor EphB2 ante ligandos (efrinas) con una distribuci贸n nanom茅trica variada. Para alcanzar la resoluci贸n espacio-temporal necesaria y ser capaces de distinguir entre los diferentes olig贸meros formados por el receptor, se emple贸 una innovadora t茅cnica que analiza las fluctuaciones en intensidad de cada uno de los pixeles de im谩genes de fluorescencia. En combinaci贸n con un modelo matem谩tico, se demostr贸 que la agregaci贸n de receptores para formar hex谩meros y oct谩meros impulsa la activaci贸n m谩xima del receptor EphB2.
Anteriormente, se hab铆a descrito que los ligandos solubles individuales eran incapaces de activar el receptor y de promover su oligomerizaci贸n. En cambio, la
presentaci贸n controlada de ligandos en grupos nanom茅tricos, no s贸lo fomenta la activaci贸n del receptor, sino que adem谩s acelera la formaci贸n de cl煤sters, demostrando nuevamente la efectividad de los ligandos nanoagrupados como moduladores y potenciadores del din谩mico proceso de oligomerizaci贸n.
A la vista de los resultados obtenidos, se puede concluir que hemos sido capaces de desarrollar una plataforma nanoestrucuturada mediante copol铆meros en bloque para su posterior modificaci贸n covalente con ligandos celulares cuya distribuci贸n en nanoagregados favorece las interacciones multivalentes con los receptores.
De este modo, estas plataformas tienen potenciales aplicaciones a la hora de promover una respuesta concreta de los receptores, en funci贸n del tama帽o del grupo de ligandos y del espaciado entre ellos. Este tipo de ligandos multivalentes se presentan como una atractiva estrategia para activar los complejos receptor-ligando de manera m谩s potente, y por lo tanto, menos costosa. Por lo tanto, las posibles aplicaciones de estos sistemas de presentaci贸n de ligandos comprenden desde aplicaciones biotecnol贸gicas a aplicaciones biom茅dicas, incluyendo sistemas de cultivo celular, materiales bioactivos y administraci贸n de f谩rmaco
Study of cell response over nanopatterned ligands on diblock copolymer surfaces
Cells in tissues are exposed to extracellular signals that integrate and appropriately translate into specific responses. Receptors at the cell membrane recognize a variety of soluble ligands, extracellular matrix proteins and molecules presented by the neighboring cells. Ligand-receptor recognition event triggers intracellular signal transduction pathways modulating the resulting cell function. Some receptors do not function individually as signaling units but require interactions and associations with other receptors in multimolecular complexes. This process is known as receptor clustering and is an evolutionarily preserved mechanism responsible for the integration of highly complex signals. Increasing evidences suggest that this exceptional integration is subjected to spatially controlled ligand distribution at the nanoscale. Recent developments in highly sophisticated nanofabrication approaches have allowed to experimentally address this detailed spatial regulation on cell signaling. However, it is still unclear how the nanoscale distribution of ligands can impact on the dynamics of receptor activation and signaling processes.
Herein we present a nanostructured platform to create patterns of ligands in regular nanosized (< 30 nm) clusters. We based our platform in self-assembled diblock copolymers composed of poly(styrene) (PS) and poly(methyl methacrylate) (PMMA) that tend to segregate into nanodomains. The hexagonal arrangement of the PMMA domains acts as template to be replicated by the ligand distribution. Thanks to the versatile functionalization strategy developed, any amine-bearing molecule can be covalently immobilized. The spatial distribution of ligand was analyzed by Atomic force microscopy (AFM) and stochastic reconstruction microscopy (STORM), unveiling the high level of fidelity between the nanopatterned ligands and the underlying polymeric template.
