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In JoVE (1)
Other Publications (11)
- Microcirculation (New York, N.Y. : 1994)
- Biophysical Journal
- American Journal of Physiology. Heart and Circulatory Physiology
- Journal of Biomedical Optics
- Biomacromolecules
- Journal of Biomedical Optics
- Current Protocols in Microbiology
- Current Protocols in Microbiology
- Journal of Biomedical Optics
- Journal of Molecular Recognition : JMR
- Experimental Cell Research
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Articles by Andreea Trache in JoVE
JoVE General
लाइव सेल यांत्रिक उत्तेजना उत्तर एकीकृत ऑप्टिकल और परमाणु शक्ति माइक्रोस्कोपी द्वारा पढ़ाई
1Department of Systems Biology and Translational Medicine, College of Medicine, Cardiovascular Research Institute, Texas A&M Health Science Center, 2Department of Biomedical Engineering, Texas A&M University
इस कागज संस्कृति में जीवित कोशिकाओं के यांत्रिक उत्तेजना के लिए एक एकीकृत परमाणु के आपरेशन में पाठक बल ऑप्टिकल इमेजिंग खुर्दबीन हिदायत करना है. एक कदम-by-कदम प्रोटोकॉल प्रस्तुत किया है. एक प्रतिनिधि डेटा सेट है कि रहते सेल यांत्रिक उत्तेजना के जवाब से पता चलता है प्रस्तुत किया है.
Other articles by Andreea Trache on PubMed
Integrins and Regulation of the Microcirculation: from Arterioles to Molecular Studies Using Atomic Force Microscopy
Integrins are an important class of receptors for extracellular matrix proteins that can mediate both force transmission, by virtue of their connections with the cell matrix and cytoskeleton; and signal transduction, resulting from the assemblages of signaling proteins that associate with focal contacts. Consequently, integrins have been proposed to be the mechanosensor in vascular smooth muscle and endothelial cells and to play a central role in mechanotransduction. In this regard, mechanical force is an important stimulus for many vascular functions, including contractile and relaxation processes,proliferation, migration, attachment, and cell phenotype determination. Collectively, these functions define physiological properties of the vasculature such as control of blood flow, capillary pressure,permeability, and peripheral vascular resistance, and play a role in pathophysiological processes like hypertension, diabetes, and arteriosclerosis. Our knowledge concerning how integrins sense and transduce physical forces into cellular signals and which integrins are involved is incomplete. Compared to other cell surface receptors, integrins have a relatively low affinity for their binding sites on the extracellular matrix and their affinity can be regulated. These characteristics of integrin-ligand interaction may facilitate dynamic processes such as cell migration, cell remodeling, and contractile activation in response to external forces. Important questions remain concerning the nature and origin of integrin-mediated signaling in the vascular wall.
Histamine Effects on Endothelial Cell Fibronectin Interaction Studied by Atomic Force Microscopy
Atomic force microscopy was used to investigate the cellular response to histamine, one of the major inflammatory mediators that cause endothelial hyperpermeability and vascular leakage. AFM probes were labeled with fibronectin and used to measure binding strength between alpha5beta1 integrin and fibronectin by quantifying the force required to break single fibronectin-integrin bonds. The cytoskeletal changes, binding probability, and adhesion force before and after histamine treatment on endothelial cells were monitored. Cell topography measurements indicated that histamine induces cell shrinkage. Local cell stiffness and binding probability increased twofold after histamine treatment. The force necessary to rupture single alpha5beta1-fibronectin bond increased from 34.0 +/- 0.5 pN in control cells to 39 +/- 1 pN after histamine treatment. Experiments were also conducted to confirm the specificity of the alpha5beta1-fibronectin interaction. In the presence of soluble GRGDdSP the probability of adhesion events decreased >50% whereas the adhesion force between alpha5beta1 and fibronectin remained unchanged. These data indicate that extracellular matrix-integrin interactions play an important role in the endothelial cell response to changes of external chemical mediators. These changes can be recorded as direct measurements on live endothelial cells by using atomic force microscopy.
