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Objective: Beta cell destruction in type 1 diabetes (T1D) results from the combined effect of inflammation and recurrent autoimmunity. In recent years, the role played by beta cells in the development of T1D has evolved from passive victims of the immune system to active contributors in their own destruction. We and others have demonstrated that perturbations in the islet microenvironment promote endoplasmic reticulum (ER) stress in beta cells, leading to enhanced immunogenicity. Among the underlying mechanisms, secretion of extracellular vesicles (EVs) by beta cells has been suggested to mediate the crosstalk with the immune cell compartment. Methods: To study the role of cellular stress in the early events of T1D development, we generated a novel cellular model for constitutive ER stress by modulating the expression of HSPA5, which encodes BiP/GRP78, in EndoC-βH1 cells. To investigate the role of EVs in the interaction between beta cells and the immune system, we characterized the EV miRNA cargo and evaluated their effect on innate immune cells. Results: Analysis of the transcriptome showed that HSPA5 knockdown resulted in the upregulation of signaling pathways involved in the unfolded protein response (UPR) and changes the miRNA content of EVs, including reduced levels of miRNAs involved in IL-1β signaling. Treatment of primary human monocytes with EVs from stressed beta cells resulted in increased surface expression of CD11b, HLA-DR, CD40 and CD86 and upregulation of IL-1β and IL-6. Conclusion: These findings indicate that the content of EVs derived from stressed beta cells can be a mediator of islet inflammation. [ABSTRACT FROM AUTHOR]

Programmed cell death and consecutive removal of cellular remnants is essential for development. During late stages of Drosophila melanogaster oogenesis, the small somatic follicle cells that surround the large nurse cells promote non-apoptotic nurse cell death, subsequently engulf them, and contribute to the timely removal of nurse cell corpses. Here, we identify a role for Vps13 in the timely removal of nurse cell corpses downstream of developmental programmed cell death. Vps13 is an evolutionarily conserved peripheral membrane protein associated with membrane contact sites and lipid transfer. It is expressed in late nurse cells, and persistent nurse cell remnants are observed when Vps13 is depleted from nurse cells but not from follicle cells. Microscopic analysis revealed enrichment of Vps13 in close proximity to the plasma membrane and the endoplasmic reticulum in nurse cells undergoing degradation. Ultrastructural analysis uncovered the presence of an underlying Vps13-dependent membranous structure in close association with the plasma membrane. The newly identified structure and function suggests the presence of a Vps13-dependent process required for complete degradation of bulky remnants of dying cells. [ABSTRACT FROM AUTHOR]

Large-scale electron microscopy ('nanotomy') allows straight forward ultrastructural examination of tissue, cells, organelles, and macromolecules in a single data set. Such data set equals thousands of conventional electron microscopy images and is freely accessible (www.nanotomy.org). The software allows zooming in and out of the image from total overview to nanometer scale resolution in a 'Google Earth' approach. We studied the life-threatening human autoimmune blistering disease pemphigus, using nanotomy. The pathomechanism of cell-cell separation (acantholysis) that underlies the blistering is poorly understood. Ultrastructural examination of pemphigus tissue revealed previously unreported findings: (i) the presence of double-membrane structures between cells in all pemphigus types; (ii) the absence of desmosomes around spontaneous blisters in pemphigus foliaceus (PF); (iii) lower level blistering in PF when force induced; and (iv) intercellular widening at non-acantholytic cell layers. Thus, nanotomy delivers open-source electron microscopic maps of patient tissue, which can be analyzed for additional anomalies from any computer by experts from different fields. [ABSTRACT FROM AUTHOR]

Membrane type 1 matrix metalloproteinase (MT1-MMP) plays a critical role in cancer cell biology by proteolytically remodeling the extracellular matrix. Utilizing fluorescence resonance energy transfer (FRET) imaging, we have developed a novel biosensor, with its sensing element anchoring at the extracellular surface of cell membrane, to visualize MT1-MMP activity dynamically in live cells with subcellular resolution. Epidermal growth factor (EGF) induced significant FRET changes in cancer cells expressing MT1-MMP, but not in MT1-MMP-deficient cells. EGF-induced FRET changes in MT1-MMP-deficient cells could be restored after reconstituting with wild-type MT1-MMP, but not MMP-2, MMP-9, or inactive MT1-MMP mutants. Deletion of the transmembrane domain in the biosensor or treatment with tissue inhibitor of metalloproteinase-2, a cell-impermeable MT1-MMP inhibitor, abolished the EGF-induced FRET response, indicating that MT1-MMP acts at the cell surface to generate FRET changes. In response to EGF, active MT1-MMP was directed to the leading edge of migrating cells along micropatterned fibronectin stripes, in tandem with the local accumulation of the EGF receptor, via a process dependent upon an intact cytoskeletal network. Hence, the MT1-MMP biosensor provides a powerful tool for characterizing the molecular processes underlying the spatiotemporal regulation of this critical class of enzymes. [ABSTRACT FROM AUTHOR]