To validate these substrates as platforms for systematic study of receptor clustering processes, an adhesive peptide which promotes focal adhesion formation, was immobilized on the nanopatterned surfaces. While the overall ligand surface density was maintained constant, the spatial distribution of ligands showed a remarkable impact on focal adhesion formation. Cells on nanopatterns showed an increased number of focal contacts, which were, in turn, more matured than those found in cells cultured on randomly presenting ligands. These findings suggest that ligand presentation in a clustered format might promote multivalent ligand-receptor interactions which can help to shed light on receptor oligomerization processes.
In addition, the nanopatterned substrates developed were used to investigate the dynamics of the process of Eph receptor assembly into oligomeric clusters upon stimulation with ephrin ligands. It is known that Eph receptor oligomer composition is crucial in the fine-tuning of receptor signaling, as it will trigger intracellular signals feedback which will modulate cell response. Oligomerization processes, which imply resolving the temporal evolution of nanometric size objects in diffusive environments such as cell membranes are beyond the reach of live-imaging tools. We in here resolve the oligomerization kinetics of the Eph receptor in live cells with the required spatial and temporal resolution by using an enhanced version of the Number and Brightness (eN&B) technique, which can discriminate with molecular sensitivity the oligomeric species. The results demonstrated that stimulation through surface-bound ligands with a random distribution was not sufficient to activate the receptor signaling. Conversely, when nanopatterned on our substrates, ligands effectively induced receptor oligomerization. In addition, surface-induced ligand clustering by our nanopatterning approach accelerated the dynamics of receptor oligomerization process when compared to antibody-induced ligand clustering. Such an efficiency was induced even when ligand surface coverage was 9-fold lower in the nanopatterned presentation. Therefore, our ligand presenting platform is thought to induce multivalent ligand-receptor interactions, and might be a useful strategy to precisely tune and potentiate receptor responses. It has promising applications in biotechnology and biomedicine, such as cell culture systems to provide insight into relevant receptor clustering processes and design of new bioactive materials and drug-delivery systems.En los tejidos, las c茅lulas reciben m煤ltiples se帽ales tanto de naturaleza f铆sica como qu铆mica del entorno que las rodea. Inmersas en un entorno tridimensional, las c茅lulas interact煤an entre s铆 y con la matriz proteica que las envuelve. Adem谩s, hasta ellas difunden diversos factores solubles que transmiten se帽ales qu铆micas revelantes implicadas en el correcto funcionamiento celular. Ante tan complejo entorno, las c茅lulas son capaces de reconocer de manera diferencial los est铆mulos que reciben y responder a todos ellos a trav茅s de complejos mecanismos intracelulares de se帽alizaci贸n.
Recientemente, se han desarrollado herramientas altamente sofisticadas que permiten estudiar el comportamiento celular ante una presentaci贸n definida de ligandos. Se ha demostrado que fen贸menos tan relevantes como la adhesi贸n, la proliferaci贸n o la diferenciaci贸n celular son sensibles a la distribuci贸n espacial nanom茅trica de ligandos en superficie. M煤ltiples receptores celulares, cuando son estimulados por sus correspondientes ligandos, necesitan agruparse y formar cl煤sters que modulan la transmisi贸n de la se帽al. Desafortunadamente, todav铆a se desconocen los pormenores de la activaci贸n y la din谩mica de agregaci贸n de los mismos ante las m煤ltiples combinaciones espaciales de ligandos.
Por este motivo, este trabajo tiene como objetivo el desarrollo de superficies que permitan la presentaci贸n controlada de ligandos en grupos nanom茅tricos para analizar el efecto de los mismos en los procesos de se帽alizaci贸n intracelular. Para abordar este ambicioso objetivo, se desarroll贸 una plataforma a partir de copol铆meros en bloque cuya principal particularidad es que se autoensamblan, generando estructuras nanom茅tricas. El copol铆mero en bloque m谩s utilizado en este 谩mbito es el compuesto por poliestireno y poli(metil metacrilato) (PS-b-PMMA). En este estudio se utilizaron dos copol铆meros en bloque con distinta fracci贸n volum茅trica de cada uno de los componentes, de manera que se autoensamblan generando cilindros nanom茅tricos de PMMA inmersos en una matriz de PS. Cuando se depositan en una capa fina sobre un sustrato de silicio o de vidrio, y se controla tanto el grosor de la capa como la energ铆a superficial del sustrato, se puede conseguir que los cilindros se posicionen de forma perpendicular y ordenada sobre la superficie.
