Elucidating the Functional Mechanism of PTK7 in Cancer Development through Spatial Assembly Analysis Using Super Resolution Imaging.

Protein tyrosine kinase-7 (PTK7) has been reported as a vital participant in the Wnt signaling pathway, influencing tumorigenesis and metastasis. However, their specific roles in the mechanisms underlying cancer development and progression remain elusive. Here, using direct stochastic optical reconstruction microscopy (dSTORM) with aptamer-probe labeling, we first revealed that a weakening clustering distribution of PTK7 on the basal membranes happened as cellular migration increased during cancer progression. This correspondence was further supported by a diminished aggregated state of PTK7 caused by direct enhancement of cell migration. By comparing the alterations in PTK7 distribution with activation or inhibition of specific Wnt signaling pathway, we speculated that PTK7 could modulate cell migration by participating in the interplay between canonical Wnt (in MCF7 cells) and noncanonical Wnt signals (in MDA-MB-231 cells). Furthermore, we discovered that the spatial distribution morphology of PTK7 was also subject to the hydrolysis ability and activation state of the related hydrolase Matrix metallopeptidase14 (MMP14). This function-related specific assembly of PTK7 reveals a clear relationship between PTK7 and cancer. Meanwhile, potential molecular interactions predicted by the apparent assembly morphology can promote a deep understanding of the functional mechanism of PTK7 in cancer progress.

An Agrobacterium-Mediated Transient Expression Method for Functional Assay of Genes Promoting Disease in Monocots.

Agrobacterium-mediated transient expression (AMTE) has been widely used for high-throughput assays of gene function in diverse plant species. However, its application in monocots is still limited due to low expression efficiency. Here, by using histochemical staining and a quantitative fluorescence assay of ß-glucuronidase (GUS) gene expression, we investigated factors affecting the efficiency of AMTE on intact barley plants. We found prominent variation in GUS expression levels across diverse vectors commonly used for stable transformation and that the vector pCBEP produced the highest expression. Additionally, concurrent treatments of plants with one day of high humidity and two days of darkness following agro-infiltration also significantly increased GUS expression efficiency. We thus established an optimized method for efficient AMTE on barley and further demonstrated its efficiency on wheat and rice plants. We showed that this approach could produce enough proteins suitable for split-luciferase assays of protein-protein interactions on barley leaves. Moreover, we incorporated the AMTE protocol into the functional dissection of a complex biological process such as plant disease. Based on our previous research, we used the pCBEP vector to construct a full-length cDNA library of genes upregulated during the early stage of rice blast disease. A subsequent screen of the library by AMTE identified 15 candidate genes (out of ~2000 clones) promoting blast disease on barley plants. Four identified genes encode chloroplast-related proteins: OsNYC3, OsNUDX21, OsMRS2-9, and OsAk2. These genes were induced during rice blast disease; however, constitutive overexpression of these genes conferred enhanced disease susceptibility to Colletotrichum higginsianum in Arabidopsis. These observations highlight the power of the optimized AMTE approach on monocots as an effective tool for facilitating functional assays of genes mediating complex processes such as plant-microbe interactions.

Regulatory Mechanisms of SNAP-25-Associated Insulin Release Revealed by Live-Cell Confocal and Single-Molecule Localization Imaging.

Impaired insulin release is the key feature of type 2 diabetes. Insulin secretion, mainly mediated by SNARE proteins, is closely related to the blood glucose level. However, the mechanism underlying how glucose controls SNARE proteins to regulate insulin release is largely unexplained. Herein, we investigated the effects of glucose on the subcellular localization and spatial distribution on the plasma membrane (PM) of t-SNAREs (SNAP-25 and STX-1A) using a live-cell confocal microscope and the single-molecule localization imaging technique. Live-cell confocal and dSTORM imaging first revealed that SNAP-25 was mostly localized to the PM as clusters under the basal glucose concentration condition and demonstrated significant colocalization with STX-1A clusters. Furthermore, our data showed that the elevated glucose concentration increased the expression of SNAP-25 and induced more and larger SNAP-25 clusters on the PM, whereas glucotoxicity severely inhibited SNAP-25 transport to the PM and caused fewer and smaller SNAP-25 clusters on the PM. Additionally, we found that glucotoxicity also had an inhibitory effect on the colocalization between SNAP-25 and STX-1A, indicating a decrease of their interactions. Our study sheds light on the regulatory effects of glucose on the functional organization of t-SNAREs at a subcellular and molecular level, thus providing new insights into the mechanisms by which SNAREs regulate insulin release.

