Development of FRET Biosensor to Characterize CSK Subcellular Regulation.

C-terminal Src kinase (CSK) is the major inhibitory kinase for Src family kinases (SFKs) through the phosphorylation of their C-tail tyrosine sites, and it regulates various types of cellular activity in association with SFK function. As a cytoplasmic protein, CSK needs be recruited to the plasma membrane to regulate SFKs' activity. The regulatory mechanism behind CSK activity and its subcellular localization remains largely unclear. In this work, we developed a genetically encoded biosensor based on fluorescence resonance energy transfer (FRET) to visualize the CSK activity in live cells. The biosensor, with an optimized substrate peptide, confirmed the crucial Arg107 site in the CSK SH2 domain and displayed sensitivity and specificity to CSK activity, while showing minor responses to co-transfected Src and Fyn. FRET measurements showed that CSK had a relatively mild level of kinase activity in comparison to Src and Fyn in rat airway smooth muscle cells. The biosensor tagged with different submembrane-targeting signals detected CSK activity at both non-lipid raft and lipid raft microregions, while it showed a higher FRET level at non-lipid ones. Co-transfected receptor-type protein tyrosine phosphatase alpha (PTPα) had an inhibitory effect on the CSK FRET response. The biosensor did not detect obvious changes in CSK activity between metastatic cancer cells and normal ones. In conclusion, a novel FRET biosensor was generated to monitor CSK activity and demonstrated CSK activity existing in both non-lipid and lipid raft membrane microregions, being more present at non-lipid ones.

Deficiency of Integrin ß4 Results in Increased Lung Tissue Stiffness and Responds to Substrate Stiffness via Modulating RhoA Activity.

The interaction between extracellular matrix (ECM) and epithelial cells plays a key role in lung development. Our studies found that mice with conditional integrin ß4 (ITGB4) knockout presented lung dysplasia and increased stiffness of lung tissues. In accordance with our previous studies regarding the functions of ITGB4 in bronchial epithelial cells (BECs), we hypothesize that the decreased ITGB4 expression during embryonic stage leads to abnormal ECM remodeling and increased tissue stiffness, thus impairing BECs motility and compromising lung development. In this study, we examined lung tissue stiffness in normal and ITGB4 deficiency mice using Atomic Force Microscopy (AFM), and demonstrated that ITGB4 deficiency resulted in increased lung tissue stiffness. The examination of ECM components collagen, elastin, and lysyl oxidase (LOX) family showed that the expression of type VI collagen, elastin and LOXL4 were significantly elevated in the ITGB4-deficiency mice, compared with those in normal groups. Airway epithelial cell migration and proliferation capacities on normal and stiff substrates were evaluated through video-microscopy and flow cytometry. The morphology of the cytoskeleton was detected by laser confocal microscopy, and RhoA activities were determined by fluorescence resonance energy transfer (FRET) microscopy. The results showed that migration and proliferation of ITGB4 deficiency cells were noticeably inhibited, along decreased cytoskeleton stabilization, and hampered RhoA activity, especially for cells cultured on the stiff substrate. These results suggest that decreased ITGB4 expression results in increased lung tissue stiffness and impairs the adaptation of bronchial epithelial cells to substrate stiffness, which may be related to the occurrence of broncho pulmonary dysplasia.

Integrin-ß4 regulates the dynamic changes of phenotypic characteristics in association with epithelial-mesenchymal transition (EMT) and RhoA activity in airway epithelial cells during injury and repair.

