RESUMEN
The alarming growth of the antibiotic-resistant superbugs methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus (VRE) is driving the development of new technologies to investigate antibiotics and their modes of action. We report the label-free detection of vancomycin binding to bacterial cell wall precursor analogues (mucopeptides) on cantilever arrays, with 10 nM sensitivity and at clinically relevant concentrations in blood serum. Differential measurements have quantified binding constants for vancomycin-sensitive and vancomycin-resistant mucopeptide analogues. Moreover, by systematically modifying the mucopeptide density we gain new insights into the origin of surface stress. We propose that stress is a product of a local chemical binding factor and a geometrical factor describing the mechanical connectivity of regions activated by local binding in terms of a percolation process. Our findings place BioMEMS devices in a new class of percolative systems. The percolation concept will underpin the design of devices and coatings to significantly lower the drug detection limit and may also have an impact on our understanding of antibiotic drug action in bacteria.
Asunto(s)
Técnicas Biosensibles/tendencias , Viabilidad Microbiana/efectos de los fármacos , Mucoproteínas/química , Nanotecnología/tendencias , Vancomicina/química , Bacterias/citología , Bacterias/efectos de los fármacos , Sitios de Unión/fisiología , Membrana Celular/efectos de los fármacos , Farmacorresistencia Bacteriana/efectos de los fármacos , Proteínas de la Membrana/metabolismo , Técnicas Analíticas Microfluídicas/tendencias , Microscopía de Fuerza Atómica/tendencias , Mucoproteínas/metabolismo , Sensibilidad y Especificidad , Estrés Fisiológico , Resistencia a la Tracción , Vancomicina/metabolismo , Vancomicina/uso terapéuticoRESUMEN
Mechanical stress affects and regulates many aspects of the cell, including morphology, growth, differentiation, gene expression and apoptosis. In this study we show how mechanical stress perturbs the intracellular structures of the cell and induces mechanical responses. In order to correlate mechanical perturbations to cellular responses, we used a combined fluorescence-atomic force microscope (AFM) to produce well defined nanomechanical perturbations of 10 nN while simultaneously tracking the real-time motion of fluorescently labelled mitochondria in live cells. The spatial displacement of the organelles in response to applied loads demonstrates the highly dynamic mechanical response of mitochondria in fibroblast cells. The average displacement of all mitochondrial structures analysed showed an increase of approximately 40%, post-perturbation ( approximately 160 nm in comparison to basal displacements of approximately 110 nm). These results show that local forces can produce organelle displacements at locations far from the initial point of contact (up to approximately 40 microm). In order to examine the role of the cytoskeleton in force transmission and its effect on mitochondrial displacements, both the actin and microtubule cytoskeleton were disrupted using Cytochalasin D and Nocodazole, respectively. Our results show that there is no significant change in mitochondrial displacement following indentation after such treatments. These results demonstrate the role of the cytoskeleton in force transmission through the cell and on mitochondrial displacements. In addition, it is suggested that care must be taken when performing mechanical experiments on living cells with the AFM, as these local mechanical perturbations may have significant structural and even biochemical effects on the cell.
Asunto(s)
Microscopía de Fuerza Atómica/métodos , Mitocondrias/metabolismo , Nanotecnología/métodos , Animales , Fenómenos Biomecánicos , Membrana Celular/efectos de los fármacos , Citocalasina D/farmacología , Citoesqueleto/efectos de los fármacos , Ratones , Mitocondrias/efectos de los fármacos , Mitocondrias/ultraestructura , Células 3T3 NIH , Nocodazol/farmacologíaRESUMEN
Free-standing cantilevers, which directly translate specific biochemical reactions into micromechanical motion, have recently attracted much attention as label-free biosensors and micro/nano robotic devices. To exploit this mechanochemical sensing technology, it is essential to develop a fundamental understanding of the origins of surface stress. Here we report a detailed study into the molecular basis of stress generation in aqueous environments focusing on the pH titration of model mercaptohexadecanoic acid self-assembled monolayers (SAMs), using in situ reference cantilevers coated with nonionizable hexadecanethiol SAMs. Semiautomated data analysis and a statistical model were developed to quantify cyclic deprotonation/protonation reactions on multiple arrays. In-plane force titrations were found to have the sensitivity to detect ionic hydrogen bond formation between protonated and nonprotonated carboxylic acid groups in the proximity of the surface pK1/2, which generated a mean tensile differential surface stress of +1.2 +/- 0.3 mN/m at pH 6.0, corresponding to 1 pN attractive force between two adjacent MHA molecules. Conversely, the magnitude of compressive differential surface stress was found to increase progressively with pH >/= 7.0, reaching a maximum of -14.5 +/- 0.5 mN/m at pH 9.0, attributed to enhanced electrostatic repulsion between deprotonated carboxylic acid groups. However, striking differences were observed in the micromechanical responses to different ionic strength and ion species present in the aqueous environment, highlighting the critical role of counter- and co-ions on surface stress. Our findings provide fundamental insights into the molecular mechanisms of in-plane mechanochemistry, which may be exploited for biosensing and nanoactuation applications.
