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Background Adipose-derived mesenchymal stem cells (ADSC)-based therapy is an outstanding treatment strategy for ischaemic disease. However, the therapeutic efficacy of this strategy is not ideal due to the poor paracrine function and low survival rate of ADSCs in target regions. Platelet extracellular vesicles (PEVs) are nanoparticles derived from activated platelets that can participate in communication between cells. Accumulating evidence indicates that PEVs can regulate the biological functions of several cell lines. In the present study, we aimed to investigate whether PEVs can modulate the proangiogenic potential of ADSCs in vitro and in vivo. Methods PEVs were identified using scanning electron microscope (SEM), flow cytometry (FCM) and nanoparticle tracking analysis (NTA). The CCK8 assay was performed to detect proliferation of cells. Transwell and wound healing assays were performed to verify migration capacity of cells. AnnexinV-FITC/PI apoptosis kit and live/dead assay were performed to assess ADSCs apoptosis under Cocl2-induced hypoxia condition. The underlying mechanisms by which PEVs affected ADSCs were explored using real time-PCR(RT-PCR) and Western blot. In addition, matrigel plug assays were conducted and mouse hindlimb ischaemic models were established to investigate the proangiogenic potential of PEV-treated ADSCs in vivo. Results We demonstrated that ADSC could internalize PEVs, which lead to a series of biological reactions. In vitro, dose-dependent effects of PEVs on ADSC proliferation, migration and antiapoptotic capacity were observed. Western blotting results suggested that multiple proteins such as ERK, AKT, FAK, Src and PLCγ1 kinase may contribute to these changes. Furthermore, PEVs induced upregulation of several growth factors expression in ADSCs and amplified the proliferation, migration and tube formation of HUVECs induced by ADSC conditioned medium (CM). In in vivo experiments, compared with control ADSCs, the injection of PEV-treated ADSCs resulted in more vascularization in matrigel plugs, attenuated tissue degeneration and increased blood flow and capillary density in ischaemic hindlimb tissues. Conclusion Our data demonstrated that PEVs could enhance the proangiogenic potential of ADSCs in mouse hindlimb ischaemia. The major mechanisms of this effect included the promotion of ADSC proliferation, migration, anti-apoptosis ability and paracrine secretion.

The limited sensitivity of Förster Resonance Energy Transfer (FRET) biosensors hinders their broader applications. Here, we develop an approach integrating high-throughput FRET sorting and next-generation sequencing (FRET-Seq) to identify sensitive biosensors with varying substrate sequences from large-scale libraries directly in mammalian cells, utilizing the design of self-activating FRET (saFRET) biosensor. The resulting biosensors of Fyn and ZAP70 kinases exhibit enhanced performance and enable the dynamic imaging of T-cell activation mediated by T cell receptor (TCR) or chimeric antigen receptor (CAR), revealing a highly organized ZAP70 subcellular activity pattern upon TCR but not CAR engagement. The ZAP70 biosensor elucidates the role of immunoreceptor tyrosine-based activation motif (ITAM) in affecting ZAP70 activation to regulate CAR functions. A saFRET biosensor-based high-throughput drug screening (saFRET-HTDS) assay further enables the identification of an FDA-approved cancer drug, Sunitinib, that can be repurposed to inhibit ZAP70 activity and autoimmune-disease-related T-cell activation., Existing Förster Resonance Energy Transfer (FRET) biosensors are often limited in their sensitivity. Here the authors report FRET-seq which they use to identify Fyn and ZAP70 kinase biosensors with enhanced performance, and use them to image T-cell activation and screen drugs.

