Increased cell-free DNA is associated with oxidative damage in patients with schizophrenia.

Cell-free DNA (cfDNA) has been found to be elevated in patients with schizophrenia (SZ), potentially derived from activated apoptosis, but the underlying mechanisms remain unknown. Moreover, whether the concentrations of cfDNA are altered with disease stage has not been investigated, which limits its clinical application as an auxiliary diagnostic marker for SZ. Using an improved fluorescence correlation spectroscopy (FCS) method that does not require DNA extraction, we measured the molar concentrations of cfDNA in plasma samples of 191 patients with SZ, 78 patients with mood disorders (MD) and 65 healthy controls (HC). We also analyzed the cfDNA composition from either the nucleus or mitochondria, oxidation markers and biochemical indexes to explore the potential mechanistic associations of the increased cfDNA levels. We found that in SZ patients, the cfDNA levels were significantly increased (P = 0.003) regardless of the different disease stages or antipsychotic medication use. Furthermore, qPCR revealed that cell-free nuclear DNA (cf-nDNA) (P = 0.041) but not cell-free mitochondrial DNA (cf-mtDNA) was elevated in SZ patients. Moreover, decreased SOD activity in SZ patients (P = 0.005) was negatively correlated with cfDNA levels (P = 0.047), and fasting blood glucose was positively correlated with cfDNA levels in SZ patients (P = 0.013). Our study provides evidence to support that the elevated cfDNA may be a convenient, effective and stable trait indicator of SZ. Further analysis showed that it mainly came from nucleus, suggesting increased apoptosis, and potentially related to oxidative stress and high blood glucose levels in patients.

Simultaneously Monitoring Multiple Autophagic Processes and Assessing Autophagic Flux in Single Cells by In Situ Fluorescence Cross-Correlation Spectroscopy.

Autophagy is a widely conserved and multistep cellular catabolic process and maintains cellular homeostasis and normal cellular functions via the degradation of some harmful intracellular components. It was reported that high basal autophagic activity may be closely related to tumorigenesis. So far, the fluorescence imaging technique has been widely used to study autophagic processes, but this method is only suitable for distinguishing autophagosomes and autolysosomes. Simultaneously monitoring multiple autophagic processes remains a significant challenge due to the lack of an efficient detection method. Here, we demonstrated a new method for simultaneously monitoring multiple autophagic processes and assessing autophagic flux in single cells based on in situ fluorescence cross-correlation spectroscopy (FCCS). In this study, microtubule-associated protein 1A/1B-light chain 3B (LC3B) was fused with two tandem fluorescent proteins [mCherry red fluorescent protein (mCherry) and enhanced green fluorescent protein (EGFP)] to achieve the simultaneous labeling and distinguishing of multiple autophagic structures based on the differences in characteristic diffusion time (τD). Furthermore, we proposed a new parameter "delivery efficiency of autophagosome (DEAP)" to assess autophagic flux based on the cross correlation (CC) value. Our results demonstrate that FCCS can efficiently distinguish three autophagic structures, assess the induced autophagic flux, and discriminate different autophagy regulators. Compared with the commonly used fluorescence imaging technique, the resolution of FCCS remains unaffected by Brownian motion and fluorescent monomers in the cytoplasm and is well suitable to distinguishing differently colored autophagic structures and monitoring autophagy.

Objective scanning-based fluorescence cross-correlation spectroscopy (Scan-FCCS) for studying the fusion dynamics of protein phase separation.