Background: In airway disease such as asthma a hyperactive cellular event of epithelial-mesenchymal transition (EMT) is considered as the mechanism of pathological airway tissue remodeling after injury to the airway epithelium. And the initiation of EMT in the airways depends on the epithelial disruption involving dissolution and/or destabilization of the adhesive structures between the cells and ECM. Previously, we have shown that integrin-ß4, an epithelial adhesion molecule in bronchial epithelium is an important regulator of cell proliferation and wound repair in human airway epithelial cells. Therefore, in this study we aimed to investigate whether integrin-ß4 also regulates EMT phenotypes during injury and repair in airway epithelial cells of both wild type/integrin-ß4-/- mice in vivo and cultured cells treated with integrin-ß4/nonsense siRNA in vitro. Methods: We induced injury to the airway epithelial cells by either repeated exposure to ozone and mechanical scratch wound, and subsequently examined the EMT-related phenotypic features in the airway epithelial cells including biomarkers expression, adhesion and cytoskeleton reorganization and cell stiffness. Results: The results show that in response to injury (ozone exposure/scratch wound) and subsequent spontaneous repair (ozone withdrawal/wound healing) both in vivo and in vitro, the airway epithelial cells underwent dynamic changes in the epithelial and mesenchymal biomarkers expression, adhesion and cytoskeleton structures as well as cell stiffness, all together exhibiting enhanced EMT phenotypic features after injury and reversal of the injury-induced effects during repair. Importantly, these injury/repair-associated EMT phenotypic changes in airway epithelial cells appeared to be dependent on integrin-ß4 expression. More specifically, when integrin-ß4 was deficient in mice (integrin-ß4-/-) the repair of ozone-injured airway epithelium was impaired and the recovery of ozone-enhanced EMT biomarkers expression in the airway epithelium was delayed. Similarly, in the scratch wounded airway epithelial cells with integrin-ß4 knockdown, the cells were impaired in all aspects related to EMT during wound and repair including cell proliferation, wound closure rate, adhesion and cytoskeleton protein expression (vinculin and vimentin), mesenchymal-like F-actin reorganization, cell stiffness and RhoA activation. Conclusion: Taken together, these results suggested that integrin-ß4 may be essential in regulating the effects of injury and repair on EMT in airway epithelial cells via influencing both the cell adhesion to ECM and cells' physical phenotypes through RhoA signaling pathway.

Sensing Traction Force on the Matrix Induces Cell-Cell Distant Mechanical Communications for Self-Assembly.

The long-range biomechanical force propagating across a large scale may reserve the capability to trigger coordinative responses within cell population such as during angiogenesis, epithelial tubulogenesis, and cancer metastasis. How cells communicate in a distant manner within the group for self-assembly remains largely unknown. Here, we found that airway smooth muscle cells (ASMCs) rapidly self-assembled into a well-constructed network on 3D Matrigel containing type I collagen (COL), which relied on long-range biomechanical force across the matrix to direct cell-cell distant interactions. Similar results happened by HUVEC cells to mimic angiogenesis. Interestingly, single ASMCs initiated multiple extended protrusions precisely pointing to neighboring cells in distance (100-300 µm away or 5-10 folds of the diameter of a round single cell), depending on traction force sensing. Individual ASMCs mechanosensed each other to move directionally on both nonfibrous Matrigel only and Matrigel containing fibrous COL but lost mutual sensing on the cross-linked gel or coated glass due to no long-range force transmission. The bead tracking assay demonstrated distant transmission of traction force (up to 400 µm) during the matrix deformation, and finite element method modeling confirmed the consistency between maximum strain distribution on the matrix and cell directional movements in experiments. Furthermore, ASMCs recruited COL from the hydrogel to build a fibrous network to mechanically stabilize the cell network. Our results revealed principally that cells can sense traction force transmitted through the matrix to initiate cell-cell distant mechanical communications, resulting in cell directional migration and coordinated cell and COL self-assembly with active matrix remodeling. As an interesting phenomenon, cells seem to be able to "make a phone call" via long-range biomechanics, which implicates physiological importance such as for tissue pattern formation.

Increased intracellular Cl- concentration improves airway epithelial migration by activating the RhoA/ROCK Pathway.

In the airway, Cl- is the most abundant anion and is critically involved in transepithelial transport. The correlation of the abnormal expression and activation of chloride channels (CLCs), such as cystic fibrosis transmembrane conductance regulators (CFTRs), anoctamin-1, and CLC-2, with cell migration capability suggests a relationship between defective Cl- transport and epithelial wound repair. However, whether a correlation exists between intracellular Cl- and airway wound repair capability has not been explored thus far, and the underlying mechanisms involved in this relationship are not fully defined. Methods: In this work, the alteration of intracellular chloride concentration ([Cl-]i) was measured by using a chloride-sensitive fluorescent probe (N-[ethoxycarbonylmethyl]-6-methoxyquinolium bromide). Results: We found that clamping with high [Cl-]i and 1 h of treatment with the CLC inhibitor CFTR blocker CFTRinh-172 and chloride intracellular channel inhibitor IAA94 increased intracellular Cl- concentration ([Cl-]i) in airway epithelial cells. This effect improved epithelial cell migration. In addition, increased [Cl-]i in cells promoted F-actin reorganization, decreased cell stiffness, and improved RhoA activation and LIMK1/2 phosphorylation. Treatment with the ROCK inhibitor of Y-27632 and ROCK1 siRNA significantly attenuated the effects of increased [Cl-]i on LIMK1/2 activation and cell migration. In addition, intracellular Ca2+ concentration was unaffected by [Cl-]i clamping buffers and CFTRinh-172 and IAA94. Conclusion: Taken together, these results suggested that Cl- accumulation in airway epithelial cells could activate the RhoA/ROCK/LIMK cascade to induce F-actin reorganization, down-regulate cell stiffness, and improve epithelial migration.

