Transmembrane channel-like 4 and 5 proteins at microvillar tips are potential ion channels and lipid scramblases.

Microvilli-membrane bound actin protrusions on the surface of epithelial cells-are sites of critical processes including absorption, secretion, and adhesion. Increasing evidence suggests microvilli are mechanosensitive, but underlying molecules and mechanisms remain unknown. Here, we localize transmembrane channel-like proteins 4 and 5 (TMC4 and 5) and calcium and integrin binding protein 3 (CIB3) to microvillar tips in intestinal epithelial cells, near glycocalyx insertion sites. We find that TMC5 colocalizes with CIB3 in cultured cells and that a TMC5 fragment forms a complex with CIB3 in vitro. Homology and AlphaFold2 models reveal a putative ion permeation pathway in TMC4 and 5, and molecular dynamics simulations predict both proteins can conduct ions and perform lipid scrambling. These findings raise the possibility that TMC4 and 5 interact with CIB3 at microvillar tips to form a mechanosensitive complex, akin to TMC1 and 2, and CIB2 and 3, within the mechanotransduction channel complex at the tips of inner ear stereocilia.

Analysis of beta-cell maturity and mitochondrial morphology in juvenile non-human primates exposed to maternal Western-style diet during development.

Introduction: Using a non-human primate (NHP) model of maternal Western-style diet (mWSD) feeding during pregnancy and lactation, we previously reported altered offspring beta:alpha cell ratio in vivo and insulin hyper-secretion ex vivo. Mitochondria are known to maintain beta-cell function by producing ATP for insulin secretion. In response to nutrient stress, the mitochondrial network within beta cells undergoes morphological changes to maintain respiration and metabolic adaptability. Given that mitochondrial dynamics have also been associated with cellular fate transitions, we assessed whether mWSD exposure was associated with changes in markers of beta-cell maturity and/or mitochondrial morphology that might explain the offspring islet phenotype. Methods: We evaluated the expression of beta-cell identity/maturity markers (NKX6.1, MAFB, UCN3) via florescence microscopy in islets of Japanese macaque pre-adolescent (1 year old) and peri-adolescent (3-year-old) offspring born to dams fed either a control diet or WSD during pregnancy and lactation and weaned onto WSD. Mitochondrial morphology in NHP offspring beta cells was analyzed in 2D by transmission electron microscopy and in 3D using super resolution microscopy to deconvolve the beta-cell mitochondrial network. Results: There was no difference in the percent of beta cells expressing key maturity markers in NHP offspring from WSD-fed dams at 1 or 3 years of age; however, beta cells of WSD-exposed 3 year old offspring showed increased levels of NKX6.1 per beta cell at 3 years of age. Regardless of maternal diet, the beta-cell mitochondrial network was found to be primarily short and fragmented at both ages in NHP; overall mitochondrial volume increased with age. In utero and lactational exposure to maternal WSD consumption may increase mitochondrial fragmentation. Discussion: Despite mWSD consumption having clear developmental effects on offspring beta:alpha cell ratio and insulin secretory response to glucose, this does not appear to be mediated by changes to beta-cell maturity or the beta-cell mitochondrial network. In general, the more fragmented mitochondrial network in NHP beta cells suggests greater ability for metabolic flexibility.

FapR regulates HssRS-mediated heme homeostasis in Bacillus anthracis.

Bacillus anthracis, a Gram-positive facultative anaerobe and the causative agent of anthrax, multiplies to extraordinarily high numbers in vertebrate blood, resulting in considerable heme exposure. Heme is an essential nutrient and the preferred iron source for bacteria during vertebrate colonization, but its high redox potential makes it toxic in excess. To regulate heme homeostasis, many Gram-positive bacteria, including B. anthracis, rely on the two-component signaling system HssRS. HssRS comprises the heme sensing histidine kinase HssS, which modulates the activity of the HssR transcription factor to enable bacteria to circumvent heme toxicity. However, the regulation of the HssRS system remains unclear. Here we identify FapR, the transcriptional regulator of fatty acid biosynthesis, as a key factor in HssRS function. FapR plays an important role in maintaining membrane integrity and the localization of the histidine kinase HssS. Specifically, disruption of fapR leads to increased membrane rigidity, which hinders the penetration of HssRS inducers, resulting in the inactivation of HssRS. Furthermore, deletion of fapR affects the loading of HssS onto the cell membrane, compromising its heme sensing function and subsequently reducing endogenous heme biosynthesis. These findings shed light on the molecular mechanisms governing bacterial adaptation to heme stress and provide potential targets for antimicrobial intervention strategies.