A profound characteristic of field cancerization is alterations in chromatin packing. This study aimed to quantify these alterations using electron microscopy image analysis of buccal mucosa cells of laryngeal, esophageal, and lung cancer patients. Analysis was done on normal-appearing mucosa, believed to be within the cancerization field, and not tumor itself. Large-scale electron microscopy (nanotomy) images were acquired of cancer patients and controls. Within the nuclei, the chromatin packing of euchromatin and heterochromatin was characterized. Furthermore, the chromatin organization was quantified through chromatin packing density scaling. A significant difference was found between the cancer and control groups in the chromatin packing density scaling parameter for length scales below the optical diffraction limit (200 nm) in both the euchromatin (p = 0.002) and the heterochromatin (p = 0.006). The chromatin packing scaling analysis also indicated that the chromatin organization of cancer patients deviated significantly from the control group. They might allow for novel strategies for cancer risk stratification and diagnosis with high sensitivity. This could aid clinicians in personalizing screening strategies for high-risk patients and follow-up strategies for treated cancer patients.

Large-scale electron microscopy (EM) allows analysis of both tissues and macromolecules in a semi-automated manner, but acquisition rate forms a bottleneck. We reasoned that a negative bias potential may be used to enhance signal collection, allowing shorter dwell times and thus increasing imaging speed. Negative bias potential has previously been used to tune penetration depth in block-face imaging. However, optimization of negative bias potential for application in thin section imaging will be needed prior to routine use and application in large-scale EM. Here, we present negative bias potential optimized through a combination of simulations and empirical measurements. We find that the use of a negative bias potential generally results in improvement of image quality and signal-to-noise ratio (SNR). The extent of these improvements depends on the presence and strength of a magnetic immersion field. Maintaining other imaging conditions and aiming for the same image quality and SNR, the use of a negative stage bias can allow for a 20-fold decrease in dwell time, thus reducing the time for a week long acquisition to less than 8 h. We further show that negative bias potential can be applied in an integrated correlative light electron microscopy (CLEM) application, allowing fast acquisition of a high precision overlaid LM-EM dataset. Application of negative stage bias potential will thus help to solve the current bottleneck of image acquisition of large fields of view at high resolution in large-scale microscopy., Competing Interests: R.L., Y.V., A.H.G.W., L.K., S.E.C., N.L., J.K., and B.N.N.G. declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. The integrated microscope used in this study is a product of Delmic BV. of which J.P.H. is a co-founder and shareholder., (© 2021 The Author(s).)

The nuclear pore complex (NPC) is embedded in the nuclear envelope and forms the main gateway to the nuclear interior including the inner nuclear membrane (INM). Two INM proteins in yeast are selectively imported. Their sorting signals consist of a nuclear localization signal, separated from the transmembrane domain by a long intrinsically disordered (ID) linker. We used computational models to predict the dynamic conformations of ID linkers and analyzed the INM targeting efficiency of proteins with linker regions with altered Stokes radii and decreased flexibilities. We find that flexibility, Stokes radius, and the frequency at which the linkers are at an extended end-to-end distance larger than 25 nm are good predictors for the targeting of the proteins. The data are consistent with a transport mechanism in which INM targeting of Heh2 is dependent on an ID linker that facilitates the crossing of the approximately 25-nm thick NPC scaffold., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Aims: Iron deficiency is common in patients with heart failure and associated with a poor cardiac function and higher mortality. How iron deficiency impairs cardiac function on a cellular level in the human setting is unknown. This study aims to determine the direct effects of iron deficiency and iron repletion on human cardiomyocytes., Methods and Results: Human embryonic stem cell-derived cardiomyocytes were depleted of iron by incubation with the iron chelator deferoxamine (DFO). Mitochondrial respiration was determined by Seahorse Mito Stress test, and contractility was directly quantified using video analyses according to the BASiC method. The activity of the mitochondrial respiratory chain complexes was examined using spectrophotometric enzyme assays. Four days of iron depletion resulted in an 84% decrease in ferritin (P < 0.0001) and significantly increased gene expression of transferrin receptor 1 and divalent metal transporter 1 (both P < 0.001). Mitochondrial function was reduced in iron-deficient cardiomyocytes, in particular ATP-linked respiration and respiratory reserve were impaired (both P < 0.0001). Iron depletion affected mitochondrial function through reduced activity of the iron-sulfur cluster containing complexes I, II and III, but not complexes IV and V. Iron deficiency reduced cellular ATP levels by 74% (P < 0.0001) and reduced contractile force by 43% (P < 0.05). The maximum velocities during both systole and diastole were reduced by 64% and 85%, respectively (both P < 0.001). Supplementation of transferrin-bound iron recovered functional and morphological abnormalities within 3 days., Conclusion: Iron deficiency directly affects human cardiomyocyte function, impairing mitochondrial respiration, and reducing contractility and relaxation. Restoration of intracellular iron levels can reverse these effects., (© 2018 The Authors. European Journal of Heart Failure published by John Wiley & Sons Ltd on behalf of European Society of Cardiology.)

