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Electron microscopy paired with immunogold labeling is the most precise tool for protein localization. However, these methods are either cumbersome, resulting in small sample numbers and restricted quantification, or limited to identifying protein epitopes external to the membrane. Here, we introduce SUB-immunogold-SEM, a scanning electron microscopy technique that detects intracellular protein epitopes proximal to the membrane. We identify four critical sample preparation factors contributing to the method's sensitivity. We validate its efficacy through precise localization and high-powered quantification of cytoskeletal and transmembrane protein distribution. We evaluate the capabilities of SUB-immunogold-SEM on cells with highly differentiated apical surfaces: (i) auditory hair cells, revealing the presence of nanoscale MYO15A-L rings at the tip of stereocilia; and (ii) respiratory multiciliate cells, mapping the distribution of the SARS-CoV-2 receptor ACE2 along the motile cilia. SUB-immunogold-SEM extends the application of SEM-based nanoscale protein localization to the detection of intracellular epitopes on the exposed surfaces of any cell. Nanoscale protein localisation by immunogold-scanning electron microscopy has been restricted to extracellular epitopes. Here, the authors extend this method to sub-membranous epitopes, revealing how transmembrane and cytoplasmic proteins distribute along the surfaces of exposed cells. [ABSTRACT FROM AUTHOR]

Background: Vascular endothelial growth factor (VEGF) is an important proangiogenic factor and has been considered as a key target of antiangiogenetic therapy in oral squamous cell carcinoma (OSCC). However, clinical application of bevacizumab, a specific VEGF antibody, didn't improve the survival rate of OSCC patients. One possible explanation is that VEGF gene expresses diverse isoforms, which associate with extracellular vesicles (EVs), and EVs potentially contribute to VEGF resistance to bevacizumab. However, clear solution is lacking in addressing this issue. Methods: Expression of VEGF isoforms in OSCC cells was confirmed by reverse transcription and polymerase chain reaction (RT-PCR) and western blot. EVs isolated from OSCC cell's conditioned medium (CM) were characterized by western blot, transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA). Flow cytometry, immunogold labeling and western blot were applied to study the VEGF on EVs. Tube formation assay and Matrigel plug angiogenesis assay were used for analyzing the angiogenesis capacity of EV-VEGF. Results: The most popular isoforms expressed by VEGF gene are VEGF121, VEGF165 and VEGF189. In this study, we demonstrated that all three isoforms of mRNA could be detected at varying levels in OSCC cells, while only VEGF165 and VEGF189 proteins were found. CM derived from OSCC cells, both soluble and non-soluble forms of VEGF could be detected. We further confirmed the presence of VGEF189 bound to EVs as a non-soluble form. EV-bound VEGF189 presented angiogenic activity, which could not be neutralized by bevacizumab. It was found that VEGF189 bound to EVs by heparan sulfate proteoglycans (HSPG). In addition, the angiogenic effect of EV-VEGF could be reversed by surfen, a kind of HSPG antagonist both in vitro and in vivo. Conclusion: Antagonists targeting HSPG might potentially overcome the resistance of EV-VEGF to bevacizumab and serve as an alternative for anti-VEGF therapy in OSCC. [ABSTRACT FROM AUTHOR]

This article is a personal reflection on the author's 20-year professional relationship and friendship with a consultant named Tony, who is highly regarded in the field of cosmetic science. The author highlights Tony's contributions to their company, including the development of the "Corneocare" concept and the creation of the SquameScan device for measuring the amount of stratum corneum protein. They also discuss their collaborative research on serine proteases in the stratum corneum and the impact of protease activity on different skin types. Additionally, the article mentions Tony's achievements as an editor and his collaborations with various universities. The author expresses their admiration for Tony's scientific expertise, pragmatism, and dedication to teaching. The article concludes with a personal anecdote about their friendship and Tony's appreciation for the author's culinary knowledge. [Extracted from the article]