HIF-2α expression and metabolic signaling require ACSS2 in clear cell renal cell carcinoma.

Clear cell renal cell carcinoma (ccRCC) is an aggressive cancer driven by VHL loss and aberrant HIF-2α signaling. Identifying means to regulate HIF-2α thus has potential therapeutic benefit. Acetyl-CoA synthetase 2 (ACSS2) converts acetate to acetyl-CoA and is associated with poor patient prognosis in ccRCC. Here we tested the effects of ACSS2 on HIF-2α and cancer cell metabolism and growth in ccRCC models and clinical samples. ACSS2 inhibition reduced HIF-2α levels and suppressed ccRCC cell line growth in vitro, in vivo, and in cultures of primary ccRCC patient tumors. This treatment reduced glycolytic signaling, cholesterol metabolism, and mitochondrial integrity, all of which are consistent with loss of HIF-2α. Mechanistically, ACSS2 inhibition decreased chromatin accessibility and HIF-2α expression and stability. While HIF-2α protein levels are widely regulated through pVHL-dependent proteolytic degradation, we identify a potential pVHL-independent pathway of degradation via the E3 ligase MUL1. We show that MUL1 can directly interact with HIF-2α and that overexpression of MUL1 decreased HIF-2α levels in a manner partially dependent on ACSS2. These findings identify multiple mechanisms to regulate HIF-2α stability and ACSS2 inhibition as a strategy to complement HIF-2α-targeted therapies and deplete pathogenically stabilized HIF-2α.

Intestinal tuft cells assemble a cytoskeletal superstructure composed of co-aligned actin bundles and microtubules.

Background & Aims: All tissues consist of a distinct set of cell types, which collectively support organ function and homeostasis. Tuft cells are a rare epithelial cell type found in diverse epithelia, where they play important roles in sensing antigens and stimulating downstream immune responses. Exhibiting a unique polarized morphology, tuft cells are defined by an array of giant actin filament bundles that support ∼2 µm of apical membrane protrusion and extend over 7 µm towards the cell's perinuclear region. Despite their established roles in maintaining intestinal epithelial homeostasis, tuft cells remain understudied due to their rarity (e.g. ∼ 1% in the small intestinal epithelium). Details regarding the ultrastructural organization of the tuft cell cytoskeleton, the molecular components involved in building the array of giant actin bundles, and how these cytoskeletal structures support tuft cell biology remain unclear. Methods: To begin to answer these questions, we used advanced light and electron microscopy to perform quantitative morphometry of the small intestinal tuft cell cytoskeleton. Results: We found that tuft cell core bundles consist of actin filaments that are crosslinked in a parallel "barbed-end out" configuration. These polarized structures are also supported by a unique group of tuft cell enriched actin-binding proteins that are differentially localized along the giant core bundles. Furthermore, we found that tuft cell actin bundles are co-aligned with a highly ordered network of microtubules. Conclusions: Tuft cells assemble a cytoskeletal superstructure that is well positioned to serve as a track for subcellular transport along the apical-basolateral axis and in turn, support the dynamic sensing functions that are critical for intestinal epithelial homeostasis. SYNOPSIS: This research leveraged advanced light and electron microscopy to perform quantitative morphometry of the intestinal tuft cell cytoskeleton. Three-dimensional reconstructions of segmented image data revealed a co-aligned actin-microtubule superstructure that may play a fundamental role in tuft cell function.

Enhanced Optic Nerve Expansion and Altered Ultrastructure of Elastic Fibers Induced by Lysyl Oxidase Inhibition in a Mouse Model of Marfan Syndrome.

