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.

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.

The structure and function of cell membranes studied by atomic force microscopy.

The cell membrane, involved in almost all communications of cells and surrounding matrix, is one of the most complicated components of cells. Lack of suitable methods for the detection of cell membranes in vivo has sparked debates on the biochemical composition and structure of cell membranes over half a century. The development of single molecule techniques, such as AFM, SMFS, and TREC, provides a versatile platform for imaging and manipulating cell membranes in biological relevant environments. Here, we discuss the latest developments in AFM and the progress made in cell membrane research. In particular, we highlight novel structure models and dynamic processes, including the mechanical properties of the cell membranes.

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.

The role of resveratrol in bone marrow-derived mesenchymal stem cells from patients with osteoporosis.

The aim of the present study was to investigate the effects of resveratrol on BMSCs from patients with osteoporosis. The cell viability and proliferation of BMSCs after treatment with different concentrations of resveratrol was respectively observed by MTT assay and EdU staining. The apoptosis was assessed using by TUNEL staining and the pluripotency was analyzed by quantitative reverse transcription-PCR (qRT-PCR). The osteogenic differentiation and adipogenic differentiation were determined by alkaline phosphatase (ALP) staining, alizarin red S (ARS) staining, oil red O (ORO) staining and qRT-PCR analysis. MTT assay showed that Res at 40, 80, 100 µM markedly improved the cell proliferation of BMSCs from patients with osteoporosis. EdU staining indicated that Res treatment significantly accelerated the proliferation of BMSCs. In addition, the results of TUNEL staining revealed that Res at 40, 80, 100 µM inhibited the osteoporosis-related apoptosis of BMSCs. qRT-PCR analysis explored that Res treatment played a positive role in the pluripotency in BMSCs. ALP, ARS staining and qRT-PCR demonstrated that Res promoted the differentiation of BMSCs into osteoblasts, especially at 80 µM. ORO staining and qRT-PCR analysis proved that treatment of Res inhibited the adipogenesis of BMSCs isolated from patients with osteoporosis. Our findings suggested that Res can play a vital role in the cell viability, proliferation, apoptosis, pluripotency, osteogenesis and adipogenesis of BMSCs. And Res might be an efficient therapeutic approach for treating patients with osteoporosis.

Treatment strategies for traumatic cervico-cranial pseudoaneurysms: A single institution experience.

AIM: Limited clinical and angiographic data exists for patients with traumatic cervico-cerebral pseudoaneurysms. In this paper, we present our limited experience with various management strategies for traumatic cervico-cranial pseudoaneurysms. MATERIALS AND METHODS: We retrospectively analyzed 37 consecutive cases of traumatic pseudoaneurysms involving the cervico-cranial or the cerebral arteries diagnosed at our center from September 2009 to December 2014. The demographic data, etiology, clinical presentation, lesion location, treatment modality, and follow-up outcomes of these patients were reviewed. Among these 37 patients, 5 patients were treated by surgery, while 29 patients were treated by the endovascular approach and 3 received conservative treatment. RESULTS: During the study period, 42 pseudoaneurysms were identified in 37 patients with a history of head or neck injury. Five patients underwent surgical exploration of the lesion with an uneventful postoperative course. Twenty-nine patients were treated by endovascular interventions with various embolization materials including coils, stents, detachable balloons, liquid embolic agents, and a combination of these agents. The angiographic follow-up imaging demonstrated complete exclusion of the aneurysm from the circulation with the patient being free from any additional neurological deficits. CONCLUSION: Proper selection of an appropriate approach is essential to address the management of traumatic cervico-cerebral pseudoaneurysms. The treatment of traumatic cervico-cerebral pseudoaneurysms should be selected according to the location and the clinical features of the pseudoaneurysms. The endovascular treatment is a safe and effective modality and should be the first-line choice for treatment of traumatic pseudoaneurysms.

Progress in the Correlative Atomic Force Microscopy and Optical Microscopy.

Atomic force microscopy (AFM) has evolved from the originally morphological imaging technique to a powerful and multifunctional technique for manipulating and detecting the interactions between molecules at nanometer resolution. However, AFM cannot provide the precise information of synchronized molecular groups and has many shortcomings in the aspects of determining the mechanism of the interactions and the elaborate structure due to the limitations of the technology, itself, such as non-specificity and low imaging speed. To overcome the technical limitations, it is necessary to combine AFM with other complementary techniques, such as fluorescence microscopy. The combination of several complementary techniques in one instrument has increasingly become a vital approach to investigate the details of the interactions among molecules and molecular dynamics. In this review, we reported the principles of AFM and optical microscopy, such as confocal microscopy and single-molecule localization microscopy, and focused on the development and use of correlative AFM and optical microscopy.

Enantiomeric Effect of d-Amino Acid Substitution on the Mechanism of Action of α-Helical Membrane-Active Peptides.