Therapeutic angiogenesis by transplantation of autologous/allogeneic adipose stem cells (ADSCs) is a potential method for the treatment of critical limb ischemia (CLI). However, the therapeutic efficiency is limited by poor viability, adhesion, migration and differentiation after cell transplantation into the target area. Astragaloside IV (AS-IV), one of the main active components of Astragalus, has been widely used in the treatment of ischemic diseases and can promote cell proliferation and angiogenesis. However, there is no report on the effect of AS-IV on ADSCs and its effect on hindlimb ischemia through cell transplantation.The purpose of this study was to elucidate that AS-IV pretreatment enhances the therapeutic effect of ADSC on critical limb ischemia, and to characterize the underlying molecular mechanisms.ADSCs were obtained and pretreated with the different concentration of AS-IV. In vitro, we analyzed the influence of AS-IV on ADSC proliferation, migration, angiogenesis and recruitment of human umbilical vein endothelial cells (HUVECs) and analyzed the relevant molecular mechanism. In vivo, we injected drug-pretreated ADSCs into a Matrigel or hindlimb ischemia model and evaluated the therapeutic effect by the laser Doppler perfusion index, immunofluorescence, and histopathology.In vitro experiments showed that AS-IV improved ADSC migration, angiogenesis and endothelial recruitment. The molecular mechanism may be related to the upregulation of CXC receptor 2 (CXCR2) to promote the phosphorylation of focal adhesion kinase (FAK). In vivo, Matrigel plug assay showed that ADSCs pretreated with AS-IV have stronger angiogenic potential. The laser Doppler perfusion index of the hindlimbs of mice in the ADSC/AS-IV group was significantly higher than the laser Doppler perfusion index of the hindlimbs of mice of the ADSC group and the control group, and the microvessel density was significantly increased.Our results demonstrate that AS-IV pretreatment of ADSC improves their therapeutic efficacy in ameliorating severe limb exclusion symptomology through CXCR2 induced FAK phosphorylation, which will bring new insights into the treatment of severe limb ischemia.

Genetically-encoded biosensors based on FRET have been widely used to dynamically monitor the activity of protein tyrosine kinases (PTKs) in living cell with high spatiotemporal resolution. However, the limitation in sensitivity, specificity, and dynamic range of FRET biosensors have hindered their broader applications. Here, we introduced a systematic platform, FRET-Seq, which integrates high-throughput FRET sorting and next-generation sequencing, to identify FRET biosensors with better performance from large-scale libraries directly in mammalian cells.

Background Peptide probes can be applied for biomarker targeting to improve the diagnostic accuracy. Cluster of differentiation 44 (CD44) is up-regulated in gastric cancer (GC). Among all the variants of CD44, CD44v6 is reported the most promising biomarker for GC. The purpose of this study was generating and identification a peptide ligand specific to CD44v6. Methods A 12-mer phage peptide library was screened on CD44v overexpressed HEK-293 cells with an improved subtractive method. Five candidate sequences emerged. Candidate phages were selected using enzyme-linked immunosorbent assay and competitive inhibition assays. Then the sequence (designated ELT) was chosen for further study. Its binding affinity and specificity were verified on recombinant protein, GC cells, GC tissues and xenograft models based on BALB/c-nu/nu mice using dissociation constant calculation, immunofluorescence, immunohistochemistry and in vivo imaging separately. Results The dissociation constant of ELT with recombinant protein was 611.2 nM. ELT stained CD44v overexpressed HEK-293 but not the cell expressing wild-type CD44s. On GC cell lines, ELT co-stained with anti-CD44v6 antibody. ELT binding on tumor tissues significantly increased compared with that of paracancer tissues, also showed a linear positive correlation with CD44v6 expression. ELT specifically accumulated in tumor and eliminated in short time in vivo. Conclusions ELT can target GC in vitro and in vivo via CD44v6, indicating its potential to serve as a probe for GC targeting diagnosis and therapy.

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.