KCNQ4 is a voltage-gated K+ channel was reported to distribute over the basolateral surface of type 1 vestibular hair cell and/or inner surface of calyx and heminode of the vestibular nerve connected to the type 1 vestibular hair cells of the inner ear. However, the precise localization of KCNQ4 is still controversial and little is known about the vestibular phenotypes caused by KCNQ4 dysfunction or the specific role of KCNQ4 in the vestibular organs. To investigate the role of KCNQ4 in the vestibular organ, 6-g hypergravity stimulation for 24 h, which represents excessive mechanical stimulation of the sensory epithelium, was applied to p.W277S Kcnq4 transgenic mice. KCNQ4 was detected on the inner surface of calyx of the vestibular afferent in transmission electron microscope images with immunogold labelling. Vestibular function decrease was more severe in the Kcnq4p.W277S/p.W277S mice than in the Kcnq4+/+ and Kcnq4+/p.W277S mice after the stimulation. The vestibular function loss was resulted from the loss of type 1 vestibular hair cells, which was possibly caused by increased depolarization duration. Retigabine, a KCNQ activator, prevented hypergravity-induced vestibular dysfunction and hair cell loss. Patients with KCNQ4 mutations also showed abnormal clinical vestibular function tests. These findings suggest that KCNQ4 plays an essential role in calyx and afferent of type 1 vestibular hair cell preserving vestibular function against excessive mechanical stimulation. [ABSTRACT FROM AUTHOR]

Gut microbiome dysbiosis is a major contributing factor to several pathological conditions. However, the mechanistic understanding of the communication between gut microbiota and extra‐intestinal organs remains largely elusive. Extracellular vesicles (EVs), secreted by almost every form of life, including bacteria, could play a critical role in this inter‐kingdom crosstalk and are the focus of present study. Here, we present a novel approach for isolating lipopolysaccharide (LPS)+ bacterial extracellular vesicles (bEVLPS) from complex biological samples, including faeces, plasma and the liver from lean and diet‐induced obese (DIO) mice. bEVLPS were extensively characterised using nanoparticle tracking analyses, immunogold labelling coupled with transmission electron microscopy, flow cytometry, super‐resolution microscopy and 16S sequencing. In liver tissues, the protein expressions of TLR4 and a few macrophage‐specific biomarkers were assessed by immunohistochemistry, and the gene expressions of inflammation‐related cytokines and their receptors (n = 89 genes) were measured using a PCR array. Faecal samples from DIO mice revealed a remarkably lower concentration of total EVs but a significantly higher percentage of LPS+ EVs. Interestingly, DIO faecal bEVLPS showed a higher abundance of Proteobacteria by 16S sequencing. Importantly, in DIO mice, a higher number of total EVs and bEVLPS consistently entered the hepatic portal vein and subsequently reached the liver, associated with increased expression of TLR4, macrophage markers (F4/80, CD86 and CD206), cytokines and receptors (Il1rn, Ccr1, Cxcl10, Il2rg and Ccr2). Furthermore, a portion of bEVLPS escaped liver and entered the peripheral circulation. In conclusion, bEV could be the key mediator orchestrating various well‐established biological effects induced by gut bacteria on distant organs. [ABSTRACT FROM AUTHOR]

Species in the genus Utricularia are carnivorous plants that prey on invertebrates using traps of leaf origin. The traps are equipped with numerous different glandular trichomes. Trichomes (quadrifids) produce digestive enzymes and absorb the products of prey digestion. The main aim of this study was to determine whether arabinogalactan proteins (AGPs) occur in the cell wall ingrowths in the quadrifid cells. Antibodies (JIM8, JIM13, JIM14, MAC207, and JIM4) that act against various groups of AGPs were used. AGP localization was determined using immunohistochemistry techniques and immunogold labeling. AGPs localized with the JIM13, JIM8, and JIM14 epitopes occurred in wall ingrowths of the pedestal cell, which may be related to the fact that AGPs regulate the formation of wall ingrowths but also, due to the patterning of the cell wall structure, affect symplastic transport. The presence of AGPs in the cell wall of terminal cells may be related to the presence of wall ingrowths, but processes also involve vesicle trafficking and membrane recycling, in which these proteins participate. [ABSTRACT FROM AUTHOR]

The genus Utricularia (bladderworts) species are carnivorous plants that prey on invertebrates using traps with a high-speed suction mechanism. The outer trap surface is lined by dome-shaped glands responsible for secreting water in active traps. In terminal cells of these glands, the outer wall is differentiated into several layers, and even cell wall ingrowths are covered by new cell wall layers. Due to changes in the cell wall, these glands are excellent models for studying the specialization of cell walls (microdomains). The main aim of this study was to check if different cell wall layers have a different composition. Antibodies against arabinogalactan proteins (AGPs) were used, including JIM8, JIM13, JIM14, MAC207, and JIM4. The localization of the examined compounds was determined using immunohistochemistry techniques and immunogold labeling. Differences in composition were found between the primary cell wall and the cell secondary wall in terminal gland cells. The outermost layer of the cell wall of the terminal cell, which was cuticularized, was devoid of AGPs (JIM8, JIM14). In contrast, the secondary cell wall in terminal cells was rich in AGPs. AGPs localized with the JIM13, JIM8, and JIM14 epitopes occurred in wall ingrowths of pedestal cells. Our research supports the hypothesis of water secretion by the external glands. [ABSTRACT FROM AUTHOR]

