Distinct NF-kappaB regulation by shear stress through Ras-dependent IkappaBalpha oscillations: real-time analysis of flow-mediated activation in live cells.
ABSTRACT NF-kappaB, a transcription factor central to inflammatory regulation during development of atherosclerosis, is activated by soluble mediators and through biomechanical inputs such as flow-mediated shear- stress. To investigate the molecular mechanisms underlying shear stress mediated signal transduction in vascular cells we have developed a system that applies flow-mediated shear stress in a controlled manner, while inserted in a confocal microscope. In combination with GFP-based methods, this allows continuous monitoring of flow induced signal transduction in live cells and in real time. Flow-mediated shear stress, induced using the system, caused a successive increase in NF-kappaB-regulated gene activation. Experiments assessing the mechanisms underlying the NF-kappaB induced activity showed time and flow rate dependent effects on the inhibitor, IkappaBalpha, involving nuclear translocation characterized by a biphasic or cyclic pattern. The effect was observed in both endothelial- and smooth muscle cells, demonstrated to impact noncomplexed IkappaBalpha, and to involve mechanisms distinct from those mediating cytokine signals. In contrast, effects on the NF-kappaB subunit relA were similar to those observed during cytokine stimulation. Further experiments showed the flow induced inter-compartmental transport of IkappaBalpha to be regulated through the Ras GTP-ase, demonstrating a pronounced reduction in the effects following blocking of Ras activity. These studies show that flow-mediated shear stress, regulated by the Ras GTP-ase, uses distinct mechanisms of NF-kappaB control at the molecular level. The oscillatory pattern, reflecting inter-compartmental translocation of IkappaBetaalpha, is likely to have fundamental impact on pathway regulation and on development of shear stress-induced distinct vascular cell phenotypes.
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ABSTRACT: Fluorescence proteins (FPs) have been widely used for live-cell imaging in the past decade. This review summarizes the recent advances in FP development and imaging technologies using FPs to monitor molecular localization and activities and gene expressions in live cells. We also discuss the utilization of FPs to develop molecular biosensors and the principles and application of advanced technologies such as fluorescence resonance energy transfer (FRET), fluorescence recovery after photobleaching (FRAP), fluorescence lifetime imaging microscopy (FLIM), and chromophore-assisted light inactivation (CALI). We present examples of the application of FPs and biosensors to visualize mechanotransduction events with high spatiotemporal resolutions in live cells. These live-cell imaging technologies, which represent a frontier area in biomedical engineering, can shed new light on the mechanisms regulating mechanobiology at cellular and molecular levels in normal and pathophysiological conditions.Annual Review of Biomedical Engineering 08/2008; 10:1-38. · 12.45 Impact Factor
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ABSTRACT: Endothelial cells (ECs) are constantly exposed to chemical and mechanical microenvironment in vivo. In mechanotransduction, cells can sense and translate the extracellular mechanical cues into intracellular biochemical signals, to regulate cellular processes. This regulation is crucial for many physiological functions, such as cell adhesion, migration, proliferation, and survival, as well as the progression of disease such as atherosclerosis. Here, we overview the current molecular understanding of mechanotransduction in ECs associated with atherosclerosis, especially those in response to physiological shear stress. The enabling technology of live-cell imaging has allowed the study of spatiotemporal molecular events and unprecedented understanding of intracellular signaling responses in mechanotransduction. Hence, we also introduce recent studies on mechanotransduction using single-cell imaging technologies.Progress in molecular biology and translational science 01/2014; 126:25-51. · 3.11 Impact Factor
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ABSTRACT: Src kinase, the first tyrosine kinase discovered, has been shown to play critical roles in a variety of cellular processes, including cell motility/migration, mechanotranduction, and cancer development. Based on fluorescent resonance energy transfer (FRET), we have developed and characterized a genetically encoded single-molecule Src biosensor, which enables the imaging and quantification of temporal-spatial activation of Src in live cells. In this paper, we summarize the application of this biosensor to study a variety of cellular functions. First, we introduced a local mechanical stimulation by applying laser-tweezer-induced traction on fibronectin-coated beads adhered to the cells. Using a membrane-anchored Src biosensor, we observed a wave propagation of Src activation in a direction opposite to the applied force. This Src reporter was also applied to visualize the interplays between cell-cell and cell-ECM adhesions. The results indicate that integrin-ligation can induce Src activation around cell-cell junctions and cause the disruption of adherens junctions. Lastly, the flow-induced dynamic Src activation at subcellular levels was visualized by the FRET biosensor simultaneously with actin-fused mCherry, a red fluorescence protein. Our results indicate that shear stress induced a moderate up-regulation of Src activation in the whole cell, but a significant translocation of active Src from perinuclear regions toward cell periphery. In summary, our novel Src biosensor has made it possible to monitor key signaling transduction cascades involving Src in live cells with temporal-spatial characterization in mechanobiology.Proceedings of SPIE - The International Society for Optical Engineering 02/2008; · 0.20 Impact Factor