Two major constituents of exfoliation material, fibrillin-1 and lysyl oxidase-like 1 (encoded by FBN1 and LOXL1), are implicated in exfoliation glaucoma, yet their individual contributions to ocular phenotype are minor. To test the hypothesis that a combination of FBN1 mutation and LOXL1 deficiency exacerbates ocular phenotypes, the pan-lysyl oxidase inhibitor ß-aminopropionitrile (BAPN) was used to treat adult wild-type (WT) mice and mice heterozygous for a missense mutation in Fbn1 (Fbn1C1041G/+) for 8 weeks and their eyes were examined. Although intraocular pressure did not change and exfoliation material was not detected in the eyes, BAPN treatment worsened optic nerve and axon expansion in Fbn1C1041G/+ mice, an early sign of axonal damage in rodent models of glaucoma. Disruption of elastic fibers was detected only in Fbn1C1041G/+ mice, which increased with BAPN treatment, as shown by histologic and immunohistochemical staining of the optic nerve pia mater. Transmission electron microscopy showed that Fbn1C1041G/+ mice had fewer microfibrils, smaller elastin cores, and a lower density of elastic fibers compared with WT mice in control groups. BAPN treatment led to elastin core expansion in both WT and Fbn1C1041G/+ mice, but an increase in the density of elastic fiber was confined to Fbn1C1041G/+ mice. LOX inhibition had a stronger effect on optic nerve and elastic fiber parameters in the context of Fbn1 mutation, indicating the Marfan mouse model with LOX inhibition warrants further investigation for exfoliation glaucoma pathogenesis.

Mammalian orthoreovirus can exit cells in extracellular vesicles.

Several egress pathways have been defined for many viruses. Among these pathways, extracellular vesicles (EVs) have been shown to function as vehicles of non-lytic viral egress. EVs are heterogenous populations of membrane-bound structures released from cells as a form of intercellular communication. EV-mediated viral egress may enable immune evasion and collective viral transport. Strains of nonenveloped mammalian orthoreovirus (reovirus) differ in cell lysis phenotypes, with T3D disrupting cell membranes more efficiently than T1L. However, mechanisms of reovirus egress and the influence of transport strategy on infection are only partially understood. To elucidate reovirus egress mechanisms, we infected murine fibroblasts (L cells) and non-polarized human colon epithelial (Caco-2) cells with T1L or T3D reovirus and enriched cell culture supernatants for large EVs, medium EVs, small EVs, and free reovirus. We found that both reovirus strains exit cells in association with large and medium EVs and as free virus particles, and that EV-enriched fractions are infectious. While reovirus visually associates with large and medium EVs, only medium EVs offer protection from antibody-mediated neutralization. EV-mediated protection from neutralization is virus strain- and cell type-specific, as medium EVs enriched from L cell supernatants protect T1L and T3D, while medium EVs enriched from Caco-2 cell supernatants largely fail to protect T3D and only protect T1L efficiently. Using genetically barcoded reovirus, we provide evidence that large and medium EVs can convey multiple particles to recipient cells. Finally, T1L or T3D infection increases the release of all EV sizes from L cells. Together, these findings suggest that in addition to exiting cells as free particles, reovirus promotes egress from distinct cell types in association with large and medium EVs during lytic or non-lytic infection, a mode of exit that can mediate multiparticle infection and, in some cases, protection from antibody neutralization.

Clostridioides difficile ferrosome organelles combat nutritional immunity.

Iron is indispensable for almost all forms of life but toxic at elevated levels1-4. To survive within their hosts, bacterial pathogens have evolved iron uptake, storage and detoxification strategies to maintain iron homeostasis1,5,6. Recent studies showed that three Gram-negative environmental anaerobes produce iron-containing ferrosome granules7,8. However, it remains unclear whether ferrosomes are generated exclusively by Gram-negative bacteria. The Gram-positive bacterium Clostridioides difficile is the leading cause of nosocomial and antibiotic-associated infections in the USA9. Here we report that C. difficile undergoes an intracellular iron biomineralization process and stores iron in membrane-bound ferrosome organelles containing non-crystalline iron phosphate biominerals. We found that a membrane protein (FezA) and a P1B6-ATPase transporter (FezB), repressed by both iron and the ferric uptake regulator Fur, are required for ferrosome formation and play an important role in iron homeostasis during transition from iron deficiency to excess. Additionally, ferrosomes are often localized adjacent to cellular membranes as shown by cryo-electron tomography. Furthermore, using two mouse models of C. difficile infection, we demonstrated that the ferrosome system is activated in the inflamed gut to combat calprotectin-mediated iron sequestration and is important for bacterial colonization and survival during C. difficile infection.