Protein phase separation plays a very important role in many biological processes and is closely related to the occurrence and development of some serious diseases. So far, the fluorescence imaging method and fluorescence correlation spectroscopy (FCS) have been frequently used to study the phase separation behavior of proteins. Due to the wide size distribution of protein condensates in phase separation from nano-scale to micro-scale in solution and living cells, it is difficult for the fluorescence imaging method and conventional FCS to fully reflect the real state of protein phase separation in the solution due to the low spatio-temporal resolution of the conventional fluorescence imaging method and the limited detection area of FCS. Here, we proposed a novel method for studying the protein phase separation process by objective scanning-based fluorescence cross-correlation spectroscopy (Scan-FCCS). In this study, CRDBP proteins were used as a model and respectively fused with fluorescent proteins (EGFP and mCherry). We first compared conventional FCS and Scan-FCS methods for characterizing the CRDBP protein phase separation behaviors and found that the reproducibility of Scan-FCS is significantly improved by the scanning mode. We studied the self-fusion process of mCherry-CRDBP and EGFP-CRDBP and observed that the phase change concentration of CRDBP was 25 nM and the fusion of mCherry-CRDBP and EGFP-CRDBP at 500 nM was completed within 70 min. We studied the effects of salt concentration and molecular crowding agents on the phase separation of CRDBP and found that salt can prevent the self-fusion of CRDBP and molecular crowding agents can improve the self-fusion of CRDBP. Furthermore, we found the recruitment behavior of CRDBP to ß-catenin proteins and studied their recruitment dynamics. Compared to conventional FCS, Scan-FCCS can significantly improve the reproducibility of measurements due to the dramatic increase of detection zone, and more importantly, this method can provide information about self-fusion and recruitment dynamics in protein phase separation.

CRDBP Protein Phase Separation and Its Recruitment to ß-Catenin Protein in a Single Living Cell.

The Coding Region Determinant-Binding Protein (CRDBP) is a carcinoembryonic protein, and it is overexpressed in various cancer cells in the form of granules. We speculated the formation of CRDBP granules possibly through liquid-liquid phase separation (LLPS) processes due to the existence of intrinsically disordered regions (IDRs) in CRDBP. So far, we did not know whether or how phase separation processes of CRDBP occur in single living cells due to the lack of in vivo methods for studying intracellular protein phase separation. Therefore, to develop an in situ method for studying protein phase separation in living cells is a very urgent task. In this work, we proposed an efficient method for studying phase separation behavior of CRDBP in a single living cell by combining in situ fluorescence correlation spectroscopy (FCS) and fluorescence cross-correlation spectroscopy (FCCS) with a fluorescence protein fusion technique. We first predicted and confirmed that CRDBP has phase separation in solution by conventional fluorescence imaging and FCS methods. And then, we in situ studied the phase separation behaviors of CRDBP in living cells and observed three states of CRDBP phase separation such as monomer state, cluster state, and granule state. We studied the effects of CRDBP truncated forms and its inhibitor on the CRDBP phase separation. Furthermore, we discovered the recruitment of CRDBP to ß-catenin protein in living cells and investigated the effects of CRDBP structures and inhibitor on CRDBP recruitment behavior. This finding may help us to further understand the mechanism of CRDBP protein for regulating Wnt signaling pathway. Additionally, our results documented that FCS/FCCS is an efficient and alternative method for studying protein phase separation in situ in living cells.

Deubiquitination Detection of p53 Protein in Living Cells by Fluorescence Cross-Correlation Spectroscopy.

Deubiquitination is a reverse post-translational modification of ubiquitination and plays significant roles in various signal transduction cascades and protein stability. The p53 is a very important tumor-suppressor protein and closely implicates more than 50% of human cancers. Although extracellular studies on the deubiquitination of p53 were reported, the process of p53 deubiquitination in living cells due to the shortage of an efficient in situ method for single living cells is still not clear. In this study, we described an in situ method for studying p53 deubiquitination in living cells by combining fluorescence cross-correlation spectroscopy with a fluorescent protein labeling technique. We first constructed the stable cell line expressing EGFP-Ub-p53-mCherry as the substrate of p53 deubiquitination. Then, we established a method for in situ monitoring of the deubiquitination of p53 in living cells. Based on the amplitudes of fluorescence correlation spectroscopy and fluorescence cross-correlation spectroscopy curves from living cells, we obtained the deubiquitination percentage for evaluating the level of p53 protein deubiquitination. Furthermore, we studied the effects of ubiquitin structures on p53 deubiquitination in living cells and found that the C-terminal Gly75-Gly76 motif of ubiquitin is a key location for p53 deubiquitination and the deubiquitination cannot occur when ubiquitin lacks the C-terminal Gly75-Gly76 motif. Our results documented that the developed strategy is an efficient method for in situ study of deubiquitination of proteins in living cells.