Thermally stable gold nanorod dispersed silicone composite with plasmonic resonance in the optical communication window.

Gold nanorods (AuNRs) possess a high optical nonlinear coefficient, ultrafast optical response speed and widely tunable localized surface plasmon resonance (LSPR) wavelength covering the visible and near infrared region. Therefore, they are extensively investigated for many optical applications. However, the poor thermal stability of the AuNRs seriously restricts their practical performance. In addition, for many applications, such as optical communication or laser modulation, AuNRs have to be combined with transparent solids, for example polymers, glass or crystals to make devices. Here, we report on the preparation of 0.23 mg AuNR dispersed methyl silicone resin (MSR) with longitudinal LSPR (L-LSPR) wavelength (1450 nm) in the optical communication window. We found that AuNR-silicone composites possess high thermal stability. After calcination in ambient environment at a temperature of 250 °C for 10 h, the L-LSPR peak of the sample can remain longer than 1380 nm, implying that the NR shape of the Au particles was well maintained. Using the open-aperture Z-scan technique, the nonlinear absorption coefficient of the composites was measured as -11.71 cm GW-1, higher than many nonlinear materials. Thus, the thermally stable AuNR@SiO2-MSR composite with high nonlinearity is promising for practical applications in the optical communication window.

Insight into the Different Channel Proteins of Human Red Blood Cell Membranes Revealed by Combined dSTORM and AFM Techniques.

Membrane proteins tend to interact with each other in the cell membranes to form protein clusters and perform the corresponding physiological functions. However, because channel proteins are involved in many biological functions, their distribution and nano-organization in these protein clusters are unclear. To study the distribution patterns and relationships between the different channel proteins, we identified the locations of glucose transporter 1 (Glut1) and Band3 (anion transporter 1) precisely in the topography of the cytoplasmic side of the human red blood cell (hRBC) membranes using combined atomic force microscopy (AFM) and single-molecule localization microscopy (SMLM). The AFM results revealed that membrane proteins interacted with each other and aggregated into protein islands. The SMLM results showed that Glut1 and Band3 tended to form protein clusters in the hRBC membranes, and there was a strong colocalization between the two proteins. The results of the combined AFM and SMLM method indicated that the protein clusters of Glut1 and Band3 were mainly located in the protein islands of topography, and the protein islands in topography also interacted with each other to assemble into larger protein clusters or functional microdomains.

Mechanistic insights into GLUT1 activation and clustering revealed by super-resolution imaging.

The glucose transporter GLUT1, a plasma membrane protein that mediates glucose homeostasis in mammalian cells, is responsible for constitutive uptake of glucose into many tissues and organs. Many studies have focused on its vital physiological functions and close relationship with diseases. However, the molecular mechanisms of its activation and transport are not clear, and its detailed distribution pattern on cell membranes also remains unknown. To address these, we first investigated the distribution and assembly of GLUT1 at a nanometer resolution by super-resolution imaging. On HeLa cell membranes, the transporter formed clusters with an average diameter of ∼250 nm, the majority of which were regulated by lipid rafts, as well as being restricted in size by both the cytoskeleton and glycosylation. More importantly, we found that the activation of GLUT1 by azide or MßCD did not increase its membrane expression but induced the decrease of the large clusters. The results suggested that sporadic distribution of GLUT1 may facilitate the transport of glucose, implying a potential association between the distribution and activation. Collectively, our work characterized the clustering distribution of GLUT1 and linked its spatial structural organization to the functions, which would provide insights into the activation mechanism of the transporter.

Structural Mechanism Analysis of Orderly and Efficient Vesicle Transport by High-Resolution Imaging and Fluorescence Tracking.