Culture conditions greatly impact the levels of vesicular and extravesicular Ago2 and RNA in extracellular vesicle preparations.

Extracellular vesicle (EV)-carried miRNAs can influence gene expression and functional phenotypes in recipient cells. Argonaute 2 (Ago2) is a key miRNA-binding protein that has been identified in EVs and could influence RNA silencing. However, Ago2 is in a non-vesicular form in serum and can be an EV contaminant. In addition, RNA-binding proteins (RBPs), including Ago2, and RNAs are often minor EV components whose sorting into EVs may be regulated by cell signaling state. To determine the conditions that influence detection of RBPs and RNAs in EVs, we evaluated the effect of growth factors, oncogene signaling, serum, and cell density on the vesicular and nonvesicular content of Ago2, other RBPs, and RNA in small EV (SEV) preparations. Media components affected both the intravesicular and extravesicular levels of RBPs and miRNAs in EVs, with serum contributing strongly to extravesicular miRNA contamination. Furthermore, isolation of EVs from hollow fiber bioreactors revealed complex preparations, with multiple EV-containing peaks and a large amount of extravesicular Ago2/RBPs. Finally, KRAS mutation impacts the detection of intra- and extra-vesicular Ago2. These data indicate that multiple cell culture conditions and cell states impact the presence of RBPs in EV preparations, some of which can be attributed to serum contamination.

Adhesion-based capture stabilizes nascent microvilli at epithelial cell junctions.

To maximize solute transport, epithelial cells build an apical "brush border," where thousands of microvilli are linked to their neighbors by protocadherin-containing intermicrovillar adhesion complexes (IMACs). Previous studies established that the IMAC is needed to build a mature brush border, but how this complex contributes to the accumulation of new microvilli during differentiation remains unclear. We found that early in differentiation, mouse, human, and porcine epithelial cells exhibit a marginal accumulation of microvilli, which span junctions and interact with protrusions on neighboring cells using IMAC protocadherins. These transjunctional IMACs are highly stable and reinforced by tension across junctions. Finally, long-term live imaging showed that the accumulation of microvilli at cell margins consistently leads to accumulation in medial regions. Thus, nascent microvilli are stabilized by a marginal capture mechanism that depends on the formation of transjunctional IMACs. These results may offer insights into how apical specializations are assembled in diverse epithelial systems.

Call to action to properly utilize electron microscopy to measure organelles to monitor disease.

This review provides an overview of the current methods for quantifying mitochondrial ultrastructure, including cristae morphology, mitochondrial contact sites, and recycling machinery and a guide to utilizing electron microscopy to effectively measure these organelles. Quantitative analysis of mitochondrial ultrastructure is essential for understanding mitochondrial biology and developing therapeutic strategies for mitochondrial-related diseases. Techniques such as transmission electron microscopy (TEM) and serial block face-scanning electron microscopy, as well as how they can be combined with other techniques including confocal microscopy, super-resolution microscopy, and correlative light and electron microscopy are discussed. Beyond their limitations and challenges, we also offer specific magnifications that may be best suited for TEM analysis of mitochondrial, endoplasmic reticulum, and recycling machinery. Finally, perspectives on future quantification methods are offered.

Mammalian orthoreovirus can exit cells in extracellular vesicles.

