In situ structure of actin remodeling during glucose-stimulated insulin secretion using cryo-electron tomography.

Actin mediates insulin secretion in pancreatic ß-cells through remodeling. Hampered by limited resolution, previous studies have offered an ambiguous depiction as depolymerization and repolymerization. We report the in situ structure of actin remodeling in INS-1E ß-cells during glucose-stimulated insulin secretion at nanoscale resolution. After remodeling, the actin filament network at the cell periphery exhibits three marked differences: 12% of actin filaments reorient quasi-orthogonally to the ventral membrane; the filament network mainly remains as cell-stabilizing bundles but partially reconfigures into a less compact arrangement; actin filaments anchored to the ventral membrane reorganize from a "netlike" to a "blooming" architecture. Furthermore, the density of actin filaments and microtubules around insulin secretory granules decreases, while actin filaments and microtubules become more densely packed. The actin filament network after remodeling potentially precedes the transport and release of insulin secretory granules. These findings advance our understanding of actin remodeling and its role in glucose-stimulated insulin secretion.

In situ structure of actin remodeling during glucose-stimulated insulin secretion using cryo-electron tomography.

Actin mediates insulin secretion from the pancreatic ß-cell through a remodeling process. Previous studies have been hampered by limited resolution, providing an ambiguous depiction of actin remodeling as a process that begins with depolymerization into actin monomers, followed by repolymerization into actin filaments. Here, we report the in situ structure of actin remodeling in INS-1E ß-cells during glucose-stimulated insulin secretion at nanoscale resolution. We demonstrate that actin remodeling occurs at the cell periphery rather than in the cell interior. The actin filament network at the cell periphery exhibits three marked differences after remodeling compared to those under basal conditions. First, approximately 12%of actin filaments reorient, their angle changing from 0-45° to 45-90° relative to the plasma membrane. Second, the actin filament network remains predominantly as cell-stabilizing bundles but partially reconfigures into a less compact arrangement. Third, actin filaments anchored to the plasma membrane reorganize from a "netlike" to a "blooming" architecture, featuring radial projections emanating from their anchor points. Remodeling precedes the transport of insulin secretory granulesto the plasma membrane and their release from it. Furthermore, the density of actin filaments and microtubules around insulin secretory granules is lowered after remodeling compared to the basal conditions, as expected for the subsequent granule transport and release. Finally, actin filaments and microtubules are more densely packed than under basal conditions. These findings advance our structural and functional understanding of actin remodeling during glucose-stimulated insulin secretion in pancreatic ß-cells.

An intensity-based post-processing tool for 3D instance segmentation of organelles in soft X-ray tomograms.

Investigating the 3D structures and rearrangements of organelles within a single cell is critical for better characterizing cellular function. Imaging approaches such as soft X-ray tomography have been widely applied to reveal a complex subcellular organization involving multiple inter-organelle interactions. However, 3D segmentation of organelle instances has been challenging despite its importance in organelle characterization. Here we propose an intensity-based post-processing tool to identify and separate organelle instances. Our tool separates sphere-like (insulin vesicle) and columnar-shaped organelle instances (mitochondrion) based on the intensity of raw tomograms, semantic segmentation masks, and organelle morphology. We validate our tool using synthetic tomograms of organelles and experimental tomograms of pancreatic ß-cells to separate insulin vesicle and mitochondria instances. As compared to the commonly used connected regions labeling, watershed, and watershed + Gaussian filter methods, our tool results in improved accuracy in identifying organelles in the synthetic tomograms and an improved description of organelle structures in ß-cell tomograms. In addition, under different experimental treatment conditions, significant changes in volumes and intensities of both insulin vesicle and mitochondrion are observed in our instance results, revealing their potential roles in maintaining normal ß-cell function. Our tool is expected to be applicable for improving the instance segmentation of other images obtained from different cell types using multiple imaging modalities.

Quantitative, in situ visualization of intracellular insulin vesicles in pancreatic beta cells.