Mechanical Properties of the Interaction Between Fibronectin and Alpha5beta1-integrin on Vascular Smooth Muscle Cells Studied Using Atomic Force Microscopy
The mechanical properties of integrin-extracellular matrix (ECM) interactions are important for the mechanotransduction of vascular smooth muscle cells (VSMC), a process that is associated with focal adhesions, and can be of particular significance in cardiovascular disease. In this study, we characterized the unbinding force and binding activity of the initial fibronectin (FN)-alpha5beta1 interaction on the surface of VSMC using atomic force microscopy (AFM). It is postulated that these initial binding events are important to the subsequent focal adhesion assembly. FN-VSMC adhesions were selectively blocked by antibodies against alpha5- and beta1-integrins as well as RGD-containing peptides but not by antibodies against alpha4- and beta3-integrins, indicating that FN primarily bound to alpha5beta1. A characteristic unbinding force of 39 +/- 8 pN was observed and interpreted to represent the FN-alpha5beta1 single-bond strength. The ability of FN to adhere to VSMC (binding probability) was significantly reduced by integrin antagonists, serum starvation, and platelet-derived growth factor (PDGF)-BB, whereas lysophosphatidic acid (LPA) increased FN binding. However, no significant change in the resolved unbinding force was observed. After engagement, the force required to dislodge the FN-coated bead from VSMC increased with increasing of contact time, suggesting a time-dependent increase in number of adhesions and/or altered binding affinity. LPA enhanced this process, whereas PDGF reduced it, suggesting that these factors also affect the multimolecular process of focal contact assembly. Thus AFM is a powerful tool for the characterization of the mechanical properties of integrin-ECM interactions and their regulation. Our results indicate that the functional activity of alpha5beta1 and focal contact assembly can be rapidly regulated.
Atomic Force-multi-optical Imaging Integrated Microscope for Monitoring Molecular Dynamics in Live Cells
A novel hybrid imaging system is constructed integrating atomic force microscopy (AFM) with a combination of optical imaging techniques that offer high spatial resolution. The main application of this instrument (the NanoFluor microscope) is the study of mechanotransduction with an emphasis on extracellular matrix-integrin-cytoskeletal interactions and their role in the cellular responses to changes in external chemical and mechanical factors. The AFM allows the quantitative assessment of cytoskeletal changes, binding probability, adhesion forces, and micromechanical properties of the cells, while the optical imaging applications allow thin sectioning of the cell body at the coverslip-cell interface, permitting the study of focal adhesions using total internal reflection fluorescence (TIRF) and internal reflection microscopy (IRM). Combined AFM-optical imaging experiments show that mechanical stimulation at the apical surface of cells induces a force-generating cytoskeletal response, resulting in focal contact reorganization on the basal surface that can be monitored in real time. The NanoFluor system is also equipped with a novel mechanically aligned dual camera acquisition system for synthesized Forster resonance energy transfer (FRET). The integrated NanoFluor microscope system is described, including its characteristics, applications, and limitations.
Quantification and Confocal Imaging of Protein Specific Molecularly Imprinted Polymers
We have employed FITC--albumin as the protein template molecule in an aqueous phase molecular imprinted polymer (HydroMIP) strategy. For the first time, the use of a fluorescently labeled template is reported, with subsequent characterization of the smart material to show that the HydroMIP possesses a significant molecular memory in comparison to that of the nonimprinted control polymer (HydroNIP). The imaging of the FITC--albumin imprinted HydroMIP using confocal microscopy is described, with the in situ removal of the imprinted protein displayed in terms of observed changes in the fluorescence of the imprinted polymer, both before and after template elution (using a 10% SDS/10% AcOH (w/v) solution). We also report the imaging of a bovine hemoglobin (BHb) imprinted HydroMIP using two-photon confocal microscopy and describe the effects of template elution upon protein autofluorescence. The findings further contribute to the understanding of aqueous phase molecular imprinting protocols and document the use of fluorescence as a useful tool in template labeling/detection and novel imaging strategies.
Use of Surface-enhanced Raman Spectroscopy for the Detection of Human Integrins
Current research has revealed the importance of a class of cell surface proteins called integrins in various vital physiological functions such as blood clotting, regulation of blood pressure, tissue blood flow, and vascular remodeling. The key to integrin functionality is its ability to mediate force transmission by interacting with the extracellular matrix and cytoskeleton. In addition, they play a role in signal transduction via their connection with the proteins in focal adhesion (FA) points. To understand the complex mechanism of cell-cell and cell-extracellular matrix (ECM) adhesion that is responsible for these diverse biochemical interactions, it is necessary to identify the integrins on cells and monitor their interaction with various ligands. To this end, for the first time, we employ surface-enhanced Raman spectroscopy (SERS) to detect integrins. The results show the capability using SERS to detect the integrins to the nanomolar concentration regime and to distinguish between two different kinds of integrins, alphaVbeta3 and alpha5beta1, that are present in vascular smooth muscle cells (VSMCs). It is anticipated that the SERS approach will potentially help elucidate the mechanism of integrin-ligand interactions in a variety of phenomena of physiological importance.
Atomic Force Microscopy (AFM)
The atomic force microscope (AFM) is an important tool for studying biological samples due to its ability to image surfaces under liquids. The AFM operates by physical interaction of a cantilever tip with the molecules on the cell surface. Adhesion forces between the tip and cell surface molecules are detected as cantilever deflections. Thus, the cantilever tip can be used to image live cells with atomic resolution and to probe single molecular events in living cells under physiological conditions. Currently, this is the only technique available that directly provides structural, mechanical, and functional information at high resolution. This unit presents the basic AFM components, modes of operation, useful tips for sample preparation, and a short review of AFM applications in microbiology.