Electron microscopy (EM) remains the primary method for imaging cellular and tissue ultrastructure, although simultaneous localization of multiple specific molecules continues to be a challenge for EM. We present a method for obtaining multicolor EM views of multiple subcellular components. The method uses sequential, localized deposition of different lanthanides by photosensitizers, small-molecule probes, or peroxidases. Detailed view of biological structures is created by overlaying conventional electron micrographs with pseudocolor lanthanide elemental maps derived from distinctive electron energy-loss spectra of each lanthanide deposit via energy-filtered transmission electron microscopy. This results in multicolor EM images analogous to multicolor fluorescence but with the benefit of the full spatial resolution of EM. We illustrate the power of this methodology by visualizing hippocampal astrocytes to show that processes from two astrocytes can share a single synapse. We also show that polyarginine-based cell-penetrating peptides enter the cell via endocytosis, and that newly synthesized PKMζ in cultured neurons preferentially localize to the postsynaptic membrane., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Adult stem cells are the ultimate source for replenishment of salivary gland (SG) tissue. Self-renewal ability of stem cells is dependent on extrinsic niche signals that have not been unraveled for the SG. The ductal compartment in SG has been identified as the location harboring stem cells. Here, we report that rare SG ductal EpCAM(+) cells express nuclear β-catenin, indicating active Wnt signaling. In cell culture experiments, EpCAM(high) cells respond potently to Wnt signals stimulating self-renewal and long-term expansion of SG organoids, containing all differentiated SG cell types. Conversely, Wnt inhibition ablated long-term organoid cultures. Finally, transplantation of cells pre-treated with Wnt agonists into submandibular glands of irradiated mice successfully and robustly restored saliva secretion and increased the number of functional acini in vivo. Collectively, these results identify Wnt signaling as a key driver of adult SG stem cells, allowing extensive in vitro expansion and enabling restoration of SG function upon transplantation., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Increasing amounts of data support a role for guanine quadruplex (G4) DNA and RNA structures in various cellular processes. We stained different organisms with monoclonal antibody 1H6 specific for G4 DNA. Strikingly, immuno-electron microscopy showed exquisite specificity for heterochromatin. Polytene chromosomes from Drosophila salivary glands showed bands that co-localized with heterochromatin proteins HP1 and the SNF2 domain-containing protein SUUR. Staining was retained in SUUR knock-out mutants but lost upon overexpression of SUUR. Somatic cells in Macrostomum lignano were strongly labeled, but pluripotent stem cells labeled weakly. Similarly, germline stem cells in Drosophila ovaries were weakly labeled compared to most other cells. The unexpected presence of G4 structures in heterochromatin and the difference in G4 staining between somatic cells and stem cells with germline DNA in ciliates, flatworms, flies and mammals point to a conserved role for G4 structures in nuclear organization and cellular differentiation., (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Spinocerebellar ataxia type 3 (SCA3) is a polyglutamine (polyQ) disorder caused by a CAG repeat expansion in the ataxin-3 ( ATXN3 ) gene resulting in toxic protein aggregation. Inflammation and oxidative stress are considered secondary factors contributing to the progression of this neurodegenerative disease. There is no cure that halts or reverses the progressive neurodegeneration of SCA3. Here we show that overexpression of cystathionine γ-lyase, a central enzyme in cysteine metabolism, is protective in a Drosophila model for SCA3. SCA3 flies show eye degeneration, increased oxidative stress, insoluble protein aggregates, reduced levels of protein persulfidation and increased activation of the innate immune response. Overexpression of Drosophila cystathionine γ-lyase restores protein persulfidation, decreases oxidative stress, dampens the immune response and improves SCA3-associated tissue degeneration. Levels of insoluble protein aggregates are not altered; therefore, the data implicate a modifying role of cystathionine γ-lyase in ameliorating the downstream consequence of protein aggregation leading to protection against SCA3-induced tissue degeneration. The cystathionine γ-lyase expression is decreased in affected brain tissue of SCA3 patients, suggesting that enhancers of cystathionine γ-lyase expression or activity are attractive candidates for future therapies.