Resonance Light-Scattering Correlation Spectroscopy and Its Application in Analytical Chemistry for Life Science.

Resonance light-scattering correlation spectroscopy (RLSCS) is a new single-particle detection method with its working principle being like fluorescence correlation spectroscopy (FCS). RLSCS is obtained by autocorrelation function analysis on the measured fluctuation of the resonance light scattering (RLS) intensity occurring within a subfemtoliter volume when a single nanoparticle (such as gold nanoparticles (NPs) or silver (SNPs)) freely diffuses through the volume. The RLSCS technique can detect such parameters as concentration, diffusion coefficient (translation and rotation), etc. Compared with the FCS technique, the correlated fluorescence intensity signal in RLSCS is replaced with the RLS signal of the nanoparticles, overcoming some limits of the fluorescent probes such as photobleaching under high-intensity or long-term illumination. In this Account, we showcase RLSCS methods, theoretical models at different optical configurations, and some key applications. First, the RLSCS optical detection system was constructed based on the confocal optics, its theoretical model was proposed, and the diffusion behaviors of the nanoparticles in the solution were studied including the rotational and translational diffusion. And, methods were developed to measure the concentration, size, aspect ratio, and size distribution of the NPs. Second, based on the RLSCS methods, some detection strategies were developed for homogeneous DNA detection, immunoassay, apoptosis assay, self-thermophoresis of the nanomotor, and quantitative assay in single living cells. Meanwhile, a new fluorescence/scattering cross-correlation spectroscopy (FSCCS) method was proposed for monitoring the molecule-particle interaction. This method enriched the conventional fluorescence/fluorescence cross-correlation spectroscopy (FCCS) method. Third, using the EMCCD with high sensitivity and rapid response as an optical detector, two temporospatially resolved scattering correlation spectroscopy methods and their theoretical models were developed: total internal reflection (TIR) configuration-based spatially resolved scattering correlation spectroscopy (SRSCS) and dark-field illumination-based scattering correlation spectroscopy (DFSCS). These methods extended single-spot confocal RLSCS to imaging RLSCS, which makes RLSCS have the ability for multiple channel detection with temporospatial resolution. The method was successfully used for investigating the dynamic behaviors of gold NPs in live cells and obtained its temporospatial concentration distribution and diffusion behaviors. The final section of this Account outlines future directions in the development of RLSCS.

In vivo monitoring of the ubiquitination of newly synthesized proteins in living cells by combining a click reaction with fluorescence cross-correlation spectroscopy (FCCS).

Newly synthesized proteins are closely related to a series of biological processes, including cell growth, differentiation, and signaling. The post-translational modifications (PTMs) of newly synthesized proteins help maintain normal cellular functions. Ubiquitination is one of the PTMs and plays a prominent role in regulating cellular functions. Although great progress has been made in studying the ubiquitination of newly synthesized proteins, the in vivo monitoring of the ubiquitination of newly synthesized proteins in living cells still remains challenging. In this study, we propose a new method for measuring the ubiquitination of newly synthesized proteins in living cells by combining a click reaction with fluorescence cross-correlation spectroscopy (FCCS). In this study, a puromycin derivative (Puro-TCO) and a fluorescence probe (Bodipy-TR-Tz) were synthesized, and then, the newly synthesized proteins in living cells were labelled with Bodipy-TR via the click reaction between Puro-TCO and Tz. Ubiquitin (Ub) in living cells was labelled with the enhanced green fluorescence protein (EGFP) by fusion using a gene engineering technique. FCCS was used to quantify the newly synthesized proteins with two labels (EGFP and Bodipy-TR) in living cells. After measurements, the cross-correlation (CC) value was used to evaluate the ubiquitination degree of proteins. Herein, we established a method for monitoring the ubiquitination of newly synthesized proteins with EGFP-Ub in living cells and studied the effects of the ubiquitin E1 enzyme inhibitor on newly synthesized proteins. Our preliminary results document that the combination of FCCS with a click reaction is an efficient strategy for studying the ubiquitination of newly synthesized proteins in vivo in living cells. This new method can be applied to basic research in protein ubiquitination and drug screening at the living-cell level.

