Yingxiao Wang

Visualizing the mechanical activation of Src

The mechanical environment crucially influences many cell functions. However, it remains largely mysterious how mechanical stimuli are transmitted into biochemical signals. Src is known to regulate the integrin–cytoskeleton interaction, which is essential for the transduction of mechanical stimuli. Using fluorescent resonance energy transfer (FRET), here we develop a genetically encoded Src reporter that enables the imaging and quantification of spatio-temporal activation of Src in live cells. We introduced a local mechanical stimulation to human umbilical vein endothelial cells (HUVECs) by applying laser-tweezer traction on fibronectin-coated beads adhering to the cells. Using the Src reporter, we observed a rapid distal Src activation and a slower directional wave propagation of Src activation along the plasma membrane. This wave propagated away from the stimulation site with a speed (mean ± s.e.m.) of 18.1 ± 1.7 nm s-1. This force-induced directional and long-range activation of Src was abolished by the disruption of actin filaments or microtubules. Our reporter has thus made it possible to monitor mechanotransduction in live cells with spatio-temporal characterization. We find that the transmission of mechanically induced Src activation is a dynamic process that directs signals via the cytoskeleton to spatial destinations.

Rapid signal transduction in living cells is a unique feature of mechanotransduction

It is widely postulated that mechanotransduction is initiated at the local force–membrane interface by inducing local conformational changes of proteins, similar to soluble ligand-induced signal transduction. However, all published reports are limited in time scale to address this fundamental issue. Using a FRET-based cytosolic Src reporter in a living cell, we quantified changes of Src activities as a local stress via activated integrins was applied. The stress induced rapid (<0.3 s) activation of Src at remote cytoplasmic sites, which depends on the cytoskeletal prestress. In contrast, there was no Src activation within 12 s of soluble epidermal growth factor (EGF) stimulation. A 1.8-Pa stress over a focal adhesion activated Src to the same extent as 0.4 ng/ml EGF at long times (minutes), and the energy levels for mechanical stimulation and chemical stimulation were comparable. The effect of both stress and EGF was less than additive. Nanometer-scale cytoskeletal deformation analyses revealed that the strong activation sites of Src by stress colocalized with large deformation sites of microtubules, suggesting that microtubules are essential structures for transmitting stresses to activate cytoplasmic proteins. These results demonstrate that rapid signal transduction via the prestressed cytoskeleton is a unique feature of mechanotransduction.

Fluorescence Proteins, Live-Cell Imaging, and Mechanobiology: Seeing Is Believing

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.

The role of the dynamics of focal adhesion kinase in the mechanotaxis of endothelial cells.

The migration of vascular endothelial cells (ECs) is critical in vascular remodeling. We showed that fluid shear stress enhanced EC migration in flow direction and called this “mechanotaxis.” To visualize the molecular dynamics of focal adhesion kinase (FAK) at focal adhesions (FAs), FAK tagged with green fluorescence protein (GFP) was expressed in ECs. Within 10 min of shear stress application, lamellipodial protrusion was induced at cell periphery in the flow direction, with the recruitment of FAK at FAs. ECs under flow migrated with polarized formation of new FAs in flow direction, and these newly formed FAs subsequently disassembled after the rear of the cell moved over them. The cells migrating under flow had a decreased number of FAs. In contrast to shear stress, serum did not significantly affect the speed of cell migration. Serum induced lamellipodia and FAK recruitment at FAs without directional preference. FAK(Y397) phosphorylation colocalized with GFP-FAK at FAs in both shear stress and serum experiments. The total level of FAK(Y397) phosphorylation after shear stress was lower than that after serum treatment, suggesting that the polarized change at cell periphery rather than the total level of FAK(Y397) phosphorylation is important for directional migration. Our results demonstrate the dynamics of FAK at FAs during the directional migration of EC in response to mechanical force, and suggest that mechanotaxis is an important mechanism controlling EC migration.

Determination of hierarchical relationship of Src and Rac at subcellular locations with FRET biosensors

Genetically encoded biosensors based on FRET have enabled the visualization of signaling events in live cells with high spatiotemporal resolution. However, the limited sensitivity of these biosensors has hindered their broad application in biological studies. We have paired enhanced CFP (ECFP) with YPet, a variant of YFP. This ECFP/YPet FRET pair markedly enhanced the sensitivity of biosensors (several folds enhancement without the need of tailored optimization for each individual biosensor) for a variety of signaling molecules, including tyrosine kinase Src, small GTPase Rac, calcium, and a membrane-bound matrix metalloproteinase MT1-MMP. The application of these improved biosensors revealed that the activations of Src and Rac by PDGF displayed distinct subcellular patterns during directional cell migration on micropatterned surface. The activity of Rac is highly polarized and concentrated at the leading edge, whereas Src activity is relatively uniform. These FRET biosensors also led to the discovery that Src and Rac mutually regulate each other. Our findings indicate that molecules within the same signaling feedback loop can be differentially regulated at different subcellular locations. In summary, ECFP/YPet may serve as a general FRET pair for the development of highly sensitive biosensors to allow the determination of molecular hierarchies at subcellular locations in live cells.