Advanced glycation end products (AGEs) induce vascular smooth muscle cells (VSMCs) contractile-synthetic phenotypic conversion which plays roles in aggravated atherosclerosis in diabetes. Matrine has been proved to suppress AGEs-induced phenotypic conversion which is governed by Notch pathway. Endoplasmic reticulum stress was associated with Notch pathway. Cultured human coronary smooth muscle cells (HCSMCs) were incubated with AGE-BSA at 0, 5 and 10 μmol/l. Specific siRNA was used to silence Protein kinase RNA-like ER kinase (PERK). Matrine at 0, 0.5 and 1.0 mmol/l were used to pre-treat the cells. Immunofluorescent staining of Smooth muscle myosin heavy chain 11 (MYH11) and smooth muscle α-actin 2 (ACTA2) were used to identify the contractile phenotype of HCSMCs. Protein phosphorylation and expression levels were evaluated by Western Blotting. AGE-BSA exposure facilitated the contractile-synthetic phenotypic conversion of HCSMCs in a concentration-dependent manner. AGE-BSA exposure increased expression levels of glucose-regulated protein 78 (GRP78), Delta-like 4 (Dll4), Notch intracellular domain (NICD1), Hes family basic helix-loop-helix (bHLH) transcriptional factor 1 (HES1), as well as the phosphorylation level of PERK. Specific perk-siRNA transfection dramatically lowered PERK phosphorylation and resulted in down-regulation of Dll4, NICD1 and HES1 in HCSMCs exposed to AGE-BSA. Pre-treatment of matrine suppressed AGE-BSA-induced phenotypic conversion of HCSMCs in a concentration-dependent manner. Moreover, matrine pre-treatment reduced expression level of GRP78, NICD1, HES1 and the phosphrylation level of PERK in AGE-BSA-exposed HCSMCs in a concentration-dependent manner. These results suggested that matrine suppressed AGE-BSA-induced HCSMCs phenotypic conversion via attenuating ER stress PERK signaling-dependent Dll4- Notch pathway activation.

Substantial data from preclinical studies have revealed the biphasic effects of statins on cardiovascular angiogenesis. Although some have reported the anti‐angiogenic potential of statins in malignant tumors, the underlying mechanism remains poorly understood. The aim of this study is to elucidate the mechanism by which simvastatin, a member of the statin family, inhibits tumor angiogenesis. Simvastatin significantly suppressed tumor cell‐conditioned medium‐induced angiogenic promotion in vitro, and resulted in dose‐dependent anti‐angiogenesis in vivo. Further genetic silencing of hypoxia‐inducible factor‐1α (HIF‐1α) reduced vascular endothelial growth factor and fibroblast growth factor‐2 expressions in 4T1 cells and correspondingly ameliorated HUVEC proliferation facilitated by tumor cell‐conditioned medium. Additionally, simvastatin induced angiogenic inhibition through a mechanism of post‐transcriptional downregulation of HIF‐1α by increasing the phosphorylation level of AMP kinase. These results were further validated by the fact that 5‐aminoimidazole‐4‐carboxamide ribonucleotide reduced HIF‐1α protein levels and ameliorated the angiogenic ability of endothelial cells in vitro and in vivo. Critically, inhibition of AMPK phosphorylation by compound C almost completely abrogated simvastatin‐induced anti‐angiogenesis, which was accompanied by the reduction of protein levels of HIF‐1α and its downstream pro‐angiogenic factors. These findings reveal the mechanism by which simvastatin induces tumor anti‐angiogenesis, and therefore identifies the target that explains the beneficial effects of statins on malignant tumors.

A new biosensor was engineered to visualize Lck kinase preactivation and regulation in live T cells., 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.

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.

CD44, especially the isoforms with variable exons (CD44v), is a promising biomarker for the detection of cancer. To develop a CD44v-specific probe, we screened a 7-mer phage peptide library against the CD44v3-v10 protein using an improved subtractive method. The consensus sequences with the highest frequency (designated CV-1) emerged after four rounds of panning. The binding affinity and specificity of the CV-1 phage and the synthesized peptide for the region of CD44 encoded by the variable exons were confirmed using enzyme-linked immunosorbent assay and competitive inhibition assays. Furthermore, the binding of the CV-1 probe to gastric cancer cells and tissues was validated using immunofluorescence and immunohistochemistry assays. CV-1 sensitively and specifically bound to CD44v on cancer cells and tissues. Thus, CV-1 has the potential to serve as a promising probe for cancer molecular imaging and target therapy.