The orderly organelle interaction network is essential for normal biological activity of cells. However, the mechanism of orderly organelle interaction remains elusive. In this report, we analyzed the structure characteristics of the cell membrane, endocytic vesicles, and the Golgi membrane through a high-resolution imaging technique and further comprehensively investigated the vesicle-transport process via epidermal growth factor receptor endocytosis and a recycling pathway using a real-time fluorescence tracing method. Our data suggest that orderly vesicle transport is due to protein protrusion from the outer surface of endocytic vesicles and that full membrane fusion between homotypic endocytic vesicles is a result of the rough outer surface. Finally, the kiss-and-run method, which is utilized by endocytic vesicles to communicate with the trans-Golgi network (TGN) is attributed to a dense protein layer at the outer surface of the TGN. In summary, by combining static structural analysis with dynamic tracing, we elucidate the mechanism of orderly vesicle transport from the overall structural features of the membrane. This work provides insight into the structural mechanisms underlying vital biological processes involving organelle interactions at the molecular level.

Quantitatively Mapping the Assembly Pattern of EpCAM on Cell Membranes with Peptide Probes.

Epithelial cell adhesion molecule (EpCAM) is an important type I transmembrane protein that is overexpressed on the surfaces of most cancer cells and involved in various biological processes such as cell adhesion and cell signaling. Although it plays crucial roles in cell functions and tumorigenesis, questions concerning the detailed morphology, molecular stoichiometry, and the assembly mechanisms of EpCAM on cell membranes have not been fully elucidated. Here, we used direct stochastic optical reconstruction microscopy (dSTORM) and relied on fluorophore-conjugated peptides to quantitatively analyze the assembly pattern of EpCAM with single-molecule precision. EpCAM was found to organize heterogeneous clusters with different sizes, which contain different numbers of EpCAM molecules on MCF-7 cell membranes. Moreover, dual-color dSTORM imaging revealed a significant correlation between EpCAM and tetraspanin CD9, and part of the EpCAM clusters could be disrupted by knockdown of CD9, which indicated that EpCAM might localize in tetraspanin-enriched microdomains (TEMs) and function cooperatively with CD9 on cell membranes. In addition, the assembly of the membrane EpCAM was found to be limited by both cytoskeleton and glycosylation. Overall, our work clarified the clustered distribution of EpCAM and revealed the potential mechanisms of its clustering at the molecular level, promoting a deeper understanding of the nano-organization of membrane proteins.

Ultrafast Tracking of a Single Live Virion During the Invagination of a Cell Membrane.

The first step in most viral infections is the penetration of the cell membrane via endocytosis. However, the underlying mechanism of this important process has not been quantitatively characterized; for example, the velocity and force of a single virion during invagination remain unknown. Here, the endocytosis of a single live virion (Singapore grouper iridovirus, SGIV) through the apical membranes of a host cell is monitored by developing and using a novel ultrafast (at the microsecond level) tracking technique: force tracing. For the first time, these results unambiguously reveal that the maximum velocity during the cell entry of a single SGIV by membrane invagination is approximately 200 nm s(-1), the endocytic force is approximately 60.8 ± 18.5 pN, and the binding energy density increases with the engulfment depth. This report utilizing high temporospatial resolution (subnanometer and microsecond levels) approaches provides new insight into the dynamic process of viral infection via endocytosis and the mechanism of membrane invagination at the single-particle level.

Entry of a novel marine DNA virus, Singapore grouper iridovirus, into host cells occurs via clathrin-mediated endocytosis and macropinocytosis in a pH-dependent manner.