In vivo measurement of autophagic flux by fluorescence correlation spectroscopy.

Autophagy is a fundamental and phylogenetically conserved self-degradation process and plays a very important role in the selective degradation of deleterious proteins, organelles, and other macromolecules. Although flow cytometry and fluorescence imaging techniques have been used to assess autophagic flux, we remain less able to in vivo monitor autophagic flux in a highly sensitive, robust, and well-quantified manner. Here, we reported a new method for real-time and quantitatively monitoring autophagosomes and assessing autophagic flux in living cells based on fluorescence correlation spectroscopy (FCS). In this study, microtubule-associated protein 1A/1B-light chain 3B (LC3B) fused with an enhanced green fluorescent protein (EGFP-LC3B) was used as a biomarker to label autophagosomes in living cells, and FCS was used to monitor EGFP-LC3B labeled autophagosomes by using the characteristic diffusion time (τD) value and brightness per particle (BPP) value. By analyzing the distribution frequency of the τD values in living cells stably expressing EGFP-LC3B, mutant EGFP-LC3B (EGFP-LC3BΔG) and enhanced green fluorescent protein (EGFP), we found that the τD value greater than 10 ms was attributed to the signal of EGFP-LC3B labeled autophagosomes. So, we proposed a parameter PAP as an indicator to assess the basal autophagic activity and induced autophagic flux. This new method was able to evaluate autophagy inducers, early-stage autophagy inhibitors, and late-stage autophagy inhibitors. Compared with current methods, our method shows high spatiotemporal resolution and very high sensitivity for autophagosomes in low EGFP-LC3B expressing cells and will become an attractive and alternative method for biological and medical studies, some drug screening, and disease treatment.

In situ quantitative measurements on MMP-9 activity in single living cells by single molecule fluorescence correlation spectroscopy.

Matrix metalloproteinase-9 (MMP-9) plays an important role in tumor progression. It is of great significance to establish a sensitive in situ assay strategy for MMP-9 activity in single living cells. Here a novel in situ single molecule spectroscopy method based on the fluorescence correlation spectroscopy (FCS) technique was proposed for measuring the MMP-9 activity at different locations within single living cells, using a fluorescent specific peptide and a reference dye as dual probes. The measurement principle is based on the decrease of the ratiometric translational diffusion time of dual probes in the detection volume due to the peptide cleavage caused by MMP-9. The peptide probe was designed to be composed of an MMP-9 cleavage and cell-penetrating peptide sequence that was labeled with a fluorophore and conjugated with a streptavidin (SAV) molecule. The ratiometric translational diffusion time was used as the measurement parameter to eliminate the effect of intracellular uncertain viscosity. The linear relationship between the ratiometric diffusion time and MMP-9 activity was established, and applied to the determination of enzymatic activity in cell lysates as well as the evaluation of the inhibitory effects of different inhibitors on MMP-9. More importantly, the method was successfully used to dynamically determine MMP-9 activity in single living cells or under the stimulation with phorbol 12-myristate 13-acetate (PMA) and inhibitors.