Several egress pathways have been defined for many viruses. Among these pathways, extracellular vesicles (EVs) have been shown to function as vehicles of non-lytic viral egress. EVs are heterogenous populations of membrane-bound structures released from cells as a form of intercellular communication. EV-mediated viral egress may enable immune evasion and collective viral transport. Strains of nonenveloped mammalian orthoreovirus (reovirus) differ in cell lysis phenotypes, with T3D disrupting cell membranes more efficiently than T1L. However, mechanisms of reovirus egress and the influence of transport strategy on infection are only partially understood. To elucidate reovirus egress mechanisms, we infected murine fibroblasts (L cells) and non-polarized human colon epithelial (Caco-2) cells with T1L or T3D reovirus and enriched cell culture supernatants for large EVs, medium EVs, small EVs, and free reovirus. We found that both reovirus strains exit cells in association with large and medium EVs and as free virus particles, and that EV-enriched fractions are infectious. While reovirus visually associates with large and medium EVs, only medium EVs offer protection from antibody-mediated neutralization. EV-mediated protection from neutralization is virus strain- and cell type-specific, as medium EVs enriched from L cell supernatants protect T1L and T3D, while medium EVs enriched from Caco-2 cell supernatants largely fail to protect T3D and only protect T1L efficiently. Using genetically barcoded reovirus, we provide evidence that large and medium EVs can convey multiple particles to recipient cells. Finally, T1L or T3D infection increases the release of all EV sizes from L cells. Together, these findings suggest that in addition to exiting cells as free particles, reovirus promotes egress from distinct cell types in association with large and medium EVs during lytic or non-lytic infection, a mode of exit that can mediate multiparticle infection and, in some cases, protection from antibody neutralization.

Apposition of Fibroblasts With Metaplastic Gastric Cells Promotes Dysplastic Transition.

BACKGROUND & AIMS: Elements of field cancerization, including atrophic gastritis, metaplasia, and dysplasia, promote gastric cancer development in association with chronic inflammation. However, it remains unclear how stroma changes during carcinogenesis and how the stroma contributes to progression of gastric preneoplasia. Here we investigated heterogeneity of fibroblasts, one of the most important elements in the stroma, and their roles in neoplastic transformation of metaplasia. METHODS: We used single-cell transcriptomics to evaluate the cellular heterogeneity of mucosal cells from patients with gastric cancer. Tissue sections from the same cohort and tissue microarrays were used to identify the geographical distribution of distinct fibroblast subsets. We further evaluated the role of fibroblasts from pathologic mucosa in dysplastic progression of metaplastic cells using patient-derived metaplastic gastroids and fibroblasts. RESULTS: We identified 4 subsets of fibroblasts within stromal cells defined by the differential expression of PDGFRA, FBLN2, ACTA2, or PDGFRB. Each subset was distributed distinctively throughout stomach tissues with different proportions at each pathologic stage. The PDGFRα+ subset expanded in metaplasia and cancer compared with normal, maintaining a close proximity with the epithelial compartment. Co-culture of metaplasia- or cancer-derived fibroblasts with gastroids showing the characteristics of spasmolytic polypeptide-expressing metaplasia-induced disordered growth, loss of metaplastic markers, and increases in markers of dysplasia. Culture of metaplastic gastroids with conditioned media from metaplasia- or cancer-derived fibroblasts also promoted dysplastic transition. CONCLUSIONS: These findings indicate that fibroblast associations with metaplastic epithelial cells can facilitate direct transition of metaplastic spasmolytic polypeptide-expressing metaplasia cell lineages into dysplastic lineages.

Serial Block Face-Scanning Electron Microscopy as a Burgeoning Technology.

Serial block face scanning electron microscopy (SBF-SEM), also referred to as serial block-face electron microscopy, is an advanced ultrastructural imaging technique that enables three-dimensional visualization that provides largerx- and y-axis ranges than other volumetric EM techniques. While SEM is first introduced in the 1930s, SBF-SEM is developed as a novel method to resolve the 3D architecture of neuronal networks across large volumes with nanometer resolution by Denk and Horstmann in 2004. Here, the authors provide an accessible overview of the advantages and challenges associated with SBF-SEM. Beyond this, the applications of SBF-SEM in biochemical domains as well as potential future clinical applications are briefly reviewed. Finally, the alternative forms of artificial intelligence-based segmentation which may contribute to devising a feasible workflow involving SBF-SEM, are also considered.

Adhesion-based capture stabilizes nascent microvilli at epithelial cell junctions.