HIV-1 gp120 glycan binding to C-type lectin adhesion receptor L-selectin/CD62L on CD4 T cells facilitates viral attachment and entry. Paradoxically, the adhesion receptor impedes HIV-1 budding from infected T cells and the viral release requires the shedding of CD62L. To systematically investigate CD62Lshedding mediated viral release and its potential inhibition, we screened compounds specific for serine-, cysteine-, aspartyl-, and Zn-dependent proteases for CD62L shedding inhibition and found that a subclass of Znmetalloproteinase inhibitors, including BB-94, TAPI, prinomastat, GM6001, and GI25423X, suppressed CD62L shedding. Their inhibition of HIV-1 infections correlated with enzymatic suppression of both ADAM10 and 17 activities and expressions of these ADAMs were transiently induced during the viral infection. These metalloproteinase inhibitors are distinct from the current antiretroviral drug compounds. Using immunogold labeling of CD62L, we observed association between budding HIV-1 virions and CD62L by transmission electron microscope, and the extent of CD62L-tethering of budding virions increased when the receptor shedding is inhibited. Finally, these CD62L shedding inhibitors suppressed the release of HIV-1 virions by CD4 T cells of infected individuals and their virion release inhibitions correlated with their CD62L shedding inhibitions. Our finding reveals a new therapeutic approach targeted at HIV-1 viral release. [ABSTRACT FROM AUTHOR]

There is accumulating evidence that pathogenic T cells in T1D recognize epitopes formed by post-translational modifications of β-cell antigens, including hybrid insulin peptides (HIPs). The ligands for several CD4 T-cell clones derived from the NOD mouse are HIPs composed of a fragment of proinsulin joined to peptides from endogenous β-cell granule proteins. The diabetogenic T-cell clone BDC-6.9 reacts to a fragment of C-peptide fused to a cleavage product of pro-islet amyloid polypeptide (6.9HIP). In this study, we used a monoclonal antibody (MAb) to the 6.9HIP to determine when and where HIP antigens are present in NOD islets during disease progression and with which immune cells they associate. Immunogold labeling of the 6.9HIP MAb and organelle-specific markers for electron microscopy were employed to map the subcellular compartment(s) in which the HIP is localized within β-cells. While the insulin B9-23 peptide was present in nearly all islets, the 6.9HIP MAb stained infiltrated islets only in NOD mice at advanced stages of T1D development. Islets co-stained with the 6.9HIP MAb and antibodies to mark insulin, macrophages, and dendritic cells indicate that 6.9HIP co-localizes within insulin-positive β-cells as well as intra-islet antigen-presenting cells (APCs). In electron micrographs, the 6.9HIP co-localized with granule structures containing insulin alone or both insulin and LAMP1 within β-cells. Exposing NOD islets to the endoplasmic reticulum (ER) stress inducer tunicamycin significantly increased levels of 6.9HIP in subcellular fractions containing crinosomes and dense-core granules (DCGs). This work demonstrates that the 6.9HIP can be visualized in the infiltrated islets and suggests that intra-islet APCs may acquire and present HIP antigens within islets. [ABSTRACT FROM AUTHOR]

The etiology of minimal change disease (MCD) remains a mystery as the only characteristic findings are the diffuse effacement of foot processes seen on electron microscopy (EM). Punctate IgG staining found floating outside glomerular capillary loops in MCD cases was recently identified as autoimmune antibodies against nephrin of podocytes. We hypothesized that the punctate IgG staining is located on budding ballooning clusters (BBC) of reactive foot processes in Bowman's space found on EM. We identified seven patients with MCD cases showing IgG staining that were subsequently evaluated for BBC on EM. We concurrently examined 12 negative controls, either unremarkable cases or tubulointerstitial diseases, by EM. Immunogold labeling was performed to confirm the presence of IgG and determine localization. In seven MCD cases, there were positive punctate IgG staining particles outside of the glomerular basement membranes (GBM) along with concurrent punctate staining for C3, kappa, and lambda. By EM, all seven (100%) MCD cases revealed BBC that was characterized by ballooning foot processes ranging from 1 to 6 µm and was either budding or detached from the GBM in 3–7 clusters; no electron-dense materials were seen in BBC. BBC was also seen in only 1 of 12 (8%) negative controls. Immunogold labeling identified IgG particles within BBC of MCD by EM, but not in the negative control. Our data suggest that BBC are EM structures of reactive foot processes that are most likely correlated with punctate IgG staining seen in cases of MCD, supported by immunogold labeling for IgG. [ABSTRACT FROM AUTHOR]