Probing the Protein Corona of Nanoparticles in a Fluid Flow by Single-Particle Differenced Resonance Light Scattering Correlation Spectroscopy.

The protein corona of nanoparticles (NPs) plays a crucial role in determining NPs' biological fates. Here, a novel measurement strategy was proposed to in situ investigate the protein corona formed in the NPs with the home-built dual-wavelength laser-irradiated differenced resonance light scattering correlation spectroscopy (D-RLSCS) technique, combined with the modified generation method of the D-RLSCS curve. With the measurement strategy, the dissociation constants and the binding rates between proteins and gold nanoparticles (GNPs) were determined based on the binding-induced ratiometric diffusion change of NPs (the ratio of characteristic rotational diffusion time to translational one), using the formation of the protein corona of bovine serum albumin (BSA) or fibrinogen (FIB) on gold nanoparticles as a model. It was found that BSA shows a stronger binding constant and faster binding rate to gold nanospheres (GNSs) compared with those of FIB. Meanwhile, the dynamic behavior of the protein corona in a fluid flow mimicking biological vessels was further studied based on the combination of the D-RLSCS technique with a microfluidic channel. The measurement results indicated that some "loose" protein corona layers would strip off the surface of NPs within the microchannel due to the fluid sheath force. This method can provide the comprehensive information of a protein corona by averaging the diffusion behavior of many particles different from some conventional methods and overcome the shortcomings of conventional correlation spectroscopy methods.

Quantification of mRNA in Single Cells Based on Dimerization-Induced Photoluminescence Nonblinking of Quantum Dots.

Photoluminescence (PL) intermittency (or "blinking") is a unique characteristic of single quantum dot (QD) emission. Here, we report a novel single-molecule detection strategy for the intracellular mRNA of interest using the mRNA-induced nonblinking QD dimers as probes. The working principle of the method is that the DNA hybrid of the target DNA (or mRNA) with a biotin-modified ssDNA probe can induce two blinking streptavidin-modified QDs (SAV-QDs) conjugated. The formed QD dimer as a bright spot showed a nonblinking emission property, observed with total inner reflection fluorescence microscopy (TIRFM). In theory, one nonblinking spot indicated a target DNA (or mRNA). The experimental results from single-spot fluorescence trajectory analysis and single-particle brightness analysis based on TIRFM and fluorescence correlation spectroscopy (FCS) techniques verified this dimerization process of QDs or its induced nonblinking emission. Employing a target DNA with the same base sequences to Survivin mRNA as a model, the detection strategy was used to detect the target DNA concentration based on the linear relationship between the percentage of the nonblinking spots and the target DNA concentration. This single-molecule detection strategy was also successfully used for determining Survivin mRNA in a single HeLa cell. The method can simplify the hybridization steps, eliminate self-quenching and photobleaching of fluorophores, and reduce the influence of unspecific binding on the detection.

Brightness Analysis per Moving Particle: In Situ Analysis of Alkaline Phosphatase in Living Cells.

In situ quantitative analysis of enzymes such as phosphatase is important to understand a number of involved biological processes ranging from various metabolisms to signal transduction and cellular regulation. In this paper, a novel in situ measurement strategy was proposed to detect alkaline phosphatase (ALP) activity in different locations within single living cells. The principle is based on the measurement of the resonance light scattering brightness ratio (SBR) per moving nanoparticle that forms in an ALP-related chemical reaction. In the method, a novel resonance light scattering correlation spectroscopy (RLSCS) system was developed using two lasers for illumination or two detection channels. Using the gold nanoparticles (AuNPs) as probes, the Au@Ag nanoparticles (Au@Ag NPs) formed due to the ALP-catalyzed hydrolysis of ascorbic acid 2-phosphate (AAP) and the subsequent reduction-deposition reaction of Ag ions that occurred on the AuNPs. The SBR value per moving particle was determined based on the obtained RLS intensity traces and RLSCS curves. The SBR value was found to be not influenced by the intracellular viscosity and size that was confirmed in the experiments. The linear relation between the SBR and ALP activity was established and applied to detect ALP activity and evaluate the inhibition of different drugs. Finally, the method was successfully used to in situ measure ALP activity within living cells. The method overcomes the shortcoming of conventional methods that lack quantitative analysis and are susceptible to intracellular viscosity.