Effects of Flow Patterns on the Localization and Expression of VE-Cadherin at Vascular Endothelial Cell Junctions: In vivo and in vitro Investigations

Atherosclerosis occurs preferentially at vascular curvature and branch sites where the vessel walls are exposed to fluctuating shear stress and have high endothelial permeability. Endothelial permeability is modulated by intercellular adhesion molecules such as VE-cadherin. This study was designed to elucidate the effects of different flow patterns on the localization and expression of VE-cadherin in endothelial cells (ECs) both in vivo and in vitro. VE-cadherin staining at EC borders was much stronger in the descending thoracic aorta and abdominal aorta, where the pulsatile flow has a strong net forward component than in the aortic arch and the poststenotic dilatation site beyond an experimental constriction, where the flow near the wall is complex and reciprocating with little net flow. With the use of flow chambers the effects of pulsatile flow (12 ± 4 dyn/cm2 at 1 Hz) and reciprocating flow (0.5 ± 4 dyn/cm2 at 1 Hz) on VE-cadherin organization in endothelial monolayers were studied in vitro. VE-cadherin staining was continuous along cell borders in static controls. Following 6 h of either pulsatile or reciprocating flow, the VE-cadherin staining at cell borders became intermittent. When the pulsatile flow was extended to 24, 48 or 72 h the staining around the cell borders became continuous again, but the staining was still intermittent when the reciprocating flow was similarly extended. Exposure to pulsatile or reciprocating flow for 6 and 24 h neither change the expression level of VE-cadherin nor its distribution between membrane and cytosol fractions as determined by Western blot and compared with static controls. These findings suggest that the cell junction remodeling induced by different flow patterns may result from a redistribution of VE-cadherin within the cell membrane. Both the in vivo and in vitro data indicate that pulsatile and reciprocating flow patterns have different effects on cell junction remodeling. The lack of junction reorganization in regions of reciprocating flow in vivo and in vitro may provide a mechanistic basis for the high permeability and the preferential localization of atherosclerosis in regions of the arterial stress with complex flow patterns and fluctuating shear stress.

Substrate rigidity regulates Ca2+ oscillation via RhoA pathway in stem cells.

Substrate rigidity plays crucial roles in regulating cellular functions, such as cell spreading, traction forces, and stem cell differentiation. However, it is not clear how substrate rigidity influences early cell signaling events such as calcium in living cells. Using highly sensitive Ca2+ biosensors based on fluorescence resonance energy transfer (FRET), we investigated the molecular mechanism by which substrate rigidity affects calcium signaling in human mesenchymal stem cells (HMSCs). Spontaneous Ca2+ oscillations were observed inside the cytoplasm and the endoplasmic reticulum (ER) using the FRET biosensors targeted at subcellular locations in cells plated on rigid dishes. Lowering the substrate stiffness to 1 kPa significantly inhibited both the magnitudes and frequencies of the cytoplasmic Ca2+ oscillation in comparison to stiffer or rigid substrate. This Ca2+ oscillation was shown to be dependent on ROCK, a downstream effector molecule of RhoA, but independent of actin filaments, microtubules, myosin light chain kinase, or myosin activity. Lysophosphatidic acid, which activates RhoA, also inhibited the frequency of the Ca2+ oscillation. Consistently, either a constitutive active mutant of RhoA (RhoA‐V14) or a dominant negative mutant of RhoA (RhoA‐N19) inhibited the Ca2+ oscillation. Further experiments revealed that HMSCs cultured on gels with low elastic moduli displayed low RhoA activities. Therefore, our results demonstrate that RhoA and its downstream molecule ROCK may mediate the substrate rigidity‐regulated Ca2+ oscillation, which determines the physiological functions of HMSCs. J. Cell. Physiol. 218: 285–293, 2009.