Asunto(s)
Técnicas Biosensibles/métodos , Ácidos Palmíticos/química , Protones , Enlace de Hidrógeno , Concentración de Iones de Hidrógeno , Concentración Osmolar , Sensibilidad y Especificidad , Factores de TiempoRESUMEN
Current models for protrusive motility in animal cells focus on cytoskeleton-based mechanisms, where localized protrusion is driven by local regulation of actin biochemistry. In plants and fungi, protrusion is driven primarily by hydrostatic pressure. For hydrostatic pressure to drive localized protrusion in animal cells, it would have to be locally regulated, but current models treating cytoplasm as an incompressible viscoelastic continuum or viscous liquid require that hydrostatic pressure equilibrates essentially instantaneously over the whole cell. Here, we use cell blebs as reporters of local pressure in the cytoplasm. When we locally perfuse blebbing cells with cortex-relaxing drugs to dissipate pressure on one side, blebbing continues on the untreated side, implying non-equilibration of pressure on scales of approximately 10 microm and 10 s. We can account for localization of pressure by considering the cytoplasm as a contractile, elastic network infiltrated by cytosol. Motion of the fluid relative to the network generates spatially heterogeneous transients in the pressure field, and can be described in the framework of poroelasticity.
Asunto(s)
Extensiones de la Superficie Celular/fisiología , Citoplasma/fisiología , Actinas/metabolismo , Animales , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Línea Celular , Extensiones de la Superficie Celular/efectos de los fármacos , Citoplasma/efectos de los fármacos , Citoesqueleto/efectos de los fármacos , Citoesqueleto/fisiología , Elasticidad , Presión Hidrostática , Microscopía por Video , Modelos Biológicos , Movimiento/efectos de los fármacos , Movimiento/fisiología , Perfusión , Estaurosporina/farmacología , Tiazoles/farmacología , Tiazolidinas , Factores de TiempoRESUMEN
Bone adapts to its environment by a process in which osteoblasts and osteocytes sense applied mechanical strain. One possible pathway for the detection of strain involves mechanosensitive channels and we sought to determine their sensitivity to membrane strain and tension. We used a combination of experimental and computational modeling techniques to gain new insights into cell mechanics and the regulation of mechanosensitive channels. Using patch-clamp electrophysiology combined with video microscopy, we recorded simultaneously the evolution of membrane extensions into the micropipette, applied pressure, and membrane currents. Nonselective mechanosensitive cation channels with a conductance of 15 pS were observed. Bleb aspiration into the micropipette was simulated using finite element models incorporating the cytoplasm, the actin cortex, the plasma membrane, cellular stiffening in response to strain, and adhesion between the membrane and the micropipette. Using this model, we examine the relative importance of the different cellular components in resisting suction into the pipette and estimate the membrane strains and tensions needed to open mechanosensitive channels. Radial membrane strains of 800% and tensions of 5 10(-4) N.m(-1) were needed to open 50% of mechanosensitive channels. We discuss the relevance of these results in the understanding of cellular reactions to mechanical strain and bone physiology.
Asunto(s)
Membrana Celular/fisiología , Membrana Celular/ultraestructura , Canales Iónicos/fisiología , Mecanotransducción Celular/fisiología , Modelos Biológicos , Osteoblastos/citología , Osteoblastos/fisiología , Animales , Animales Recién Nacidos , Permeabilidad de la Membrana Celular/fisiología , Células Cultivadas , Simulación por Computador , Elasticidad , Activación del Canal Iónico/fisiología , Fluidez de la Membrana/fisiología , Micromanipulación/métodos , Presión , Ratas , Estrés Mecánico , Resistencia a la TracciónRESUMEN
Many organs adapt to their mechanical environment as a result of physiological change or disease. Cells are both the detectors and effectors of this process. Though many studies have been performed in vitro to investigate the mechanisms of detection and adaptation to mechanical strains, the cellular strains remain unknown and results from different stimulation techniques cannot be compared. By combining experimental determination of cell profiles and elasticities by atomic force microscopy with finite element modeling and computational fluid dynamics, we report the cellular strain distributions exerted by common whole-cell straining techniques and from micromanipulation techniques, hence enabling their comparison. Using data from our own analyses and experiments performed by others, we examine the threshold of activation for different signal transduction processes and the strain components that they may detect. We show that modulating cell elasticity, by increasing the F-actin content of the cytoskeleton, or cellular Poisson ratio are good strategies to resist fluid shear or hydrostatic pressure. We report that stray fluid flow in some substrate-stretch systems elicits significant cellular strains. In conclusion, this technique shows promise in furthering our understanding of the interplay among mechanical forces, strain detection, gene expression, and cellular adaptation in physiology and disease.
Asunto(s)
Microscopía de Fuerza Atómica/métodos , Osteoblastos/citología , Actinas/metabolismo , Animales , Fenómenos Biofísicos , Biofisica , Células Cultivadas , Citoesqueleto/metabolismo , Magnetismo , Microscopía Confocal , Modelos Teóricos , Distribución de Poisson , Ratas , Transducción de Señal , Programas Informáticos , Estrés MecánicoRESUMEN
The skeleton adapts to its mechanical usage, although at the cellular level, the distribution and magnitude of strains generated and their detection are ill-understood. The magnitude and nature of the strains to which cells respond were investigated using an atomic force microscope (AFM) as a microindentor. A confocal microscope linked to the setup enabled analysis of cellular responses. Two different cell response pathways were identified: one, consequent upon contact, depended on activation of stretch-activated ion channels; the second, following stress relaxation, required an intact microtubular cytoskeleton. The cellular responses could be modulated by selectively disrupting cytoskeletal components thought to be involved in the transduction of mechanical stimuli. The F-actin cytoskeleton was not required for responses to mechanical strain, whereas the microtubular and vimentin networks were. Treatments that reduced membrane tension, or its transmission, selectively reduced contact reactions. Immunostaining of the cell cytoskeleton was used to interpret the results of the cytoskeletal disruption studies. We provide an estimate of the cellular strain magnitude needed to elicit intracellular calcium responses and propose a model that links single cell responses to whole bone adaptation. This technique may help to understand adaptation to mechanical usage in other organs.