V13K, a 26-residue peptide, has been shown to have strong antimicrobial activity, negligible hemolytic activity, and significant anticancer activity. In the present work, V13K was used as the framework to investigate the influence of helicity, as influenced by d-amino acid substitutions in the center of the peptide polar and non-polar faces of the amphipathic helix, on biological activity. The antibacterial and anticancer activities of the peptides were investigated. Atomic force microscopy and other biophysical methods were used to investigate the effect of peptide helicity on biological activity. The results showed the importance of suitable and rational modification of membrane-active peptides, based on helicity, in optimizing potential biological activity.

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.

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.

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.

Mechanistic insights into CDCP1 clustering on non-small-cell lung cancer membranes revealed by super-resolution fluorescent imaging.

CDCP1 is a transmembrane protein that is involved in a variety of important biological processes and upregulated in a variety of human solid malignancies; however, its spatial distribution and variation at the molecular level remain unclear. To solve this problem, we first analyzed its expression level and prognostic implications in lung cancer. Then, we used super-resolution microscopy to reveal the spatial organization of CDCP1 at different levels, and found that cancer cells generated more and larger CDCP1 clusters than normal cells. Furthermore, we found that CDCP1 can be integrated into larger and denser clusters as functional domains upon activation. Our findings elucidated the significant differences of CDCP1 clustering characteristics between cancer and normal cells, and revealed the relationship between its distribution and function, which will contribute to a comprehensive understanding of its oncogenic mechanism, and will be of great help for the development of CDCP1-targeted drugs for lung cancer.

Erratum: Mechanistic insights into CDCP1 clustering on non-small-cell lung cancer membranes revealed by super-resolution fluorescent imaging.

[This corrects the article DOI: 10.1016/j.isci.2023.106103.].

Using a 3D Navigation Template to Increase the Accuracy of Thoracic Pedicle Screws in Patients with Scoliosis.

This study compares the accuracy and safety of pedicle screw placement using a 3D navigation template with the free-hand fluoroscopy technique in scoliotic patients. Fifteen scoliotic patients were recruited and divided into a template group (eight cases) and a free-hand group (seven cases). All patients received posterior corrective surgeries, and the pedicle screw was placed using a 3D navigation template or a free-hand technique. After surgery, the positions of the pedicle screws were evaluated using CT. A total of 264 pedicle screws were implanted in 15 patients. Both the two techniques were found to achieve satisfactory safety of screw insertion in scoliotic patients (89.9% vs. 90.5%). In the thoracic region, the 3D navigation template was able to achieve a much higher accuracy of screw than the free-hand technique (75.3% vs. 60.4%). In the two groups, the accuracy rates on the convex side were slightly higher than on the concave side, while no significance was seen. In terms of rotational vertebrae, no significant differences were seen in Grades I or II vertebrae between the two groups. In conclusion, the 3D navigation template technique significantly increased the accuracy of thoracic pedicle screw placement, which held great potential for extensively clinical application.

Direct evidence of lipid rafts by in situ atomic force microscopy.

Lipid rafts are membrane microdomains enriched with cholesterol, glycosphingolipids, and proteins. Although they are broadly presumed to play a pivotal role in various cellular functions, there are still fierce debates about the composition, functions, and even existence of lipid rafts. Here high-resolution and time-lapse in situ atomic force microscopy is used to directly confirm the existence of lipid rafts in native erythrocyte membranes. The results indicate some important aspects of lipid rafts: most of the lipid rafts are in the size range of 100-300 nm and have irregular shape; the detergent-resistant membranes consist of cholesterol microdomains and are not likely the same as the lipid rafts; cholesterol contributes significantly to the formation and stability of the protein domains; and Band III is an important protein of lipid rafts in the inner leaflet of erythrocyte membranes, indicating that lipid rafts are exactly the functional domains in plasma membrane. This work provides direct evidence of the presence, size, and main constitutive protein of lipid rafts at a resolution of a few nanometers, which will pave the way for studying their structure and functions in detail.

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.

Cell membrane sample preparation method of combined AFM and dSTORM analysis.

A major role of cell membranes is to provide an ideal environment for the constituent proteins to perform their biological functions. A deep understanding of the membrane proteins assembly process under physiological conditions is quite important to elucidate both the structure and the function of the cell membranes. Along these lines, in this work, a complete workflow of the cell membrane sample preparation and the correlated AFM and dSTORM imaging analysis methods are presented. A specially designed, angle-controlled sample preparation device was used to prepare the cell membrane samples. The correlated distributions of the specific membrane proteins with the topography of the cytoplasmic side of the cell membranes can be obtained by performing correlative AFM and dSTORM measurements. These methods are ideal for systematically studying the structure of the cell membranes. The proposed method of the sample characterization was not only limited to the measurement of the cell membrane but also can be applied for both biological tissue section analysis and detection.