Introduction: Leptoids, the food-conducting cells of polytrichaceous mosses, share key structural features with sieve elements in tracheophytes, including an elongated shape with oblique end walls containing modified plasmodesmata or pores. In tracheophytes, callose is instrumental in developing the pores in sieve elements that enable efficient photoassimilate transport. Aside from a few studies using aniline blue fluorescence that yielded confusing results, little is known about callose in moss leptoids. Methods: Callose location and abundance during the development of leptoid cell walls was investigated in the moss Polytrichum commune using aniline blue fluorescence and quantitative immunogold labeling (label density) in the transmission electron microscope. To evaluate changes during abiotic stress, callose abundance in leptoids of hydrated plants was compared to plants dried for 14 days under field conditions. A bioinformatic study to assess the evolution of callose within and across bryophytes was conducted using callose synthase (CalS) genes from 46 bryophytes (24 mosses, 15 liverworts, and 7 hornworts) and one representative each of five tracheophyte groups. Results: Callose abundance increases around plasmodesmata from meristematic cells to end walls in mature leptoids. Controlled drying resulted in a significant increase in label density around plasmodesmata and pores over counts in hydrated plants. Phylogenetic analysis of the CalS protein family recovered main clades (A, B, and C). Different from tracheophytes, where the greatest diversity of homologs is found in clade A, the majority of gene duplication in bryophytes is in clade B. Discussion: This work identifies callose as a crucial cell wall polymer around plasmodesmata from their inception to functioning in leptoids, and during water stress similar to sieve elements of tracheophytes. Among bryophytes, mosses exhibit the greatest number of multiple duplication events, while only two duplications are revealed in hornwort and none in liverworts. The absence in bryophytes of the CalS 7 gene that is essential for sieve pore development in angiosperms, reveals that a different gene is responsible for synthesizing the callose associated with leptoids in mosses. [ABSTRACT FROM AUTHOR]

We have previously established that 670 nm energy induces relaxation of blood vessels via an endothelium derived S-nitrosothiol (RSNO) suggested to be embedded in vesicles. Here, we confirm that red light facilitates the exocytosis of this vasodilator from cultured endothelial cells and increases ex vivo blood vessel diameter. Ex vivo pressurized and pre-constricted facial arteries from C57Bl6/J mice relaxed 14.7% of maximum diameter when immersed in the medium removed from red-light exposed Bovine Aortic Endothelial Cells. In parallel experiments, 0.49 nM RSNO equivalent species was measured in the medium over the irradiated cells vs dark control. Electron microscopy of light exposed endothelium revealed significant increases in the size of the Multi Vesicular Body (MVB), a regulator of exosome trafficking, while RSNO accumulated in the MVBs as detected with immunogold labeling electron microscopy (1.8-fold of control). Moreover, red light enhanced the presence of F-actin related stress fibers (necessary for exocytosis), and the endothelial specific marker VE-cadherin levels suggesting an endothelial origin of the extracellular vesicles. Flow cytometry coupled with DAF staining, an indirect sensor of nitric oxide (NO), indicated significant amounts of NO within the extracellular vesicles (1.4-fold increase relative to dark control). Therefore, we further define the mechanism on the 670 nm light mediated traffic of endothelial vasodilatory vesicles and plan to leverage this insight into the delivery of red-light therapies. [ABSTRACT FROM AUTHOR]