Optogenetic Control for Investigating Subcellular Localization of Fyn Kinase Activity in Single Live Cells.

Previous studies with various Src family kinase biosensors showed that the nuclear kinase activities are much suppressed compared to those in the cytosol, suggesting that these kinases are regulated differently in the nucleus and in the cytosol. In this study, using Fyn as an example, we first engineered a Fyn biosensor with a light-inducible nuclear localization signal to demonstrate that the Fyn kinase activity is significantly lower in the nucleus than in the cytosol. To understand how different equilibrium states between Fyn and the corresponding phosphatases are maintained in the cytosol and nucleus, we further engineered a Fyn kinase domain with light-inducible nuclear localization signal. The results revealed that the Fyn kinase can be actively transported into the nucleus upon light activation and upregulate the biosensor signals in the nucleus. Our results suggest that there is limited transport or diffusion of Fyn kinase between the cytosol and nucleus in the cells, which is important for the maintenance of different equilibrium states of Fyn in situ.

Cell motion-coordinated fibrillar assembly of soluble collagen I to promote MDCK cell branching formation.

Extracellular Matrix (ECM) assembly and remodeling are critical physiological events in vivo, and abnormal ECM assembly or remodeling is related to pathological conditions such as osteoarthritis, fibrosis, cancers, and genetic diseases. ECM assembly/remodeling driven by cells represents more physiological processes. Collagen I (COL) is very abundant in tissues, which assembly/remodeling is mediated by biochemical and mechanical factors. How cells regulate COL assembly biomechanically still remains to be well understood. Here we used fluorescent COL in the medium to study how cells assembled ECM which represents more physiological structures. The results showed that MDCK cells actively recruited COL from the medium and helped assemble the fibers, which in turn facilitated cell branching morphogenesis, both displaying highly spatial associations and mutual dependency. Inhibition of cellular contraction force by ROCK and Myosin II inhibitors attenuated but did not block the COL fiber formation, while cell motion showed high consistency with the fiber assembly. Under ROCK or Myosin II inhibition, further analysis indicated high correlation between local cell movement and COL fiber strength as quantified from different regions of the same groups. Blocking cell motion by actin cytoskeleton disruption completely inhibited the fiber formation. These suggest that cell motion coordinated COL fiber assembly from the medium, possibly through generated strain on deposited COL to facilitate the fiber growth.

Biophysical basis underlying dynamic Lck activation visualized by ZapLck FRET biosensor.

Lck plays crucial roles in TCR signaling. We developed a new and sensitive FRET biosensor (ZapLck) to visualize Lck kinase activity with high spatiotemporal resolutions in live cells. ZapLck revealed that 62% of Lck signal was preactivated in T-cells. In Lck-deficient JCam T-cells, Lck preactivation was abolished, which can be restored to 51% by reconstitution with wild-type Lck (LckWT) but not a putatively inactive mutant LckY394F. LckWT also showed a stronger basal Lck-Lck interaction and a slower diffusion rate than LckY394F. Interestingly, aggregation of TCR receptors by antibodies in JCam cells led to a strong activation of reconstituted LckY394F similar to LckWT. Both activated LckY394F and LckWT diffused more slowly and displayed increased Lck-Lck interaction at a similar level. Therefore, these results suggest that a phosphorylatable Y394 is necessary for the basal-level interaction and preactivation of LckWT, while antibody-induced TCR aggregation can trigger the full activation of LckY394F.

Sensitive FRET Biosensor Reveals Fyn Kinase Regulation by Submembrane Localization.

Fyn kinase plays crucial roles in hematology and T cell signaling; however, there are currently limited tools to visualize the dynamic Fyn activity in live cells. Here we developed and characterized a highly sensitive Fyn biosensor based on fluorescence resonance energy transfer (FRET) to monitor Fyn kinase activity in live cells. Our results show that Fyn kinase activity can be induced in both mouse embryonic fibroblasts (MEFs) and T cells by ligand engagement. Two different motifs were further introduced to target the biosensor at the cellular membrane microdomains in MEFs, revealing that the Fyn-tagged biosensor had 70% greater response to growth factor stimulation than the Lyn-tagged version. This suggests that the plasma membrane microdomains can be categorized into different functional subdomains. Further experiments show that while the membrane accessibility is necessary for Fyn activation, the localization of Fyn outside of its microdomains causes its hyperactivity, indicating that membrane microdomains provide a suppressive microenvironment for Fyn regulation in MEFs. Interestingly, a relatively high Fyn activity can be observed at perinuclear regions, further supporting the notion that the membrane microenvironment has a significant impact on the local molecular functions. Our work hence highlights a novel Fyn FRET biosensor for live cell imaging and its application in revealing an intricate submembrane regulation of Fyn in live MEFs.