Para ello, en primer lugar se modific贸 la energ铆a superficial del sustrato mediante el anclaje de pol铆meros con una disposici贸n de mon贸meros aleatoria. Por otro lado, el grosor de la capa fina se control贸 mediante la concentraci贸n de la soluci贸n empleada y esta capa
fina se someti贸 a un tratamiento t茅rmico a 220掳C en vac铆o que permite equilibrar las tensiones superficiales del PS y del PMMA. De este modo, se fabricaron dos plataformas nanoestructuradas con patrones circulares compuestos de cilindros de PMMA (21 y 28 nm de di谩metro) separados por una matriz de PS.
Una vez obtenidas las plataformas nanostructuradas, se dise帽贸 un proceso de funcionalizaci贸n que permitiera la localizaci贸n de peque帽os grupos de ligandos sobre los dominios nanom茅tricos de PMMA. Para ello, se realiz贸 una hidr贸lisis superficial de los grupos metilos del PMMA, generando as铆 grupos 谩cidos m谩s reactivos que posibilitan la uni贸n covalente de cualquier mol茅cula con un grupo amino terminal. En este tipo de mol茅culas se incluyen todas las prote铆nas y peque帽os p茅ptidos, lo cual pone de manifiesto la gran versatilidad de la estrategia de funcionalizaci贸n.
La caracterizaci贸n de la disposici贸n espacial de los ligandos se realiz贸 mediante microscop铆a de fuerzas at贸micas, y se corrobor贸 utilizando una novedosa t茅cnica de alta resoluci贸n denominada microscop铆a de reconstrucci贸n 贸ptica estoc谩stica, que permite confirmar el estado de agregaci贸n de los ligandos biol贸gicamente activos.
Para validar la utilidad de estas superficies nanoestructuradas, primeramente se inmoviliz贸 un conocido ligando de adhesi贸n celular y se monitoriz贸 la respuesta celular, en concreto evaluando la formaci贸n de contactos focales. Los resultados demostraron que sobre estas superficies, los fibroblastos se expand铆an de tal manera que el 谩rea ocupada por las c茅lulas era equivalente en todos los sustratos. En cambio, cuando se analiz贸 en detalle las estructuras macromoleculares que forman los receptores en la membrana celular tras la activaci贸n por parte del ligando, se observaron diferencias significativas. El n煤mero de contactos focales formados en la superficie donde los grupos de ligandos estaban m谩s separados, era menor que en aquellos cuya distancia entre ligandos era menor. Por otro lado, aquellas superficies donde los ligandos se presentaban en grupos fomentaban la maduraci贸n de los contactos focales, revelando de este modo que este proceso puede manipularse utilizando estrategias de presentaci贸n de ligandos como la desarrollada en esta tesis.
Tras verificar el potencial de nuestras plataformas, se indag贸 en el proceso de agregaci贸n del receptor EphB2 ante ligandos (efrinas) con una distribuci贸n nanom茅trica variada. Para alcanzar la resoluci贸n espacio-temporal necesaria y ser capaces de distinguir entre los diferentes olig贸meros formados por el receptor, se emple贸 una innovadora t茅cnica que analiza las fluctuaciones en intensidad de cada uno de los pixeles de im谩genes de fluorescencia. En combinaci贸n con un modelo matem谩tico, se demostr贸 que la agregaci贸n de receptores para formar hex谩meros y oct谩meros impulsa la activaci贸n m谩xima del receptor EphB2.