Simultaneous Visualization of Protumorigenic Src and MT1-MMP Activities with Fluorescence Resonance Energy Transfer

Both Src kinase and membrane type 1 matrix metalloproteinase (MT1-MMP) play critical roles in cancer invasion and metastasis. It is not clear, however, how the spatiotemporal activation of these two critical enzymes is coordinated in response to an oncogenic epithelial growth factor (EGF) stimulation. Here, we have visualized the activities of Src and MT1-MMP concurrently in a single live cell by combining two fluorescence resonance energy transfer (FRET) pairs with distinct spectra: (a) cyan fluorescent protein (CFP) and yellow FP (YFP), and (b) orange FP (mOrange2) and red FP (mCherry). The new FRET pair, mOrange2 and mCherry, was first characterized in vitro and in cultured mammalian cells. When integrated with the CFP/YFP pair, this new pair allowed the revelation of an immediate, rapid, and relatively dispersed Src activity. In contrast, the MT1-MMP activity displayed a slow increase at the cell periphery, although Src was shown to play a role upstream to MT1-MMP globally. This difference in the activation patterns of MT1-MMP and Src in response to EGF is further confirmed using an optimized MT1-MMP biosensor capable of being rapidly cleaved by MT1-MMP. The results indicate that although Src and MT1-MMP act globally in the same signaling pathway, their activations differ in space and time upon EGF stimulation, possibly mediated by different sets of intermediates at different subcellular locations. Our results also showed the potential of mOrange2/mCherry as a new FRET pair, together with the popular variants of CFP and YFP, for the simultaneous visualization of multiple molecular activities in a single live cell.

Intravital FLIM-FRET Imaging Reveals Dasatinib-Induced Spatial Control of Src in Pancreatic Cancer

Cancer invasion and metastasis occur in a complex three-dimensional (3D) environment, with reciprocal feedback from the surrounding host tissue and vasculature-governing behavior. In this study, we used a novel intravital method that revealed spatiotemporal regulation of Src activity in response to the anti-invasive Src inhibitor dasatinib. A fluorescence lifetime imaging microscopy-fluorescence resonance energy transfer (FLIM-FRET) Src biosensor was used to monitor drug-targeting efficacy in a transgenic p53-mutant mouse model of pancreatic cancer. In contrast to conventional techniques, FLIM-FRET analysis allowed for accurate, time-dependent, live monitoring of drug efficacy and clearance in live tumors. In 3D organotypic cultures, we showed that a spatially distinct gradient of Src activity exists within invading tumor cells, governed by the depth of penetration into complex matrices. In parallel, this gradient was also found to exist within live tumors, where Src activity is enhanced at the invasive border relative to the tumor cortex. Upon treatment with dasatinib, we observed a switch in activity at the invasive borders, correlating with impaired metastatic capacity in vivo. Src regulation was governed by the proximity of cells to the host vasculature, as cells distal to the vasculature were regulated differentially in response to drug treatment compared with cells proximal to the vasculature. Overall, our results in live tumors revealed that a threshold of drug penetrance exists in vivo and that this can be used to map areas of poor drug-targeting efficiency within specific tumor microenvironments. We propose that using FLIM-FRET in this capacity could provide a useful preclinical tool in animal models before clinical translation.

Visualization of Polarized Membrane Type 1 Matrix Metalloproteinase Activity in Live Cells by Fluorescence Resonance Energy Transfer Imaging

Membrane type 1 matrix metalloproteinase (MT1-MMP) plays a critical role in cancer cell biology by proteolytically remodeling the extracellular matrix. Utilizing fluorescence resonance energy transfer (FRET) imaging, we have developed a novel biosensor, with its sensing element anchoring at the extracellular surface of cell membrane, to visualize MT1-MMP activity dynamically in live cells with subcellular resolution. Epidermal growth factor (EGF) induced significant FRET changes in cancer cells expressing MT1-MMP, but not in MT1-MMP-deficient cells. EGF-induced FRET changes in MT1-MMP-deficient cells could be restored after reconstituting with wild-type MT1-MMP, but not MMP-2, MMP-9, or inactive MT1-MMP mutants. Deletion of the transmembrane domain in the biosensor or treatment with tissue inhibitor of metalloproteinase-2, a cell-impermeable MT1-MMP inhibitor, abolished the EGF-induced FRET response, indicating that MT1-MMP acts at the cell surface to generate FRET changes. In response to EGF, active MT1-MMP was directed to the leading edge of migrating cells along micropatterned fibronectin stripes, in tandem with the local accumulation of the EGF receptor, via a process dependent upon an intact cytoskeletal network. Hence, the MT1-MMP biosensor provides a powerful tool for characterizing the molecular processes underlying the spatiotemporal regulation of this critical class of enzymes.