Total Internal Reflection Fluorescence (TIRF) Microscopy
Total internal reflection fluorescence (TIRF) microscopy represents a method of exciting and visualizing fluorophores present in the near-membrane region of live or fixed cells grown on coverslips. TIRF microscopy is based on the total internal reflection phenomenon that occurs when light passes from a high-refractive medium (e.g., glass) into a low-refractive medium (e.g., cell, water). The evanescent field produced by total internally reflected light excites the fluorescent molecules at the cell-substrate interface and is accompanied by minimal exposure of the remaining cell volume. This technique provides high-contrast fluorescence images, with very low background and virtually no out-of-focus light, ideal for visualization and spectroscopy of single-molecule fluorescence near a surface. This unit presents, in a concise manner, the principle of operation, instrument diversity, and TIRF microscopy applications for the study of biological samples.
Integrated Microscopy for Real-time Imaging of Mechanotransduction Studies in Live Cells
Mechanical force is an important stimulus and determinant of many vascular smooth muscle cell functions including contraction, proliferation, migration, and cell attachment. Transmission of force from outside the cell through focal adhesions controls the dynamics of these adhesion sites and initiates intracellular signaling cascades that alter cellular behavior. To understand the mechanism by which living cells sense mechanical forces, and how they respond and adapt to their environment, a critical first step is to develop a new technology to investigate cellular behavior at subcellular level that integrates an atomic force microscope (AFM) with total internal reflection fluorescence (TIRF) and fast-spinning disk (FSD) confocal microscopy, providing high spatial and temporal resolution. AFM uses a nanosensor to measure the cell surface topography and can apply and measure mechanical force with high precision. TIRF microscopy is an optical imaging technique that provides high-contrast images with high z-resolution of fluorescently labeled molecules in the immediate vicinity of the cell-coverslip interface. FSD confocal microscopy allows rapid 3-D imaging throughout the cell in real time. The integrated system is broadly applicable across a wide range of molecular dynamic studies in any adherent live cells, allowing direct optical imaging of cell responses to mechanical stimulation in real time.
Mg2+ Modulates Integrin-extracellular Matrix Interaction in Vascular Smooth Muscle Cells Studied by Atomic Force Microscopy
Atomic force microscopy (AFM) was used to investigate the interaction between alpha5beta1 integrin and fibronectin (FN) in the presence of divalent cations. AFM probes were labeled with FN and used to measure binding strength between alpha5beta1 integrin and FN by quantifying the force required to break single FN-integrin bonds on a physiological range of loading rates (100-10,000 pN/s). The force necessary to rupture single alpha5beta1-FN bond increased twofold over the regime of loading rates investigated. Changes in Mg(2+) and Ca(2+) concentration affected the thermodynamical parameters of the interaction and modulated the binding energy. These data indicate that the external ionic environment in which vascular smooth muscle cells reside, influences the mechanical parameters that define the interaction between the extracellular matrix and integrins. Thus, in a dynamic mechanical environment such as the vascular wall, thermodynamic binding properties between FN and alpha5beta1 integrin vary in relation to locally applied loads and divalent cations concentrations. These changes can be recorded as direct measurements on live smooth muscle cells by using AFM.
Extracellular Matrix Effect on RhoA Signaling Modulation in Vascular Smooth Muscle Cells
Morphological adaptations of vascular smooth muscle cells (VSMC) to the mechanically active environment in which they reside, are mediated by direct interactions with the extracellular matrix (ECM) which induces physiological changes at the intracellular level. This study aimed to analyze the effects of the ECM on RhoA-induced mechanical signaling that controls actin organization and focal adhesion formation. VSMC were transfected with RhoA constructs (wild type, dominant negative or constitutively active) and plated on different ECM proteins used as substrate (fibronectin, collagen IV, collagen I, and laminin) or poly-l-lysine as control. Morphological changes of the VSMC were detected by fluorescence confocal microscopy and total internal reflection fluorescence (TIRF) microscopy, and were independently verified using adhesion assays and Western blot analysis. Our results showed that the ECM has an important role in cell spreading, adhesion and morphology with a direct effect on modulating RhoA signaling. RhoA activity significantly affected the stress fibers and focal adhesions reorganization, but in a context imposed by the ECM. Thus, RhoA activity modulation in VSMC induced an increased activation of stress fibers and FA formation at 5h, while a significant inhibition was recorded at 24h after plating on the different ECM. Our findings provide biophysical evidence that ECM modulates VSMC response to mechanical stimuli inducing intracellular biochemical signaling involved in cellular adaptation to the local microenvironment.