Engineered proteins with sensing and activating modules for automated reprogramming of cellular functions.

Protein-based biosensors or activators have been engineered to visualize molecular signals or manipulate cellular functions. Here we integrate these two functionalities into one protein molecule, an integrated sensing and activating protein (iSNAP). A prototype that can detect tyrosine phosphorylation and immediately activate auto-inhibited Shp2 phosphatase, Shp2-iSNAP, is designed through modular assembly. When Shp2-iSNAP is fused to the SIRPα receptor which typically transduces anti-phagocytic signals from the 'don't eat me' CD47 ligand through negative Shp1 signaling, the engineered macrophages not only allow visualization of SIRPα phosphorylation upon CD47 engagement but also rewire the CD47-SIRPα axis into the positive Shp2 signaling, which enhances phagocytosis of opsonized tumor cells. A second SIRPα Syk-iSNAP with redesigned sensor and activator modules can likewise rewire the CD47-SIRPα axis to the pro-phagocytic Syk kinase activation. Thus, our approach can be extended to execute a broad range of sensing and automated reprogramming actions for directed therapeutics.Protein-based biosensors have been engineered to interrogate cellular signaling and manipulate function. Here the authors demonstrate iSNAP, a tool to detect tyrosine phosphorylation and activate desired protein enzymes allowing the control of phagocytosis in macrophages.

Electroporation-delivered fluorescent protein biosensors for probing molecular activities in cells without genetic encoding.

Fluorescent protein biosensors are typically implemented via genetic encoding which makes the examination of scarce cell samples impractical. By directly delivering the protein form of the biosensor into cells using electroporation, we detected intracellular molecular activity with the sample size down to ∼100 cells with high spatiotemporal resolution.

Genetically encoded fluorescent biosensors for live-cell imaging of MT1-MMP protease activity.

The proteolytic activity of Membrane-type 1 Matrix Metalloproteinase (MT1-MMP) is crucial for cancer cell invasion and metastasis. To visualize the protease activity of MT1-MMP with high spatiotemporal resolution at the extracellular plasma membrane surface of live cancer cells, a genetically encoded fluorescent biosensor of MT1-MMP has been developed. Here we describe the design principles of the MT1-MMP biosensor, the characterization of the MT1-MMP biosensor in vitro, and the live-cell imaging protocol used to visualize MT1-MMP activity in mammalian cells. We also provide brief guidelines for observing MT1-MMP subcellular activity by fluorescence resonance energy transfer (FRET) in a cell migration assay.

Antagonism between binding site affinity and conformational dynamics tunes alternative cis-interactions within Shp2.

Protein functions are largely affected by their conformations. This is exemplified in proteins containing modular domains. However, the evolutionary dynamics that define and adapt the conformation of such modular proteins remain elusive. Here we show that cis-interactions between the C-terminal phosphotyrosines and SH2 domain within the protein tyrosine phosphatase Shp2 can be tuned by an adaptor protein, Grb2. The competitiveness of two phosphotyrosines, namely pY542 and pY580, for cis-interaction with the same SH2 domain is governed by an antagonistic combination of contextual amino acid sequence and position of the phosphotyrosines. Specifically, pY580 with the combination of a favourable position and an adverse sequence has an overall advantage over pY542. Swapping the sequences of pY542 and pY580 results in one dominant form of cis-interaction and subsequently inhibits the trans-regulation by Grb2. Thus, the antagonistic combination of sequence and position may serve as a basic design principle for proteins with tunable conformations.

Long-range mechanical force enables self-assembly of epithelial tubular patterns.