UNLABELLED: Iridoviruses are nucleocytoplasmic DNA viruses which cause great economic losses in the aquaculture industry but also show significant threat to global biodiversity. However, a lack of host cells has resulted in poor progress in clarifying iridovirus behavior. We investigated the crucial events during virus entry using a combination of single-virus tracking and biochemical assays, based on the established virus-cell infection model for Singapore grouper iridovirus (SGIV). SGIV infection in host cells was strongly inhibited when cells were pretreated with drugs blocking clathrin-mediated endocytosis, including sucrose and chlorpromazine. Inhibition of key regulators of macropinocytosis, including Na(+)/H(+) exchanger, Rac1 GTPase, p21-activated kinase 1 (PAK1), protein kinase C (PKC), and myosin II, significantly reduced SGIV uptake. Cy5-labeled SGIV particles were observed to colocalize with clathrin and macropinosomes. In contrast, disruption of cellular cholesterol by methyl-ß-cyclodextrin and nystatin had no effect on virus infection, suggesting that SGIV entered grouper cells via the clathrin-mediated endocytic pathway and macropinocytosis but not via caveola-dependent endocytosis. Furthermore, inhibitors of endosome acidification such as chloroquine and bafilomycin A1 blocked virus infection, indicating that SGIV entered cells in a pH-dependent manner. In addition, SGIV particles were observed to be transported along both microtubules and actin filaments, and intracellular SGIV motility was remarkably impaired by depolymerization of microtubules or actin filaments. The results of this study for the first time demonstrate that not only the clathrin-dependent pathway but also macropinocytosis are involved in fish DNA enveloped virus entry, thus providing a convenient tactic for exploring the life cycle of DNA viruses. IMPORTANCE: Virus entry into host cells is critically important for initiating infections and is usually recognized as an ideal target for the design of antiviral strategies. Iridoviruses are large DNA viruses which cause serious threats to ecological diversity and the aquaculture industry worldwide. However, the current understanding of iridovirus entry is limited and controversial. Singapore grouper iridovirus (SGIV) is a novel marine fish DNA virus which belongs to genus Ranavirus, family Iridoviridae. Here, using single-virus tracking technology in combination with biochemical assays, we investigated the crucial events during SGIV entry and demonstrated that SGIV entered grouper cells via the clathrin-mediated endocytic pathway in a pH-dependent manner but not via caveola-dependent endocytosis. Furthermore, we propose for the first time that macropinocytosis is involved in iridovirus entry. Together, this work not only contributes greatly to understating iridovirus pathogenesis but also provides an ideal model for exploring the behavior of DNA viruses in living cells.

Reduced energy offset via substitutional doping for efficient organic/inorganic hybrid solar cells.

Charge carrier transport in bulk heterojunction that is central to the device performance of solar cells is sensitively dependent on the energy level alignment of acceptor and donor. However, the effect of energy level regulation induced by nickel ions on the primary photoexcited electron transfer and the performance of P3HT/TiO2 hybrid solar cells remains being poorly understood and rarely studied. Here we demonstrate that the introduction of the versatile nickel ions into TiO2 nanocrystals can significantly elevate the conduction and valence band energy levels of the acceptor, thus resulting in a remarkable reduction of energy level offset between the conduction band of acceptor and lowest unoccupied molecular orbital of donor. By applying transient photoluminescence and femtosecond transient absorption spectroscopies, we demonstrate that the electron transfer becomes more competitive after incorporating nickel ions. In particular, the electron transfer life time is shortened from 30.2 to 16.7 ps, i.e., more than 44% faster than pure TiO2 acceptor, thus leading to a notable increase of power conversion efficiency in organic/inorganic hybrid solar cells. This work underscores the promising virtue of engineering the reduction of 'excess' energy offset to accelerate electron transport and demonstrates the potential of nickel ions in applications of solar energy conversion and photon detectors.

Dual-functional probes for sequential thiol and redox homeostasis sensing in live cells.

A new type of resorufin-based dual-functional fluorescent probe whose fluorescence emission features are sensitive to thiol compounds and redox homeostasis was developed. Thiols-triggered nucleophilic substitution of the probes converts the nonfluorescent probe to the highly fluorescent resorufin moiety; the released resorufin not only enables fluorescence signaling specific for thiol compounds but functions as a redox indicator with sensitive colorimetric and fluorescence emission change upon redox variation. Preliminary fluorescence imaging experiments have revealed the biocompatibility of the as-prepared probes and validated their practicability for thiol sensing and redox homeostasis mapping in living cells.

Magnetic-field-enabled resolution enhancement in super-resolution imaging.

A novel strategy for modulating the photophysics of organic dyes in super-resolution fluorescence imaging using an external magnetic field was reported. The magnetic field induced increase in fluorescence intensity, localization number of probe molecules, and the number of photons emitted per molecule as compared to those acquired without a magnetic field were experimentally confirmed. Improved dSTORM localization precision and imaging resolution were consequently achieved.

Development of polymeric nanoprobes with improved lifetime dynamic range and stability for intracellular oxygen sensing.