Characterizing relationships between Zn2+, insulin, and insulin vesicles is of vital importance to the study of pancreatic beta cells. However, the precise content of Zn2+ and the specific location of insulin inside insulin vesicles are not clear, which hinders a thorough understanding of the insulin secretion process and diseases caused by blood sugar dysregulation. Here, we demonstrated the colocalization of Zn2+ and insulin in both single extracellular insulin vesicles and pancreatic beta cells by using an X-ray scanning coherent diffraction imaging (ptychography) technique. We also analyzed the elemental Zn2+ and Ca2+ contents of insulin vesicles using electron microscopy and energy dispersive spectroscopy (EDS) mapping. We found that the presence of Zn2+ is an important characteristic that can be used to distinguish insulin vesicles from other types of vesicles in pancreatic beta cells and that the content of Zn2+ is proportional to the size of insulin vesicles. By using dual-energy contrast X-ray microscopy and scanning transmission X-ray microscopy (STXM) image stacks, we observed that insulin accumulates in the off-center position of extracellular insulin vesicles. Furthermore, the spatial distribution of insulin vesicles and their colocalization with other organelles inside pancreatic beta cells were demonstrated using three-dimensional (3D) imaging by combining X-ray ptychography and an equally sloped tomography (EST) algorithm. This study describes a powerful method to univocally describe the location and quantitative analysis of intracellular insulin, which will be of great significance to the study of diabetes and other blood sugar diseases.

Auto-segmentation and time-dependent systematic analysis of mesoscale cellular structure in ß-cells during insulin secretion.

The mesoscale description of the subcellular organization informs about cellular mechanisms in disease state. However, applications of soft X-ray tomography (SXT), an important approach for characterizing organelle organization, are limited by labor-intensive manual segmentation. Here we report a pipeline for automated segmentation and systematic analysis of SXT tomograms. Our approach combines semantic and first-applied instance segmentation to produce separate organelle masks with high Dice and Recall indexes, followed by analysis of organelle localization based on the radial distribution function. We demonstrated this technique by investigating the organization of INS-1E pancreatic ß-cell organization under different treatments at multiple time points. Consistent with a previous analysis of a similar dataset, our results revealed the impact of glucose stimulation on the localization and molecular density of insulin vesicles and mitochondria. This pipeline can be extended to SXT tomograms of any cell type to shed light on the subcellular rearrangements under different drug treatments.

In vitro and in vivo characterization of insulin vesicles by electron microscopy.

Insulin is the main hypoglycemic hormone, promoting the absorption and storage of glucose and inhibiting its production. It is a hexamer composed of six insulin macromolecules and a Zn2+ and clustered in insulin vesicles of pancreatic ß cell. Most current research has focused on the in vivo imaging of whole cells while there are few detailed studies on structure of insulin vesicles. The precise content of Zn2+ in vesicles is not clear, and the aggregation state and location of insulin in insulin vesicles is not fully characterized, which hinders a thorough understanding of insulin secretion process and diseases caused by blood sugar regulation. Here, we performed electron microscopy (EM) studies on both whole cells (in vivo) and extracted isolated insulin vesicles by supercentrifugation (in vitro) to explore the location and distribution of insulin vesicles in pancreatic ß cells. Meanwhile, we analyzed the content of Zn2+ and Ca2+ through EM imaging and energy dispersive spectroscopy (EDS) mapping, and the content of Zn2+ was found to be proportional to the size of insulin vesicles. In addition, by taking advantage of TEM tomography, the three-dimensional structure of insulin vesicle was obtained by acquisition projections in different angles of insulin vesicle. This study provides a promising way to quantitative analysis of intracellular insulin, which may be of great significance to the study of diabetes and other blood sugar diseases.

Soft X-ray tomography to map and quantify organelle interactions at the mesoscale.

Inter-organelle interactions are a vital part of normal cellular function; however, these have proven difficult to quantify due to the range of scales encountered in cell biology and the throughput limitations of traditional imaging approaches. Here, we demonstrate that soft X-ray tomography (SXT) can be used to rapidly map ultrastructural reorganization and inter-organelle interactions in intact cells. SXT takes advantage of the naturally occurring, differential X-ray absorption of the carbon-rich compounds in each organelle. Specifically, we use SXT to map the spatiotemporal evolution of insulin vesicles and their co-localization and interaction with mitochondria in pancreatic ß cells during insulin secretion and in response to different stimuli. We quantify changes in the morphology, biochemical composition, and relative position of mitochondria and insulin vesicles. These findings highlight the importance of a comprehensive and unbiased mapping at the mesoscale to characterize cell reorganization that would be difficult to detect with other existing methodologies.

Bayesian metamodeling of complex biological systems across varying representations.