The Theoretical Model, Method, and Applications of Scattering Photon Burst Counting Based on an Objective Scanning Technique.

Scattering photon burst counting (SPBC) is a single-particle detection method, which is based on measuring scattering photon bursting of single nanoparticles through a detection volume of <1 fL. Although SPBC has been used for bioassays and analysis of nanoparticles, it is necessary to establish its theoretical model and develop a new detection mode in order to further enhance its sensitivity and enlarge its application fields. In this paper, we proposed a theoretical model for the confocal SPBC method and developed a novel SPBC detection mode using the fast objective scanning technique. The computer simulations and experiments documented that this model well describes the relation between photon counts and experimental parameters (such as nanoparticle concentration and diameter, temperature, and viscosity). Based on this model, we developed a novel SPBC detection mode by using the fast objective scanning technique. Compared to the current confocal SPBC method, the sensitivity of this new method was significantly increased due to the significantly increased photon counts per sampling time, the linear detection range is from 0.9 to 90 pM, and the limit of detection is reduced to 40 fM for 30 nm gold nanoparticles. Furthermore, this new method was successfully applied to determine the enzyme activity of caspase-3 and evaluate the inhibition effectiveness of some inhibitors.

Single-Particle Catalytic Analysis by a Photon Burst Counting Technique Combined with a Microfluidic Chip.

Single-particle catalytic analysis plays an important role to understand the catalytic mechanism of nanocatalysts. Currently, some methods are used to study the relationship between single-particle catalytic activity and morphology. However, there is still lack of a simple and rapid analysis method for evaluating the catalytic activity of an individual nanocatalyst that freely moves in solution. Here, we proposed a novel single-particle catalytic analysis method for investigating the catalytic activity of a free nanocatalyst. Its working principle is based on the photon burst counting analysis on fluorescent catalytic products of an individual nanocatalyst combined with a microfluidic chip. In this study, we used the reduction reaction of resazurin (RZ) to resorufin (RF) catalyzed by gold nanoparticles (GNPs) as a model. When nonfluorescent RZ molecules in one microchannel of the microfluidic chip mixed with the GNPs flowing in another channel under the control of flow rates, each individual photon burst from the catalytic product RF by GNPs was measured in real time with a constructed flow single-particle catalytic analysis (SPCA) system. With the method, the obtained intensity of each photon burst reflects the capacity of a particle to catalyze RZ molecules into RF(s). The number of photon burst within sampling time reflects the particle number of GNPs with catalytic activity. The experimental conditions including the mixing mode of the nanocatalyst and the substrate, the flow rate, RZ concentration, and detection time were optimized. Finally, the method was successfully used to study the catalytic activity of GNPs with different sizes and morphologies.

In Situ Assay of Proteins Incorporated with Unnatural Amino Acids in Single Living Cells by Differenced Resonance Light Scattering Correlation Spectroscopy.

