RESUMO
Active migration in both healthy and malignant cells requires the integration of information derived from soluble signaling molecules with positional information gained from interactions with the extracellular matrix and with other cells. How a cell responds and moves involves complex signaling cascades that guide the directional functions of the cytoskeleton as well as the synthesis and release of proteases that facilitate movement through tissues. The biochemical events of the signaling cascades occur in a spatially and temporally coordinated manner then dynamically shape the cytoskeleton in specific subcellular regions. Therefore, cell migration and invasion involve a precise but constantly changing subcellular nano-architecture. A multidisciplinary effort that combines new surface chemistry and cell biological tools is required to understand the reorganization of cytoskeleton triggered by complex signaling during migration. Here we generate a class of model substrates that modulate the dynamic environment for a variety of cell adhesion and migration experiments. In particular, we use these dynamic substrates to probe in real-time how the interplay between the population of cells, the initial pattern geometry, ligand density, ligand affinity and integrin composition affects cell migration and growth. Whole genome microarray analysis indicates that several classes of genes ranging from signal transduction to cytoskeletal reorganization are differentially regulated depending on the nature of the surface conditions.
Assuntos
Adesão Celular/fisiologia , Movimento Celular/fisiologia , Animais , Células CHO , Linhagem Celular , Cricetulus , Citoesqueleto/metabolismo , Camundongos , Modelos Biológicos , Propriedades de SuperfícieRESUMO
The ability to precisely control the interactions between materials and mammalian cells at the molecular level is crucial to understanding the fundamental chemical nature of how the local environment influences cellular behavior as well as for developing new biomaterials for a range of biotechnological and tissue engineering applications. In this report, we develop and apply for the first time a quantitative electroactive microarray strategy that can present a variety of ligands with precise control over ligand density to study human mesenchymal stem cell (hMSC) differentiation on transparent surfaces with a new method to quantitate adipogenic differentiation. We found that both the ligand composition and ligand density influence the rate of adipogenic differentiation from hMSC's. Furthermore, this new analytical biotechnology method is compatible with other biointerfacial characterization technologies (surface plasmon resonance, mass spectrometry) and can also be applied to investigate a range of protein-ligand or cell-material interactions for a variety of systems biology studies or cell behavior based assays.
Assuntos
Diferenciação Celular , Células-Tronco Mesenquimais/citologia , Análise em Microsséries/instrumentação , Desenho de Equipamento , Humanos , Ligantes , Propriedades de SuperfícieRESUMO
Coculture control: We report a combined photochemical and electroactive self-assembled monolayer (SAM)-based substrate strategy to generate a coculture platform with spatial and temporal control of cell-cell interactions. These dynamic substrates can present a variety of ligands on the surface for biospecific interactions between the ligands and cell surface receptors. Photopatterning enables the ligands to be immobilized in patterns and even gradients.
Assuntos
Técnicas de Cocultura/instrumentação , Actinas/química , Actinas/metabolismo , Animais , Benzaldeídos/química , Adesão Celular , Técnicas de Cocultura/métodos , Eletroquímica , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/metabolismo , Hidroquinonas/química , Ligantes , Camundongos , Propriedades de Superfície , Células Swiss 3T3 , Transfecção , Raios UltravioletaRESUMO
We report a combined photochemical and electrochemical method to pattern ligands and cells in complex geometries and gradients on inert surfaces. This work demonstrates: (1) the control of density of immobilized ligands within overlapping photopatterns, and (2) the attached cell culture patterned onto ligand defined gradients for studies of directional cell polarity. Our approach is based on the photochemical activation of benzoquinonealkanethiols. Immobilization of aminooxy terminated ligands in selected region of the quinone monolayer resulted in patterns on the surface. This approach is unique in that the extent of photochemical deprotection, as well as ligand immobilization can be monitored and quantified by cyclic voltammetry in situ. Furthermore, complex photochemical patterns of single or multiple ligands can be routinely generated using photolithographic masks. Finally, this methodology is completely compatible with attached cell culture and we show how the subtle interplay between cell-cell interactions and underlying peptide gradient influences cell polarization. The combined use of photochemistry, electrochemistry and well defined surface chemistry provides molecular level control of patterned ligands and gradients on surfaces.