Comprehensive modeling of a whole cell requires an integration of vast amounts of information on various aspects of the cell and its parts. To divide and conquer this task, we introduce Bayesian metamodeling, a general approach to modeling complex systems by integrating a collection of heterogeneous input models. Each input model can in principle be based on any type of data and can describe a different aspect of the modeled system using any mathematical representation, scale, and level of granularity. These input models are 1) converted to a standardized statistical representation relying on probabilistic graphical models, 2) coupled by modeling their mutual relations with the physical world, and 3) finally harmonized with respect to each other. To illustrate Bayesian metamodeling, we provide a proof-of-principle metamodel of glucose-stimulated insulin secretion by human pancreatic ß-cells. The input models include a coarse-grained spatiotemporal simulation of insulin vesicle trafficking, docking, and exocytosis; a molecular network model of glucose-stimulated insulin secretion signaling; a network model of insulin metabolism; a structural model of glucagon-like peptide-1 receptor activation; a linear model of a pancreatic cell population; and ordinary differential equations for systemic postprandial insulin response. Metamodeling benefits from decentralized computing, while often producing a more accurate, precise, and complete model that contextualizes input models as well as resolves conflicting information. We anticipate Bayesian metamodeling will facilitate collaborative science by providing a framework for sharing expertise, resources, data, and models, as exemplified by the Pancreatic ß-Cell Consortium.

Visualizing subcellular rearrangements in intact ß cells using soft x-ray tomography.

Characterizing relationships between cell structures and functions requires mesoscale mapping of intact cells showing subcellular rearrangements following stimulation; however, current approaches are limited in this regard. Here, we report a unique application of soft x-ray tomography to generate three-dimensional reconstructions of whole pancreatic ß cells at different time points following glucose-stimulated insulin secretion. Reconstructions following stimulation showed distinct insulin vesicle distribution patterns reflective of altered vesicle pool sizes as they travel through the secretory pathway. Our results show that glucose stimulation caused rapid changes in biochemical composition and/or density of insulin packing, increased mitochondrial volume, and closer proximity of insulin vesicles to mitochondria. Costimulation with exendin-4 (a glucagon-like peptide-1 receptor agonist) prolonged these effects and increased insulin packaging efficiency and vesicle maturation. This study provides unique perspectives on the coordinated structural reorganization and interactions of organelles that dictate cell responses.

miR-124 regulates cerebromicrovascular function in APP/PS1 transgenic mice via C1ql3.

Many neurodegenerative diseases, such as Alzheimer's disease (AD) and Parkinson's disease, are associated with microvascular dysfunction, but the cellular and molecular mechanisms are poorly understood. Recently, microRNAs (miRNAs) have been suggested to be involved in the microvascular dysfunction and subsequent memory impairment. MicroRNA-124 (miR-124) is one of the most abundant miRNAs in the brain that is dysregulated in the hippocampus of AD animals. To explore the role of miR-124 in AD pathology, we employed the APP/PS1 transgenic mice and found downregulation of miR-124 and upregulation of complement C1q-like protein 3 (C1ql3) in the hippocampus and cerebral cortex. Downregulation of miR-124 expression resulted in Aß deposition and a variety of cerebromicrovascular impairments, including the decline in microvascular density, reduced angiogenesis, accompanied by C1ql3 alteration. Treatment with lentivirus-mediated overexpression of miR-124 or the C1q inhibitor C1INH rescued breakdown of blood-brain barrier, promoted angiogenesis and reduced Aß deposition, and finally alleviated learning and memory deficit in APP/PS1 mice. Moreover, we found that C1ql3, a component of the classical complement, might be a potential target of miR-124. These results suggested that miR-124 was involved in the angiogenesis and vascular integrity in the hippocampus and the cerebral cortex of the AD mice by regulating the classical complement C1ql3.

miR-132 regulates the expression of synaptic proteins in APP/PS1 transgenic mice through C1q.

Cognitive impairment in Alzheimer's disease (AD) is usually accompanied by synaptic loss in both the hippocampus and neocortex. In the early stage of AD, amyloid ß-induced synapse changes is the main reason, while in the later stage, the accumulation of Tau protein promotes synapse degeneration as the key factor leading to dementia. MicroRNA (miRNA) is closely related to the expression changes of many AD-related genes. One of the most abundant brain-enriched miRNAs is miR-132, which has been shown to regulate both neuron morphogenesis and plasticity. It has been reported that miR-132 is significantly reduced in the brains of Alzheimer's patients. Genetic deletion of miR-132 in mice promotes Aß deposition, leading to impaired memory and enhanced Tau pathology, but how the miRNA-mediated gene expression dysregulation contributes to AD pathology remains unclear. Here we found the possible downstream target of miR-132 by in silico analysis, namely C1q. C1q is the primary protein of classical complement cascade, which is highly expressed in the synaptic regions of the central nervous system in Alzheimer's patients. However, it is not clear whether miR-132 plays a role in AD through regulating C1q. To address this question, the APP/PS1 transgenic mice were transfected with miR-132 and given C1 inhibitors. Behavior tests were conducted to assess memory and cognitive abilities seven days after administration. In addition, we analyzed the expression of PSD95, Synapsin-1 and phosphorylated (p)-Synapsin. We found that the expression levels of the synaptic proteins treated with miR-132 or C1INH were significantly increased compared with the AD group. Further RT-qPCR result suggested that miR-132 might regulate C1q expression in AD.