Site-specific incorporation of unnatural amino acids (UAAs) into target proteins (UAA-proteins) provides the unprecedented opportunities to study cell biology and biomedicine. However, it is a big challenge to in situ quantitatively determine the expression level of UAA-proteins due to serious interferences from autofluorescence, background scattering, and different viscosity in living cells. Here, we proposed a novel single nanoparticle spectroscopy method, differenced resonance light scattering correlation spectroscopy (D-RLSCS), to measure the UAA-proteins in single living cells. The D-RLSCS principle is based on the simultaneous measurement of the resonance scattering light fluctuation of a single gold nanoparticle (GNP) in two detection channels irradiated by two coaxial laser beams and then autocorrelation analysis on the differenced fluctuation signals between two channels. D-RLSCS can avoid the interferences from intracellular background scattering and provide the concentration and rotational and translational diffusion information of GNPs in solution or in living cells. Furthermore, we proposed a parameter, the ratiometric diffusion time and found that this parameter is proportional to the square of particle size. The theoretical and experimental results demonstrated that the ratiometric diffusion time was not influenced by the intracellular viscosity. This method was successfully applied for in situ quantification of the UAA-protein within single living cells based on the increase in the ratiometric diffusion time of nanoprobes bound with proteins. Using UAA-EGFP (enhanced green fluorescent protein) as a model, we observed the significant difference in the UAA-protein concentrations at different positions in single living cells.

Studying Homo-oligomerization and Hetero-oligomerization of MDMX and MDM2 Proteins in Single Living Cells by Using In Situ Fluorescence Correlation Spectroscopy.

Protein oligomerization plays a very important role in many physiological processes. p53 acts as a key tumor suppressor by regulating cell cycle arrest, DNA repair, and apoptosis, and its antitumor activity is regulated by the hetero- and homo-oligomerization of MDMX and MDM2 proteins. So far, some traditional methods have been utilized to study the oligomerization of MDMX and MDM2 in vitro, but they have not clarified some controversial issues or whether the extracellular results can represent the intracellular results. Here, we put forward an in situ method for studying protein homo- and hetero-oligomerization in single living cells by using fluorescence correlation spectroscopy. In this study, MDMX and MDM2 were labeled with fluorescent proteins using lentiviral transfection. Autocorrelation spectroscopy and cross-correlation spectroscopy methods were used to study the oligomerization of MDMX and MDM2 in situ and the effect of regulation of MDMX oligomerization on p53-MDMX interactions in single living cells. We observed the homo- and hetero-oligomerization of MDMX and MDM2 in living cells. Meanwhile, the levels of the homo-oligomers of MDMX and MDM2 were increased due to the lack of hetero-oligomerization. Finally, the binding affinity of MDMX for p53 was improved with an increase in the level of MDMX hetero-oligomerization.

Simultaneously monitoring endogenous MAPK members in single living cells by multi-channel fluorescence correlation spectroscopy.

The mitogen-activated protein kinase (MAPK) pathway is a major module for cellular signal transduction. The dysregulation of the MAPK pathway has been involved in the pathogenesis of multiple diseases ranging from cancers to chronic inflammations. So far, we have not fully understood the influences of external factors and signaling networks on the MAPK pathway due to the lack of in situ methods for simultaneous detection of multiple kinases in the pathway in living cells. Herein, we present a new strategy for in situ and simultaneously monitoring MAPK pathway kinases in single living cells combining multi-channel fluorescence correlation spectroscopy (FCS) with affinity fluorescent probes. We chose rapidly growing fibrosarcoma kinase (RAF), mitogen-activated protein kinase (MEK), and extracellular signal-regulated kinase (ERK) as representative members in the MAPK pathway. We designed and synthesized three fluorescent affinity probes and experimental results demonstrated that the three probes specifically targeted endogenous BRAF, MEK1/2, and ERK1/2 in living cells. Based on the multi-channel FCS system, we studied the influences of biological substances, drugs and oxidative stress on the activities of endogenous MAPK kinases and the cross-talk between the MAPK and PI3K-mTOR pathways. We have found that serum, sorafenib, and hydrogen peroxide can regulate multiple MAPK kinases and the effects of external stimuli can transmit to the MAPK pathway; furthermore, we have observed that the MAPK pathway can be activated by modulating the PI3K-mTOR pathway. Our results illustrated the complexity of a cellular signal network and the necessity of in situ and simultaneous determination of biomolecules in living cells.