The dramatic reorganization of chromatin during mitosis is perhaps one of the most fundamental of all cell processes. It remains unclear how epigenetic histone modifications, despite their crucial roles in regulating chromatin architectures, are dynamically coordinated with chromatin reorganization in controlling this process. We have developed and characterized biosensors with high sensitivity and specificity based on fluorescence resonance energy transfer (FRET). These biosensors were incorporated into nucleosomes to visualize histone H3 Lys-9 trimethylation (H3K9me3) and histone H3 Ser-10 phosphorylation (H3S10p) simultaneously in the same live cell. We observed an anticorrelated coupling in time between H3K9me3 and H3S10p in a single live cell during mitosis. A transient increase of H3S10p during mitosis is accompanied by a decrease of H3K9me3 that recovers before the restoration of H3S10p upon mitotic exit. We further showed that H3S10p is causatively critical for the decrease of H3K9me3 and the consequent reduction of heterochromatin structure, leading to the subsequent global chromatin reorganization and nuclear envelope dissolution as a cell enters mitosis. These results suggest a tight coupling of H3S10p and H3K9me3 dynamics in the regulation of heterochromatin dissolution before a global chromatin reorganization during mitosis.

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.

Peptides selected from phage display have great potential to become probes for the imaging detection of the cancer. To develop the peptide probe for diagnosis of GC, a 12-mer phage display library was used to select peptides that bind specifically to the human GC cell line SGC-7901. After four rounds of in vitro selection, five phage clones that bound specifically to the SGC-7901 cells were selected. The phage clone GP-5 had a particularly high affinity and specificity for SGC-7901 cells. This clone was identified using a series of methods. The peptide GP-5 that was displayed on phage GP-5 exhibited high specificity to SGC-7901 cells and gastric tissues. Thus, the peptide GP-5 displays excellent potential for imaging detection of human gastric cancer.

The microenvironment has been shown to regulate cellular functions including cell growth, differentiation, proliferation, migration, cancer development and metastasis. However, the underlying molecular mechanism remains largely unclear. We have integrated micro-pattern technology and molecular biosensors based on fluorescence resonance energy transfer (FRET) to visualize calcium responses in cells constrained to grow on a micro-patterned surface. Upon ATP stimulation, human umbilical vein endothelial cells (HUVECs) cultured on different surface micro-patterns had a shorter decay time and a reduced peak of a transient intracellular calcium rise compared to control cells without constraints. The decay time is regulated by the plasma membrane and the membrane calcium channels, while the peak by endoplasmic reticulum (ER) calcium release. Further results revealed that voltage operated channels (VOCs), coupling the plasma membrane and ER, can affect both the decay time and the peak of calcium response. The inhibition of VOCs can eliminate the effect of different micro-patterns on calcium signals. When two connected HUVECs were constrained to grow on a micro-pattern, drastically distinct calcium responses upon ATP stimulation can be observed, in contrast to the similar responses of two connected cells cultured without patterns. Interestingly, the inhibition of VOCs also blocked this difference of calcium responses between two connected cells on micro-patterns. These results indicate that a micro-patterned surface can have a profound effect on the calcium responses of HUVECs under ATP stimulation, largely mediated by VOCs. Therefore, our results shed new light on the molecular mechanism by which HUVECs perceive the microenvironment and regulate intracellular calcium signals.