A class of core-shell nanoparticles possessing a layer of biocompatible shell and hydrophobic core with embedded oxygen-sensitive platinum-porphyrin (PtTFPP) dyes is developed via a radical-initiated microemulsion co-polymerization strategy. The influences of host matrices and the PtTFPP incorporation manner on the photophysical properties and the oxygen-sensing performance of the nanoparticles are investigated. Self-loading capability with cells and intracellular-oxygen-sensing ability of the as-prepared nanoparticle probes in the range 0%-20% oxygen concentration are confirmed. Polymeric nanoparticles with optimized formats are characterized by their relatively small diameter (<50 nm), core-shell structures with biocompatible shells, covalent-attachment-imparted leak-free construction, improved lifetime dynamic range (up to 44 µs), excellent storage stability and photostability, and facile cell uptake. The nanoparticles' small sensor diameter and core-shell structure with biocompatible shell make them suitable for intracellular detection applications. For intracellular detection applications, the leak-free feature of the as-prepared nanoparticle sensor effectively minimizes potential chemical interferences and cytotoxicity. As a salient feature, improved lifetime dynamic range of the sensor is expected to enable precise oxygen detection and control in specific practical applications in stem-cell biology and medical research. Such a feature-packed nanoparticle oxygen sensor may find applications in precise oxygen-level mapping of living cells and tissue.

High glucose-induced glucagon resistance and membrane distribution of GCGR revealed by super-resolution imaging.

The glucagon receptor (GCGR) is a member of the class B G protein-coupled receptor family. Many research works have been carried out on GCGR structure, glucagon signaling pathway, and GCGR antagonists. However, the expression and fine distribution of GCGR proteins in response to glucagon under high glucose remain unclear. Using direct stochastic optical reconstruction microscopy (dSTORM) imaging, nanoscale GCGR clusters were observed on HepG2 cell membranes, and high glucose promoted GCGR expression and the formation of more and larger clusters. Moreover, glucagon stimulation under high glucose did not inhibit GCGR levels as significantly as that under low glucose and did not increase the downstream cyclic 3,5'-adenosine monophosphate-protein kinase A (cAMP-PKA) signal, and there were still large-size clusters on the membranes, indicating that high glucose induced glucagon resistance. In addition, high glucose induced stronger glucagon resistance in hepatoma cells compared with hepatic cells. Our work will pave a way to further our understanding of the pathogenesis of diabetes and develop more effective drugs targeting GCGR.

Effects of sequential cleavage and blastocyst embryo transfer on pregnancy outcomes in patients with poor ovarian response.

The management of patients with poor ovarian response (POR) remains a major challenge for fertility specialists in in vitro fertilization-embryo transfer (IVF-ET). In this retrospective cohort study, we aimed to evaluate the clinical effect of sequential transfer on pregnancy outcomes in patients with POR. A total of 3579 POR patients who underwent the first frozen embryo transfer (FET) cycle were enrolled from January 2018 to April 2021. The patients were divided into three groups according to the embryo transfer (ET) strategy adopted: a study group that included POR patients in whom a cleavage-stage embryo (day 3) and a blastocyst (day 5/6) were transferred (sequential transfer group), and two control groups in whom two cleavage-stage embryos (D3-dET group) or two blastocysts (D5/6-dET group) were transferred. The study group was matched with the control groups at a ratio of 1:4 by propensity score matching (PSM). The main pregnancy outcomes measured were the live birth rate and multiple pregnancy rate. After PSM, the live birth rate in the sequential transfer group was significantly higher than that in the D3-dET group (44.2% vs. 34.3%, P = 0.019), and was similar to that in the D5/6-dET group (44.2% vs. 45.3%; P = 0.90). In addition, there was no increase in the risk of multiple pregnancy among POR patients undergoing sequential transfer compared with both D3-dET (26.7% vs. 25.6%, P = 0.85) and D5/6-dET (26.7% vs. 28.4%, P = 0.97) groups. These findings imply that sequential transfer is an effective option for POR patients and could be utilized after careful consideration.

Mechanism of INSR clustering with insulin activation and resistance revealed by super-resolution imaging.

