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.

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.

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.

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.

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.

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.

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.

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.

A multidrug-resistant P-glycoprotein assembly revealed by tariquidar-probe's super-resolution imaging.

As an efflux pump, P-glycoproteins (P-gps) are over-expressed in many cancer cell types to confer them with multi-drug resistance. Many studies have focused on elucidating their molecular structure or protein expression; however, the relationship between the molecular assembly and dysfunction remains unclear. Super-resolution microscope is an excellent imaging tool to reveal the molecular biological details, but its high-quality imaging often suffers from the labeling method currently available. In this work, by exploiting its specificity and small size, tariquidar (specific inhibitor of P-gp) was modified by TAMRA to form a small chemical probe of P-gp. By direct stochastic optical reconstruction microscopic (dSTORM) imaging, tariquidar-TAMRA was first revealed to possess a higher labeling superiority and high binding specificity. Then, with the application of tariquidar-TAMRA labeling, we found that P-gps accumulate into larger and denser clusters on cancer cells and drug-resistant cells than on normal cells and drug-sensitive cells, indicating that P-gps can facilitate the pumping efficiency by aggregating together to form functional platforms. Moreover, these specific distribution patterns might serve as potential biomarkers for tumor and drug therapy screening.

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.

Quantitatively mapping the interaction of HER2 and EGFR on cell membranes with peptide probes.

Human epidermal growth factor receptor-2 (HER2) is a member of the epidermal growth factor receptor (HER) family that is involved in various biological processes such as cell proliferation, survival, differentiation, migration and invasion. It generally functions in the form of homo-/hetero-dimers or oligomers with other HER family members. Although its essential roles in cellular activities have been widely recognized, questions concerning the spatial distribution of HER2 on the membranes and the interactions between it and other ErbB family members remain obscure. Here, we obtained a high-quality dSTORM image of HER2 nanoscale clusters recognized by peptide probes, and found that HER2 forms clusters containing different numbers of molecules on cell membranes. Moreover, we found that HER2 and EGFR formed hetero-oligomers on non-stimulated cell membranes, whereas EGF stimulation reduced the degree of heteromerization, suggesting that HER2 and EGFR hetero-oligomers may inhibit the activation of EGFR. Collectively, our work revealed the clustered distribution of HER2 and quantified the changes of the interaction between HER2 and EGFR in the resting and active states at the single molecular level, which promotes a deeper understanding of the protein-protein interaction on cell membranes.

Membrane protein density determining membrane fusion revealed by dynamic fluorescence imaging.

Membrane fusion is fundamental to biological activity of cells, so disclosingits relevant mechanism is very important for understanding various cell functions. Although artificial model systems have been developed to uncover the mechanism of membrane fusion, key factors determining the mode of membrane fusion remain unclear. Based on the construction of different types of liposome vesicles, we used a dynamic fluorescence imaging method to investigate the effect of membrane protein distribution density on membrane fusion. Time-resolved imaging revealed that protein-free pure phospholipid vesicles themselves occurred full membrane fusion. Moreover, we prepared proteoliposomes with increasing protein-to-lipid ratio to better reflect the characteristic of membrane structure in vivo. Our data showed that pure phospholipid vesicles no longer fused with the proteoliposomes that in a higher protein proportion, indicating dense membrane proteins may hinder membrane fusion. A further comparative analysis of the interactions of pure phospholipid vesicles with the cell membrane / giant plasma membrane vesicles (GPMVs) / protein-free giant unilamellar vesicles (GUVs) confirmed the inhibitory effect of dense membrane proteins on membrane fusion. Our work demonstrates the membrane protein density influences the mode of membrane fusion and lays a foundation for constructing quasi-native membrane fusion models in vitro.

Development of small molecule inhibitor-based fluorescent probes for highly specific super-resolution imaging.