Recombinant Lactococcus lactis NZ9000 secretes a bioactive kisspeptin that inhibits proliferation and migration of human colon carcinoma HT-29 cells.

BACKGROUND: Proteinaceous bioactive substances and pharmaceuticals are most conveniently administered orally. However, the facing problems are the side effects of proteolytic degradation and denaturation in the gastrointestinal tract. In recent years, lactic acid bacteria (LAB) have been verified to be a promising delivery vector for susceptible functional proteins and drugs. KiSS-1 peptide, a cancer suppressor, plays a critical role in inhibiting cancer metastasis and its activity has been confirmed by direct administration. However, whether this peptide can be functionally expressed in LAB and exert activity on cancer cells, thus providing a potential alternative administration manner in the future, has not been demonstrated. RESULTS: A recombinant Lactococcus lactis strain NZ9000-401-kiss1 harboring a plasmid containing the gene of the tumor metastasis-inhibiting peptide KiSS1 was constructed. After optimization of the nisin induction conditions, the recombinant strain efficiently secreted KiSS1 with a maximum detectable amount of 27.9 µg/ml in Dulbecco's Modified Eagle medium. The 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide and would healing assays, respectively, indicated that the secreted KiSS1 peptide remarkably inhibited HT-29 cell proliferation and migration. Furthermore, the expressed KiSS1 was shown to induce HT-29 cell morphological changes, apoptosis and reduce the expression of matrix metalloproteinase 9 (MMP-9) at both mRNA and protein levels. CONCLUSIONS: A recombinant L. lactis NZ9000-401-kiss1 successfully expressing the human kiss1 was constructed. The secreted KiSS1 peptide inhibited human HT-29 cells' proliferation and migration probably by invoking, or mediating the cell-apoptosis pathway and by down regulating MMP-9 expression, respectively. Our results suggest that L. lactis is an ideal cell factory for secretory expression of tumor metastasis-inhibiting peptide KiSS1, and the KiSS1-producing L. lactis strain may serve as a new tool for cancer therapy in the future.

[Mechanism of chloroquine in promoting sensitivity of chemotherapeutics in oral squamous cell carcinoma CAL-27 cell line to cisplatin].

PURPOSE: To study the role and mechanism of autophagy in chemotherapy of oral squamous cell carcinoma, and provide theoretical evidence to improve chemotherapeutic efficacy of oral squamous cell carcinoma patients. METHODS: The cell survival rate changes induced by cisplatin (DDP) and chloroquine (CQ) in CAL-27 cells were assayed by methyl thiazolyl tetrazolium method(MTT). The LC3-II expression level was detected by laser scanning confocal microscope; The apoptotic rate was determined by flow cytometry. SPSS17.0 software package was used for statistical analysis. RESULTS: MTT results showed that compared with the control group, the cell survival rate reduced with the increasing time of DDP and CQ treatment; The optimal concentration of CAL-27 cells was 5 mg/L after treatment with CQ. IC50 of the CAL-27 cells was 5 mg/L after treatment with DDP; MTT results showed that the cell survival rate of CQ+DDP group was significantly lower than control group, CQ group and DDP group (P<0.05). With the action of CQ and DDP to CAL-27 cells for 48 hours, immunofluorescence results showed that the average fluorescence intensity of DDP group was significantly higher than the other 3 groups (P<0.05), while it was significantly lower in CQ group than the other 3 groups (P<0.05). With the action of CQ and DDP to CAL-27 cells for 48 hours, flow cytometry results showed that the cell apoptosis rate of DDP group and CQ+DDP group were significantly higher than control group and CQ group. The cell apoptosis rate of CQ+DDP group was significantly higher than DDP group (P<0.05). With the action of CQ and DDP to CAL-27 cells for 48 hours, cells in G1 phase of DDP group and CQ+DDP group increased, indicating G1 phase blockage. The cell count in G1 phase of CQ+DDP group was significantly higher than DDP group (P<0.05). CONCLUSIONS: Inhibition of autophagy can enhance the chemotherapeutic sensitivity of DDP in CAL-27 cells. Autophagy in CAL-27 cells is an important mechanism for chemotherapy resistance of oral squamous cell carcinoma. Autophagy inhibitor may have significant potential to be a novel chemotherapeutic sensitizer for oral squamous cell carcinoma.