The dynamic activation of oncogenic kinases and regulation of focal adhesions (FAs) are crucial molecular events modulating cell adhesion in cancer metastasis. However, it remains unclear how these events are temporally coordinated at single FA sites. Therefore, we targeted fluorescence resonance energy transfer (FRET)-based biosensors toward subcellular FAs to report local molecular events during cancer cell adhesion. Employing single FA tracking and cross-correlation analysis, we quantified the dynamic coupling characteristics between biochemical kinase activities and structural FA within single FAs. We show that kinase activations and FA assembly are strongly and sequentially correlated, with the concurrent FA assembly and Src activation leading focal adhesion kinase (FAK) activation by 42.6 ± 12.6 sec. Strikingly, the temporal coupling between kinase activation and individual FA assembly reflects the fate of FAs at later stages. The FAs with a tight coupling tend to grow and mature, while the less coupled FAs likely disassemble. During FA disassembly, however, kinase activations lead the disassembly, with FAK being activated earlier than Src. Therefore, by integrating subcellularly targeted FRET biosensors and computational analysis, our study reveals intricate interplays between Src and FAK in regulating the dynamic life of single FAs in cancer cells.

This paper indicates that in ventricular myocytes when the SR (sarcoplasmic reticulum) is pharmacologically inhibited, the intracellular Ca2+concentration rapidly increases during Ca2+ entrance (0-70 ms), whereas the decay of Ca2+ is slow; in the absence of fluorescent dye, large Ca2+ concentration gradients might develop near the cell membrane; intracellular Ca2+ distribution is tightly regulated by the localization of Ca2+transporter proteins along the sarcolemma and strongly relays on the presence of mobile and stationary Ca2+ buffers. These studies also imply that in ventricular cells with intact and functional SR, the Ca2+ signal most likely would spread faster along the t-tubular system, surface membrane than to the cell interior and that in the absence of Ca2+ dye high Ca2+ gradients under the surface membrane and more uniform Ca2+ distribution in the cell interior might be expected.

We developed a quantum-dot-based fluorescence resonance energy transfer (QD-FRET) nanosensor to visualize the activity of matrix metalloproteinase (MT1-MMP) at cell membrane. A bended peptide with multiple motifs was engineered to position the FRET pair at a close proximity to allow energy transfer, which can be cleaved by active MT1-MMP to result in FRET changes and the exposure of cell penetrating sequence. Via FRET and penetrated QD signals, the nanosensor can profile cancer cells.

Human telomerase reverse transcriptase (hTERT) is upregulated in most cancer cell types as well in immortalized cells. The underlying mechanism for such upregulation, however, remains largely unknown. We report here that bile acids under acidified media increase hTERT expression via c-myc activation in primary human gastric cancer cell lines. Human gastric cancer MKN28, MGC803 and SGC7901 cells were treated with 100 µM deoxycholic acid (DCA) or chenodeoxycholic acid (CDCA) with or without acidified media in the presence or absence of the c-myc inhibitor 10058-F4 for 24 h. hTERT and c-myc protein levels were determined by western blot analysis. hTERT and c-myc mRNA levels were determined by RT-PCR. The promoter activities of hTERT and c-myc transcription were determined using promoter reporter luciferase assays for both. Telomerase enzyme activity was analyzed by stretch PCR. hTERT mRNA and protein levels were significantly increased by bile acids in acidified media and were accompanied with enhanced telomerase activity. No changes were found at a pH of 7.0 or with acidified media alone. Similarly, the mRNA and protein levels of c-myc were also increased by bile acids in acidified media but not at a pH of 7.0 or with acidified media alone. Importantly, pharmacologic inhibition of c-myc using 10058-F4 prevented hTERT induction by DCA or CDCA in gastric cancer cells under acidic conditions. Bile acids (DCA and CDCA) under acidic conditions increased hTERT expression in human gastric cancer cells by activation of c-myc transcription. This suggests that acidified bile acids may promote tumorigenesis and affect cell ageing via telomerase activation.