To ensure the ultimate high-quality imaging of super-resolution fluorescence microscopy with increasingly high resolution, it is significant to use small specific fluorescent probes. Compared with the common biological fluorescent labeling technology, because of small size, strong specificity, abundance and special binding sites, single-targeted small-molecule inhibitors (SMIs) can link with organic dyes to form small fluorescent probes for various biomolecules. Herein, to confirm the feasibility of the SMI-probes, epidermal growth factor (EGF) receptor (EGFR)-targeted tyrosine kinase inhibitor Gefitinib was selected for modification with the fluorescent dye to form Gefitinib-probe. Then, the labeling superiority of Gefitinib-probe was revealed by comparing the direct stochastic optical reconstruction microscopy (dSTORM) images of EGFR labeled with different probes. Additionally, a high co-localization of fluorescent points from Gefitinib-probe and EGF-probe labeling indicated a high specificity of Gefitinib-probe to EGFR. Finally, higher co-localization of EGFR and HER3 labeled with the probe pair containing Gefitinib-probe than with the antibody-probe pair suggested that Gefitinib-probe with a cytoplasmic binding site benefited dual-color imaging. These results indicate that the SMI-probes are able to serve as versatile labeling tools for high-quality super-resolution imaging.

Correlative dual-color dSTORM/AFM reveals protein clusters at the cytoplasmic side of human bronchial epithelium membranes.

The organization of a cell membrane is vital for various functions, such as receptor signaling and membrane traffic. However, the understanding of membrane organization remains insufficient, especially the localizations of specific proteins in the cell membrane. Here, we used correlative super-resolution fluorescence/atomic force microscopy to correlate the distributions of specific proteins Na+/K+-ATPase (NKA, an integral membrane protein) and ankyrin G (AnkG, a scaffolding protein) with the topography of the cytoplasmic side of human bronchial epithelium membranes. Our data showed that NKA and AnkG proteins preferred to localize in the protein islands of membranes. Interestingly, we also found that functional domains composed of specific proteins with a few hundreds of nanometers were formed by assembling protein islands with a few tens of nanometers.

Size-Dependent Transmembrane Transport of Gold Nanocages.

Gold nanocages (Au NCs), as drug carriers, have been widely applied for cancer diagnosis and photothermal therapy (PTT). Transmembrane transporting efficacy of Au NCs is the fundamental and important issue for their use in PTT. Herein, we used a force tracing technique based on atomic force microscopy to track the dynamic transmembrane process of Au NCs at the single-particle level in real time. Meanwhile, we measured and compared the dynamic parameters of Au NCs with sizes of 50 and 100 nm usually used as nanodrug carriers of PTT. It is concluded that the 50 nm Au NC transmembrane transporting needs smaller force and shorter duration with a much faster speed. However, both the 50 and 100 nm Au NC transmembrane transporting depends on the caveolin-mediated endocytosis, clathrin-mediated endocytosis, and macropinocytosis, which was also confirmed by confocal fluorescence imaging. This report will provide a potential technique for screening nanodrug carriers from the perspective of transmembrane transporting efficacy.

Entry Dynamics of Single Ebola Virus Revealed by Force Tracing.

Infections by the Ebola virus (EBOV) rapidly cause fatal hemorrhagic fever in humans. Viral entry into host cells is the most critical step in infection and an attractive target for therapeutic intervention. Herein, the invagination behavior and entry dynamics of filamentous Ebola virus-like particles (EBO-VLPs) were investigated using a force tracing technique based on atomic force microscopy and single-particle fluorescence tracking in real time. The filamentous EBOV-VLPs might enter cells in both horizontal and vertical modes, and the virus-receptor interactions during endocytic uptake were analyzed. In addition, molecular dynamics simulations and engulfment energy analysis further depicted EBO-VLP entry in the horizontal and vertical directions and suggested that internalization in the vertical direction requires a larger force and more time. This report provides useful information for further revealing the mechanism of viral infection, which is important for understanding viral pathogenesis.

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.

Size-Independent Transmembrane Transporting of Single Tetrahedral DNA Nanostructures.

DNA nanostructures have attracted considerable attention as drug delivery carriers. However, the transmembrane kinetics of DNA nanostructures remains less explored. Herein, the dynamic process of transporting single tetrahedral DNA nanostructures (TDNs) is monitored in real time using a force-tracing technique based on atomic force microscopy. The results show that transporting single TDNs into living HeLa cells need ≈53 pN force and ≈25 ms duration with the average speed of ≈0.6 µm s-1. Interestingly, the dynamic parameters are irrelevant to the size of TDNs, while the larger TDNs rotated slightly during the transporting process. Meanwhile, both the results from single-molecule force tracing and ensemble fluorescence imaging demonstrate that the different size TDNs transmembrane transporting depends on caveolin-mediated endocytosis.