Enabling long-range transport of molecules, tubules are critical for human body homeostasis. One fundamental question in tubule formation is how individual cells coordinate their positioning over long spatial scales, which can be as long as the sizes of tubular organs. Recent studies indicate that type I collagen (COL) is important in the development of epithelial tubules. Nevertheless, how cell-COL interactions contribute to the initiation or the maintenance of long-scale tubular patterns is unclear. Using a two-step process to quantitatively control cell-COL interaction, we show that epithelial cells developed various patterns in response to fine-tuned percentages of COL in ECM. In contrast with conventional thoughts, these patterns were initiated and maintained by traction forces created by cells but not diffusive factors secreted by cells. In particular, COL-dependent transmission of force in the ECM led to long-scale (up to 600 µm) interactions between cells. A mechanical feedback effect was encountered when cells used forces to modify cell positioning and COL distribution and orientations. Such feedback led to a bistability in the formation of linear, tubule-like patterns. Using micro-patterning technique, we further show that the stability of tubule-like patterns depended on the lengths of tubules. Our results suggest a mechanical mechanism that cells can use to initiate and maintain long-scale tubular patterns.

A femtomol range FRET biosensor reports exceedingly low levels of cell surface furin: implications for the processing of anthrax protective antigen.

Furin, a specialized endoproteinase, transforms proproteins into biologically active proteins. Furin function is important for normal cells and also in multiple pathologies including malignancy and anthrax. Furin is believed to cycle between the Golgi compartment and the cell surface. Processing of anthrax protective antigen-83 (PA83) by the cells is considered thus far as evidence for the presence of substantial levels of cell-surface furin. To monitor furin, we designed a cleavage-activated FRET biosensor in which the Enhanced Cyan and Yellow Fluorescent Proteins were linked by the peptide sequence SNSRKKR / STSAGP derived from anthrax PA83. Both because of the sensitivity and selectivity of the anthrax sequence to furin proteolysis and the FRET-based detection, the biosensor recorded the femtomolar levels of furin in the in vitro reactions and cell-based assays. Using the biosensor that was cell-impermeable because of its size and also by other relevant methods, we determined that exceedingly low levels, if any, of cell-surface furin are present in the intact cells and in the cells with the enforced furin overexpression. This observation was in a sharp contrast with the existing concepts about the furin presentation on cell surfaces and anthrax disease mechanism. We next demonstrated using cell-based tests that PA83, in fact, was processed by furin in the extracellular milieu and that only then the resulting PA63 bound the anthrax toxin cell-surface receptors. We also determined that the biosensor, but not the conventional peptide substrates, allowed continuous monitoring of furin activity in cancer cell extracts. Our results suggest that there are no physiologically-relevant levels of cell-surface furin and, accordingly, that the mechanisms of anthrax should be re-investigated. In addition, the availability of the biosensor is a foundation for non-invasive monitoring of furin activity in cancer cells. Conceptually, the biosensor we developed may serve as a prototype for other proteinase-activated biosensors.

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.

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 Ca(2+) 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 Ca(2+) 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 Ca(2+) oscillation in comparison to stiffer or rigid substrate. This Ca(2+) 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 Ca(2+) oscillation. Consistently, either a constitutive active mutant of RhoA (RhoA-V14) or a dominant negative mutant of RhoA (RhoA-N19) inhibited the Ca(2+) 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 Ca(2+) oscillation, which determines the physiological functions of HMSCs.

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.

The spatiotemporal pattern of Src activation at lipid rafts revealed by diffusion-corrected FRET imaging.

Genetically encoded biosensors based on fluorescence resonance energy transfer (FRET) have been widely applied to visualize the molecular activity in live cells with high spatiotemporal resolution. However, the rapid diffusion of biosensor proteins hinders a precise reconstruction of the actual molecular activation map. Based on fluorescence recovery after photobleaching (FRAP) experiments, we have developed a finite element (FE) method to analyze, simulate, and subtract the diffusion effect of mobile biosensors. This method has been applied to analyze the mobility of Src FRET biosensors engineered to reside at different subcompartments in live cells. The results indicate that the Src biosensor located in the cytoplasm moves 4-8 folds faster (0.93+/-0.06 microm(2)/sec) than those anchored on different compartments in plasma membrane (at lipid raft: 0.11+/-0.01 microm(2)/sec and outside: 0.18+/-0.02 microm(2)/sec). The mobility of biosensor at lipid rafts is slower than that outside of lipid rafts and is dominated by two-dimensional diffusion. When this diffusion effect was subtracted from the FRET ratio images, high Src activity at lipid rafts was observed at clustered regions proximal to the cell periphery, which remained relatively stationary upon epidermal growth factor (EGF) stimulation. This result suggests that EGF induced a Src activation at lipid rafts with well-coordinated spatiotemporal patterns. Our FE-based method also provides an integrated platform of image analysis for studying molecular mobility and reconstructing the spatiotemporal activation maps of signaling molecules in live cells.

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.