Anteriormente, se hab铆a descrito que los ligandos solubles individuales eran incapaces de activar el receptor y de promover su oligomerizaci贸n. En cambio, la
presentaci贸n controlada de ligandos en grupos nanom茅tricos, no s贸lo fomenta la activaci贸n del receptor, sino que adem谩s acelera la formaci贸n de cl煤sters, demostrando nuevamente la efectividad de los ligandos nanoagrupados como moduladores y potenciadores del din谩mico proceso de oligomerizaci贸n.
A la vista de los resultados obtenidos, se puede concluir que hemos sido capaces de desarrollar una plataforma nanoestrucuturada mediante copol铆meros en bloque para su posterior modificaci贸n covalente con ligandos celulares cuya distribuci贸n en nanoagregados favorece las interacciones multivalentes con los receptores.
De este modo, estas plataformas tienen potenciales aplicaciones a la hora de promover una respuesta concreta de los receptores, en funci贸n del tama帽o del grupo de ligandos y del espaciado entre ellos. Este tipo de ligandos multivalentes se presentan como una atractiva estrategia para activar los complejos receptor-ligando de manera m谩s potente, y por lo tanto, menos costosa. Por lo tanto, las posibles aplicaciones de estos sistemas de presentaci贸n de ligandos comprenden desde aplicaciones biotecnol贸gicas a aplicaciones biom茅dicas, incluyendo sistemas de cultivo celular, materiales bioactivos y administraci贸n de f谩rmaco
Large-area biomolecule nanopatterns on diblock copolymer surfaces for cell adhesion studies
Cell membrane receptors bind to extracellular ligands, triggering intracellular signal transduction pathways that result in specific cell function. Some receptors require to be associated forming clusters for effective signaling. Increasing evidences suggest that receptor clustering is subjected to spatially controlled ligand distribution at the nanoscale. Herein we present a method to produce in an easy, straightforward process, nanopatterns of biomolecular ligands to study ligand鈥搑eceptor processes involving multivalent interactions. We based our platform in self-assembled diblock copolymers composed of poly(styrene) (PS) and poly(methyl methacrylate) (PMMA) that form PMMA nanodomains in a closed-packed hexagonal arrangement. Upon PMMA selective functionalization, biomolecular nanopatterns over large areas are produced. Nanopattern size and spacing can be controlled by the composition of the block-copolymer selected. Nanopatterns of cell adhesive peptides of different size and spacing were produced, and their impact in integrin receptor clustering and the formation of cell focal adhesions was studied. Cells on ligand nanopatterns showed an increased number of focal contacts, which were, in turn, more matured than those found in cells cultured on randomly presenting ligands. These findings suggest that our methodology is a suitable, versatile tool to study and control receptor clustering signaling and downstream cell behavior through a surface-based ligand patterning technique
Large-area biomolecule nanopatterns on diblock copolymer surfaces for cell adhesion studies
Cell membrane receptors bind to extracellular ligands, triggering intracellular signal transduction pathways that result in specific cell function. Some receptors require to be associated forming clusters for effective signaling. Increasing evidences suggest that receptor clustering is subjected to spatially controlled ligand distribution at the nanoscale. Herein we present a method to produce in an easy, straightforward process, nanopatterns of biomolecular ligands to study ligand鈥搑eceptor processes involving multivalent interactions. We based our platform in self-assembled diblock copolymers composed of poly(styrene) (PS) and poly(methyl methacrylate) (PMMA) that form PMMA nanodomains in a closed-packed hexagonal arrangement. Upon PMMA selective functionalization, biomolecular nanopatterns over large areas are produced. Nanopattern size and spacing can be controlled by the composition of the block-copolymer selected. Nanopatterns of cell adhesive peptides of different size and spacing were produced, and their impact in integrin receptor clustering and the formation of cell focal adhesions was studied. Cells on ligand nanopatterns showed an increased number of focal contacts, which were, in turn, more matured than those found in cells cultured on randomly presenting ligands. These findings suggest that our methodology is a suitable, versatile tool to study and control receptor clustering signaling and downstream cell behavior through a surface-based ligand patterning technique