While Src plays crucial roles in shear stress-induced cellular processes, little is known on the spatiotemporal pattern of high shear stress (HSS)-induced Src activation. HSS (65 dyn/cm(2)) was applied on bovine aortic endothelial cells to visualize the dynamic Src activation at subcellular levels utilizing a membrane-targeted Src biosensor (Kras-Src) based on fluorescence resonance energy transfer (FRET). A polarized Src activation was observed with higher activity at the side facing the flow, which was enhanced by a cytochalasin D-mediated disruption of actin filaments but inhibited by a benzyl alcohol-mediated enhancement of membrane fluidity. Further experiments revealed that HSS decreased RhoA activity, with a constitutively active RhoA mutant inhibiting while a negative RhoA mutant enhancing the HSS-induced Src polarity. Cytochalasin D can restore the polarity in cells expressing the active RhoA mutant. Further results indicate that HSS stimulates FAK activation with a spatial polarity similar to Src. The inhibition of Src by PP1, as well as the perturbation of RhoA activity and membrane fluidity, can block this HSS-induced FAK polarity. These results indicate that the HSS-induced Src and subsequently FAK polarity depends on the coordination between intracellular tension distribution regulated by RhoA, its related actin structures and the plasma membrane fluidity.

Cell migration requires the fine spatiotemporal integration of many proteins that regulate the fundamental processes that drive cell movement. Focal adhesion (FA) dynamics is a continuous process involving coordination between FA and actin cytoskeleton, which is essential for cell migration. We studied the spatiotemporal relationship between the dynamics of focal adhesion kinase (FAK) and paxillin at FAs in the protrusion of living endothelial cells. Concurrent dual-color imaging showed that FAK was assembled at FA first, which was followed by paxillin recruitment to the FA. By tracking and quantifying FAK and paxillin in migrating cells, the normalized FAK/Paxillin fluorescence intensity (FI) ratio is > 1 (∼4 fold) at cell front, ∼1 at cell center, and < 1 at cell rear. The significantly higher FAK FI than paxillin FI at cell front indicates that the assembly of FAK-FAs occurs ahead of paxillin at cell front. To determine the time difference between the assemblies of FAK and paxillin at nascent FAs, FAs containing both FAK and paxillin were quantified by image analysis and time correlation. The results show that FAK assembles at the nascent FAs earlier than paxillin in the protrusions at cell front. © 2014 Macmillan Publishers Limited. All rights reserved.

Focal adhesions (FAs) are dynamic subcellular structures crucial for cell adhesion, migration and differentiation. It remains an enigma how enzymatic activities in these local complexes regulate their structural remodeling in live cells. Utilizing biosensors based on fluorescence resonance energy transfer (FRET), we developed a correlative FRET imaging microscopy (CFIM) approach to quantitatively analyze the subcellular coordination between the enzymatic Src activation and the structural FA disassembly. CFIM reveals that the Src kinase activity only within the microdomain of lipid rafts at the plasma membrane is coupled with FA dynamics. FA disassembly at cell periphery was linearly dependent on this raft-localized Src activity, although cells displayed heterogeneous levels of response to stimulation. Within lipid rafts, the time delay between Src activation and FA disassembly was 1.2 min in cells seeded on low fibronectin concentration ([FN]) and 4.3 min in cells on high [FN]. CFIM further showed that the level of Src-FA coupling, as well as the time delay, was regulated by cell-matrix interactions, as a tight enzyme-structure coupling occurred in FA populations mediated by integrin αvβ₃, but not in those by integrin α₅β₁. Therefore, different FA subpopulations have distinctive regulation mechanisms between their local kinase activity and structural FA dynamics.