Pollen germination is a crucial process in the life cycle of flowering plants, signifying the transition of quiescent pollen grains into active growth. This study delves into the dynamic changes within organelles and the pivotal role of autophagy during lily pollen germination. Initially, mature pollen grains harbor undifferentiated organelles, including amyloplasts, mitochondria, and the Golgi apparatus. However, germination unveils remarkable transformations, such as the redifferentiation of amyloplasts accompanied by starch granule accumulation. We investigate the self-sustained nature of amylogenesis during germination, shedding light on its association with osmotic pressure. Employing BODIPY 493/503 staining, we tracked lipid body distribution throughout pollen germination, both with or without autophagy inhibitors (3-MA, NEM). Typically, lipid bodies undergo polarized movement from pollen grains into elongating pollen tubes, a process crucial for directional growth. Inhibiting autophagy disrupted this essential lipid body redistribution, underscoring the interaction between autophagy and lipid body dynamics. Notably, the presence of tubular endoplasmic reticulum (ER)-like structures associated with developing amyloplasts and lipid bodies implies their participation in autophagy. Starch granules, lipid bodies, and membrane remnants observed within vacuoles further reinforce the involvement of autophagic processes. Among the autophagy inhibitors, particularly BFA, significantly impede germination and growth, thereby affecting Golgi morphology. Immunogold labeling substantiates the pivotal role of the ER in forming autophagosome-like compartments and protein localization. Our proposed speculative model of pollen germination encompasses proplastid differentiation and autophagosome formation. This study advances our understanding of organelle dynamics and autophagy during pollen germination, providing valuable insights into the realm of plant reproductive physiology. [ABSTRACT FROM AUTHOR]

Brain‐derived extracellular vesicles (EVs) play an active role in Alzheimer's disease (AD), relaying important physiological information about their host tissues. The internal cargo of EVs is protected from degradation, making EVs attractive AD biomarkers. However, it is unclear how circulating EVs relate to EVs isolated from disease‐vulnerable brain regions. We developed a novel method for collecting EVs from the hippocampal interstitial fluid (ISF) of live mice. EVs (EVISF) were isolated via ultracentrifugation and characterized by nanoparticle tracking analysis, immunogold labelling, and flow cytometry. Mass spectrometry and proteomic analyses were performed on EVISF cargo. EVISF were 40–150 nm in size and expressed CD63, CD9, and CD81. Using a model of cerebral amyloidosis (e.g., APPswe, PSEN1dE9 mice), we found protein concentration increased but protein diversity decreased with Aβ deposition. Genotype, age, and Aβ deposition modulated proteostasis‐ and immunometabolic‐related pathways. Changes in the microglial EVISF proteome were sexually dimorphic and associated with a differential response of plaque associated microglia. We found that female APP/PS1 mice have more amyloid plaques, less plaque associated microglia, and a less robust‐ and diverse‐ EVISF microglial proteome. Thus, in vivo microdialysis is a novel technique for collecting EVISF and offers a unique opportunity to explore the role of EVs in AD. [ABSTRACT FROM AUTHOR]

Cell-to-cell transport of plant viruses through plasmodesmata (PD) requires viral movement proteins (MPs) often associated with cell membranes. The genome of the Hibiscus green spot virus encodes two MPs, BMB1 and BMB2, which enable virus cell-to-cell transport. BMB2 is known to localize to PD-associated membrane bodies (PAMBs), which are derived from the endoplasmic reticulum (ER) structures, and to direct BMB1 to PAMBs. This paper reports the fine structure of PAMBs. Immunogold labeling confirms the previously observed localization of BMB1 and BMB2 to PAMBs. EM tomography data show that the ER-derived structures in PAMBs are mostly cisterns interconnected by numerous intermembrane contacts that likely stabilize PAMBs. These contacts predominantly involve the rims of the cisterns rather than their flat surfaces. Using FRET-FLIM (Förster resonance energy transfer between fluorophores detected by fluorescence-lifetime imaging microscopy) and chemical cross-linking, BMB2 is shown to self-interact and form high-molecular-weight complexes. As BMB2 has been shown to have an affinity for highly curved membranes at cisternal rims, the interaction of BMB2 molecules located at rims of adjacent cisterns is suggested to be involved in the formation of intermembrane contacts in PAMBs. [ABSTRACT FROM AUTHOR]