Insulin receptor (INSR) is a key protein in the INSR signaling pathway and plays a critical role in biological processes, especially in the regulation of glucose homeostasis. Many metabolic diseases are often accompanied by abnormal INSR signaling. However, the specific effector mechanisms regulating insulin resistance and the distribution patterns of INSR during cell membrane activation remain unclear. Here, we investigated the changes in the distribution of INSR during activation using super-resolution imaging. By observing the connection between INSR activation and its distribution, we found that insulin resistance inhibits its receptor clustering. More importantly, we found that INSR has a highly co-localized relationship with the skeletal protein ßII-spectrin. Specific knockout of ßII-spectrin inhibited the interaction of INSR with GLUT4 and affected the normal metabolism of glucose. Our work elucidates the effects of insulin activation and insulin resistance on INSR distribution and reveals a potential relationship between INSR and cytoskeleton at the single molecule level, which promotes a deeper understanding of the roles associated with insulin signaling and insulin resistance.

Spatiotemporal tracking of the transport of RNA nano-drugs: from transmembrane to intracellular delivery.

The popularity of RNA nanoparticles (RNPs) has risen rapidly during the past decade due to the development of RNA nanotechnology. Understanding the fast dynamic process of cell entry and intracellular delivery of RNPs is essential for the design of intelligent therapeutic RNA nano-drugs and mRNA vaccines.How the interaction between the membrane and target ligand of RNPs influences the cell entry, and how the dynamic mechanism of RNPs takes place in different organelles remain ill-defined. Herein, the cell entry of Antimir21-RNP-Apt is monitored using a force tracing technique with a high spatiotemporal resolution at the single particle level, the specific interaction of Apt and EGFR promotes the cell entry efficiency and achieves long-lasting curative effects. Furthermore, the intracellular delivery pathway through different organelles is discovered using fluorescence tracking, and the low motility in early endosomes and the high motility in late endosomes are analyzed. This report provides key strategies for engineering RNA nanomedicines and facilitating clinical translation.

Quantification of mechanical stimuli inducing nucleoplasmic translocation of YAP and its distribution mechanism using an AFM-dSTORM coupled technique.

Cells can regulate a variety of behaviors by sensing mechanical signals, including growth, differentiation, apoptosis and so on. Yes-associated protein (YAP) is a mechanically sensitive protein that can be used as an indicator of mechanosignaling transduction. Unlike macroscopic statistical analysis, single-cell analysis is more demanding and challenging in terms of mechanistic regulation. Here, we quantified the location, amplitude and duration of single-cell mechanical stimulation by precise mechanical modulation, and simultaneously observed the mechanical force induced YAP nuclear and cytoplasmic distribution translocation using the AFM-dSTORM coupled techniques. Additionally, we investigated the regulation of YAP translocation according to the physical factors (cytoskeletal destruction and osmotic pressure) and biochemical factors (nuclear active transport protein inhibiter and starvation). Our study revealed that mechanical signals were transferred from the cytoskeleton to the nucleus through the synergistic action of microfilaments and microtubules, and then induced YAP translocation from the nucleus to the cytoplasm under the cooperation of nuclear export proteins. This conclusion deepens the understanding of the signaling pathway by which mechanical signals are transmitted from the plasma membrane to the cytoplasm and then to the nucleus to determine the cell's fate.

Mechanistic Insights into Membrane Protein Clustering Revealed by Visualizing EGFR Secretion.

Most plasmalemmal proteins are organized into clusters to modulate various cellular functions. However, the machineries that regulate protein clustering remain largely unclear. Here, with EGFR as an example, we directly and in detail visualized the entire process of EGFR from synthesis to secretion onto the plasma membrane (PM) using a high-speed, high-resolution spinning-disk confocal microscope. First, colocalization imaging revealed that EGFR secretory vesicles underwent transport from the ER to the Golgi to the PM, eventually forming different distribution forms on the apical and basal membranes; that is, most EGFR formed larger clusters on the apical membrane than the basal membrane. A dynamic tracking image and further siRNA interference experiment confirmed that fusion of secretory vesicles with the plasma membrane led to EGFR clusters, and we showed that EGFR PM clustering may be intimately related to EGFR signaling and cell proliferation. Finally, we found that the size and origin of the secretory vesicles themselves may determine the difference in the distribution patterns of EGFR on the PM. More importantly, we showed that actin influenced the EGFR distribution by controlling the fusion of secretory vesicles with the PM. Collectively, a comprehensive understanding of the EGFR secretion process helps us to unravel the EGFR clustering process and elucidate the key factors determining the differences in the spatial distribution of EGFR PM, highlighting the correlation between EGFR secretion and its PM distribution pattern.