Assuntos
Benzoquinonas/química , Polaridade Celular , Células 3T3 , Animais , Técnicas Biossensoriais , Técnicas de Cultura de Células , Eletroquímica , Fibroblastos/metabolismo , Ligantes , Camundongos , Microscopia de Fluorescência , Fotoquímica , Propriedades de SuperfícieAssuntos
Fibroblastos/química , Análise Serial de Proteínas , Células 3T3 , Adsorção , Animais , Adesão Celular , Polaridade Celular , Ouro/química , Hidroquinonas/química , Ligantes , Camundongos , Microscopia de Fluorescência/métodos , Estrutura Molecular , Nanotecnologia/métodos , Organelas/química , Compostos de Sulfidrila/química , Propriedades de SuperfícieAssuntos
Fibroblastos/efeitos dos fármacos , Nanotecnologia/métodos , Oligopeptídeos/farmacologia , Análise Serial de Proteínas/métodos , Células 3T3 , Adsorção , Animais , Adesão Celular/efeitos dos fármacos , Eletroquímica , Fibroblastos/citologia , Ouro/química , Hidroquinonas/química , Ligantes , Camundongos , Estrutura Molecular , Nanoestruturas/química , Oligopeptídeos/química , Oxirredução , Tamanho da Partícula , Sensibilidade e Especificidade , Relação Estrutura-Atividade , Compostos de Sulfidrila/química , Propriedades de SuperfícieAssuntos
Técnicas Biossensoriais/métodos , Transferência Ressonante de Energia de Fluorescência/métodos , Proteína rhoA de Ligação ao GTP/análise , Animais , Técnicas Biossensoriais/instrumentação , Adesão Celular/efeitos dos fármacos , Adesão Celular/fisiologia , Linhagem Celular , Fibronectinas/química , Transferência Ressonante de Energia de Fluorescência/instrumentação , Vidro/química , Ouro/química , Interações Hidrofóbicas e Hidrofílicas , Camundongos , Microscopia de Fluorescência , Compostos de Sulfidrila/química , Propriedades de Superfície , Proteína rhoA de Ligação ao GTP/química , Proteína rhoA de Ligação ao GTP/metabolismoAssuntos
Microeletrodos , Fenômenos Químicos , Físico-Química , Eletroquímica , Ligantes , Oxirredução , Propriedades de SuperfícieRESUMO
We report a broadly applicable surface chemistry methodology to immobilize ligands, proteins, and cells to an electroactive substrate with precise control of ligand density. This strategy is based on the coupling of soluble aminooxy terminated ligands with an electroactive quinone terminated monolayer. The surface chemistry product oxime is also redox active but at a different potential and therefore allows for real-time monitoring of the immobilization reaction. Only the quinone form of the immobilized redox pair is reactive with soluble aminooxy groups, which allows for the determination of the yield of reaction, the ability to immobilize multiple ligands at controlled densities, and the in-situ modulation of ligand activity. We demonstrate this methodology by using cyclic voltammetry to characterize the kinetics of a model interfacial reaction with aminooxy acetic acid. We also demonstrate the synthetic flexibility and utility of this method for biospecific interactions by installing aminooxy terminated FLAG peptides and characterizing their binding to soluble anti-FLAG with surface plasmon resonance spectroscopy. We further show this methodology is compatible with microarray technology by printing rhodamine-oxyamine in various size spots and characterizing the yield within the spots by cyclic voltammetry. We also show this methodology is compatible with cell culture conditions and fluorescent microscopy technology for cell biological studies. Arraying RGD-oxyamine peptides on these substrates allows for bio-specific adhesion of Swiss 3T3 Fibroblasts.
Assuntos
Ácido Amino-Oxiacético/química , Benzoquinonas/química , Técnicas Biossensoriais/métodos , Modelos Químicos , Eletroquímica , Ligantes , Oxirredução , Propriedades de SuperfícieRESUMO
[structure: see text] The parent and dipropyl-substituted anti (1a,b) and syn doublebent (2a,b) [5]phenylenes have been assembled by CpCo-catalyzed double cyclization of regiospecifically constructed appropriate hexaynes. (1)H NMR, NICS, and an X-ray structural analysis of 1a reflect the aromatizing effect of double angular fusion on the central ring of the linear [3]phenylene substructure.
RESUMO
This paper reports a simple method for the multilayer immobilization of conjugated polymers, gold nanoparticles on solid supports. Poly(phenyenevinylene) functionalized with aldehyde and aminooxy groups was chemoselectively immobilized onto both glass and gold substrates via layer-by-layer deposition. The physical properties of the thin films were characterized by grazing angle IR, TM-AFM, fluorescence, and UV-visible spectroscopy. This methodology was also successfully applied to prepare polymer/gold nanocluster alternating multilayers. The results show that this methodology provides a general route for preparing robust and functionalizable multilayer films on solid substrates with molecular-level thickness control.