Differentiated transporting epithelial cells present an extensive apical array of microvilli - a "brush border" - where neighboring microvilli are linked together by intermicrovillar adhesion complexes (IMACs) composed of protocadherins CDHR2 and CDHR5. Although loss-of-function studies provide strong evidence that IMAC function is needed to build a mature brush border, how the IMAC contributes to the stabilization and accumulation of nascent microvilli remains unclear. We found that, early in differentiation, the apical surface exhibits a marginal accumulation of microvilli, characterized by higher packing density relative to medial regions of the surface. While medial microvilli are highly dynamic and sample multiple orientations over time, marginal protrusions exhibit constrained motion and maintain a vertical orientation. Unexpectedly, we found that marginal microvilli span the junctional space and contact protrusions on neighboring cells, mediated by complexes of CDHR2/CDHR5. FRAP analysis indicated that these transjunctional IMACs are highly stable relative to adhesion complexes between medial microvilli, which explains the restricted motion of protrusions in the marginal zone. Finally, long-term live imaging revealed that the accumulation of microvilli at cell margins consistently leads to accumulation in medial regions of the cell. Collectively, our findings suggest that nascent microvilli are stabilized by a capture mechanism that is localized to cell margins and enabled by the transjunctional formation of IMACs. These results inform our understanding of how apical specializations are assembled in diverse epithelial systems.

Elevated transferrin receptor impairs T cell metabolism and function in systemic lupus erythematosus.

T cells in systemic lupus erythematosus (SLE) exhibit multiple metabolic abnormalities. Excess iron can impair mitochondria and may contribute to SLE. To gain insights into this potential role of iron in SLE, we performed a CRISPR screen of iron handling genes on T cells. Transferrin receptor (CD71) was identified as differentially critical for TH1 and inhibitory for induced regulatory T cells (iTregs). Activated T cells induced CD71 and iron uptake, which was exaggerated in SLE-prone T cells. Cell surface CD71 was enhanced in SLE-prone T cells by increased endosomal recycling. Blocking CD71 reduced intracellular iron and mTORC1 signaling, which inhibited TH1 and TH17 cells yet enhanced iTregs. In vivo treatment reduced kidney pathology and increased CD4 T cell production of IL-10 in SLE-prone mice. Disease severity correlated with CD71 expression on TH17 cells from patients with SLE, and blocking CD71 in vitro enhanced IL-10 secretion. T cell iron uptake via CD71 thus contributes to T cell dysfunction and can be targeted to limit SLE-associated pathology.

Engineering functional 3-dimensional patient-derived endocrine organoids for broad multiplatform applications.

BACKGROUND: Recent advancements in 3-dimensional patient-derived organoid models have revolutionized the field of cancer biology. There is an urgent need for development of endocrine tumor organoid models for medullary thyroid carcinoma, adrenocortical carcinoma, papillary thyroid carcinoma, and a spectrum of benign hyperfunctioning parathyroid and adrenal neoplasms. We aimed to engineer functionally intact 3-dimensional endocrine patient-derived organoids to expand the in vitro and translational applications for the advancement of endocrine research. METHODS: Using our recently developed fine needle aspiration-based methodology, we established patient-derived 3-dimensional endocrine organoid models using prospectively collected human papillary thyroid carcinoma (n = 6), medullary thyroid carcinoma (n = 3), adrenocortical carcinoma (n = 3), and parathyroid (n = 5). and adrenal (n = 5) neoplasms. Multiplatform analyses of endocrine patient-derived organoids and applications in oncoimmunology, near-infrared autofluorescence, and radiosensitization studies under 3-dimensional in vitro conditions were performed. RESULTS: We have successfully modeled and analyzed the complex endocrine microenvironment for a spectrum of endocrine neoplasms in 3-dimensional culture. The endocrine patient-derived organoids recapitulated complex tumor microenvironment of endocrine neoplasms morphologically and functionally and maintained cytokine production and near-infrared autofluorescence properties. CONCLUSION: Our novel engineered endocrine patient-derived organoid models of thyroid, parathyroid and adrenal neoplasms represent an exciting and elegant alternative to current limited 2-dimensional systems and afford future broad multiplatform in vitro and translational applications, including in endocrine oncoimmunology.