Focal adhesion kinase (FAK) and paxillin are involved in focal adhesion (FA) disassembly and formation, which can regulate cell migration, intracellular signaling, and remodeling of actin filaments. We used time-lapse double-color imaging to monitor concurrently the spatial-temporal dynamics of FAK and paxillin in live endothelial cells (ECs) co-transfected with GFP-FAK and DsRed-paxillin, and the fluorescence intensities (FI) of paxillin and FAK at FAs were determined in cell front, center and rear. The mean FAK/Paxillin FI ratio is highest at cell front (mean±SEM: 4.73±0.48), near 1 at cell center (0.95±0.11), and lowest at cell rear (0.64±0.05) (P

Mesenchymal stem cells (MSCs) are a promising candidate for cell-based therapy in regenerative medicine. These stem cells can interact with their mechanical microenvironment to control their functions. External mechanical cues can be perceived and transmitted into intracellular calcium dynamics to regulate various cellular processes. Recent studies indicate that human MSCs (hMSCs) exhibit a heterogeneous nature with a subset of hMSCs lacking spontaneous calcium oscillations. In this study, we studied whether and how external mechanical tension can be applied to trigger and restore the intracellular calcium oscillation in these hMSCs lacking spontaneous activities. Utilizing the fluorescence resonance energy transfer (FRET) based calcium biosensor, we found that this subpopulation of hMSCs can respond to a prolonged mechanical stretch (PMS). Further results revealed that the triggering of calcium oscillations in these cells is dependent on the calcium influx across the plasma membrane, as well as on both cytoskeletal supports, myosin light chain kinase (MLCK)-driven actomyosin contractility, and phospholipase C (PLC) activity. Thus, our report confirmed that mechanical tension can govern the intracellular calcium oscillation in hMSCs, possibly via the control of the calcium permeability of channels at the plasma membrane. Our results also provide novel mechanistic insights into how hMSCs sense mechanical environment to regulate cellular functions.

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.

To develop a novel diagnostic method for the assessment of drug efficacy in chronic myeloid leukemia (CML) patients individually, we generated a biosensor that enables the evaluation of BCR-ABL kinase activity in living cells using the principle of fluorescence resonance energy transfer (FRET).To develop FRET-based biosensors, we used CrkL, the most characteristic substrate of BCR-ABL, and designed a protein in which CrkL is sandwiched between Venus, a variant of YFP, and enhanced cyan fluorescent protein, so that CrkL intramolecular binding of the SH2 domain to phosphorylated tyrosine (Y207) increases FRET efficiency. After evaluation of the properties of this biosensor by comparison with established methods including Western blotting and flow cytometry, BCR-ABL activity and its response to drugs were examined in CML patient cells.After optimization, we obtained a biosensor that possesses higher sensitivity than that of established techniques with respect to measuring BCR-ABL activity and its suppression by imatinib. Thanks to its high sensitivity, this biosensor accurately gauges BCR-ABL activity in relatively small cell numbers and can also detect1% minor drug-resistant populations within heterogeneous ones. We also noticed that this method enabled us to predict future onset of drug resistance as well as to monitor the disease status during imatinib therapy, using patient cells.In consideration of its quick and practical nature, this method is potentially a promising tool for the prediction of both current and future therapeutic responses in individual CML patients, which will be surely beneficial for both patients and clinicians.

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 µm2/sec) than those anchored on different compartments in plasma membrane (at lipid raft: 0.11±0.01 µm2/sec and outside: 0.18±0.02 µm2/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., Author Summary Fluorescence biosensors have been widely used to report the spatial and temporal activity of target molecules in live cells. However, biosensors can move independently of the target molecule and carry its signal to other subcellular locations. Therefore, the observed images appear to be the combination of the target molecular activity and the artifacts introduced by the movement of the biosensors (mainly due to diffusion). The intriguing question is how to estimate and exclude the movement effect of biosensors from the observed fluorescent images and to reconstruct the real activity map of the target molecules. The Src molecule plays important roles in cell adhesion, migration, and cancer invasion. In this paper, we developed a novel computational method to analyze and simulate the movement of the Src biosensor, which was then subtracted from the original fluorescent images. With this computational method, we observed discrete clusters of high Src activity at relatively stationary locations on the plasma membrane. Therefore, our results highlight the coordination of molecular activities in space and time. In addition to Src, our computational method can be used to reconstruct the activity map of other signaling molecules.

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.