Analyses of p73 Protein Oligomerization and p73-MDM2 Interaction in Single Living Cells Using In Situ Single Molecule Spectroscopy.

Protein oligomerization and protein-protein interaction are crucial to regulate protein functions and biological processes. p73 protein is a very important transcriptional factor and can promote apoptosis and cell cycle arrest, and its transcriptional activity is regulated by p73 oligomerization and p73-MDM2 interaction. Although extracellular studies on p73 oligomerization and p73-MDM2 interaction have been carried out, it is unclear how p73 oligomerization and p73-MDM2 interaction occur in living cells. In our study, we described an in situ method for studying p73 oligomerization and p73-MDM2 interaction in living cells by combining fluorescence cross-correlation spectroscopy with a fluorescent protein labeling technique. Lentiviral transfection was used to transfect cells with a plasmid for either p73 or MDM2, each fused to a different fluorescent protein. p73 oligomerization was evaluated using brightness per particle, and the p73-MDM2 interaction was quantified using the cross-correlation value. We constructed a series of p73 mutants in three domains (transactivation domain, DNA binding domain, and oligomerization domain) and MDM2 mutants. We systematically studied p73 oligomerization and the effects of p73 oligomerization and the p73 and MDM2 structures on the p73-MDM2 interaction in single living cells. We have found that the p73 protein can form oligomers and that the p73 structure changes in the oligomerization domain significantly influence its oligomerization. p73 oligomerization and the structure changes significantly affect the p73-MDM2 interaction. Furthermore, the effects of inhibitors on p73 oligomerization and p73-MDM2 interaction were studied.

Artificial Aquaporin That Restores Wound Healing of Impaired Cells.

Artificial aquaporins are synthetic molecules that mimic the structure and function of natural aquaporins (AQPs) in cell membranes. The development of artificial aquaporins would provide an alternative strategy for treatment of AQP-related diseases. In this report, an artificial aquaporin has been constructed from an amino-terminated tubular molecule, which operates in a unimolecular mechanism. The artificial channel can work in cell membranes with high water permeability and selectivity rivaling those of AQPs. Importantly, the channel can restore wound healing of the cells that contain function-lost AQPs.

In Situ Study of Interactions between Endogenous c-myc mRNA with CRDBP in a Single Living Cell by Combining Fluorescence Cross-Correlation Spectroscopy with Molecular Beacons.

mRNA-protein interactions play key roles in facilitating various biological functions in gene expression regulations and even the progression of diseases. However, it is still a challenge to directly monitor mRNA-protein interactions in a single living cell at present. Herein, we propose a new strategy for real-time studying of mRNA-protein interactions in a single living cell using fluorescence cross-correlation spectroscopy (FCCS) and molecular beacon (MB) labeling techniques. The c-myc mRNA and coding region determinant binding protein (CRDBP) were used as models. We first evaluated the performances of unmodified (2'-deoxy) and modified (2'-O-methyl) MBs and found that the 2'-O-methyl loop MB (2'-O-methyl loop domain, 2'-deoxy stem region) has high affinity to target mRNA and good nuclease resistance. Then we constructed stable cell line expressing mCherry-CRDBP using lentivirus infection, and on the basis of FCCS, we established an efficient method for quantifying the interaction of c-myc mRNA with CRDBP in a single living cell. The RNA binding domains of CRDBP cover two RNA recognition motifs (RRM) and four K homologies (KH). Furthermore, we constructed the truncated variants and point mutants on RNA binding domains of CRDBP, systematically studied the effects of RNA binding domains of CRDBP on c-myc mRNA-CRDBP interaction in living cells, and found that KH3-4 is indispensable for c-myc mRNA binding, KH1-2 plays a supplementary role, and RRM1-2 shows no binding ability to c-myc mRNA. Our work reveals the mechanisms of c-myc mRNA-CRDBP interactions and provides a general strategy for quantifying the interactions of endogenous mRNA with protein in a single living cell.