Peptide presentation by MHC class I and MHC class II molecules plays important roles in the regulation of the immune response. One factor in these displays is the density of antigen, which must exceed a critical threshold for the effective activation of T cells. Nonrandom distribution of MHC class I and class II has already been detected at the nanometer level and at higher hierarchical levels. It is not clear how the absence and reappearance of some protein molecules can influence the nonrandom distribution. Therefore, we performed experiments on HLA II‐deficient bare lymphocyte syndrome (BLS1) cells: we created a stable transfected cell line, tDQ6‐BLS‐1, and were able to detect the effect of the appearance of HLA‐DQ6 molecules on the homo and heteroassociation of different cell surface molecules by comparing Förster resonance energy transfer (FRET) efficiency on transfected cells to that on nontransfected BLS‐1 and JY human B‐cell lines. Our FRET results show a decrease in homoassociation FRET between HLA I chains in HLA‐DQ6‐transfected tDQ6‐BLS‐1 cells compared with the parent BLS‐1 cell line and an increase in heteroassociation FRET between HLA I and HLA II (compared with JY cells), suggesting a similar pattern of antigen presentation by the HLA‐DQ6 allele. Transmission electron microscopy (TEM) revealed that both HLA class I and class II molecules formed clusters at higher hierarchical levels on the tDQ6‐BLS‐1 cells, and the de novo synthesized HLA DQ molecules did not intersperse with HLA class I islands. These observations could be important in understanding the fine tuning of the immune response. [ABSTRACT FROM AUTHOR]

Background: Arabinogalactan proteins (AGPs) are plant cell components found in the extracellular matrix that play crucial roles in fruit growth and development. AGPs demonstrate structural diversity due to the presence of a protein domain and an expanded carbohydrate moiety. Considering their molecular structure, the modification of glycosylation is a primary factor contributing to the functional variety of AGPs. Main body: Immunocytochemical methods are used for qualitative and quantitative analyses of AGPs in fruit tissues. These include in situ techniques such as immunofluorescence and immunogold labelling for visualising AGP distribution at different cellular levels and ex situ methods such as Western blotting and enzyme-linked immunoenzymatic assays (ELISA) for molecular characterisation and quantitative detection of isolated AGPs. The presented techniques were modified by considering the structure of AGPs and the changes that occur in fruit tissues during the development and ripening processes. These methods are based on antibodies that recognise carbohydrate chains, which are the only commercially available highly AGP-specific tools. These probes recognise AGP epitopes and identify structural modifications and changes in spatio-temporal distribution, shedding light on their functions in fruit. Conclusion: This paper provides a concise overview of AGP research methods, emphasising their use in fruit tissue analysis and demonstrating the accessibility gaps in other tools used in such research (e.g. antibodies against protein moieties). It underscores fruit tissue as a valuable source of AGPs and emphasises the potential for future research to understand of AGP synthesis, degradation, and their roles in various physiological processes. Moreover, the application of advanced probes for AGP visualisation is a milestone in obtaining more detailed insights into the localisation and function of these proteins within fruit. [ABSTRACT FROM AUTHOR]

Summary: We studied Arabidopsis HYPOXIA‐RESPONSIVE MODULATOR 1 (HRM1), which belongs to a group of core hypoxia‐responsive genes that are conserved among plant species across great evolutionary distance.The hrm1 mutants had lower survival rates and showed more damage than the wild‐type (WT) plants under hypoxic stress. Promoter analyses showed that HRM1 is regulated by EIN3 and RAP2.2 during hypoxia. Fluorescence tracing and immunogold labeling assays showed that HRM1 protein was enriched in mitochondria.Co‐immunoprecipitation coupled with mass spectrometry and bimolecular fluorescence complementation assays showed that HRM1 associates with the complex‐I in mitochondria. Compared with the WT plants, metabolic activities related to the mitochondrial electron transport chain (mETC) were higher in hrm1 mutants during hypoxia. Loss of HRM1 caused de‐repression of mETC complex I, II, and IV activities and higher basal and maximum respiration rates under hypoxia.Our results showed that through association with complex‐I, HRM1 attenuates mETC activity and modulates the respiratory chain under low oxygen. Compared with the regulatory system in mammalian, adjustment of mitochondrial respiration to low oxygen helps plants decrease reactive oxygen species production and is also critical for the submergence survival. [ABSTRACT FROM AUTHOR]

Various intracellular degradation organelles, including autophagosomes, lysosomes, and endosomes, work in tandem to perform autophagy, which is crucial for cellular homeostasis. Altered autophagy contributes to the pathophysiology of various diseases, including cancers and metabolic diseases. This paper aims to describe an approach to reproducibly identify and distinguish subcellular structures involved in macroautophagy. Methods are provided that help avoid common pitfalls. How to distinguish between lysosomes, lipid droplets, autolysosomes, autophagosomes, and inclusion bodies are also discussed. These methods use transmission electron microscopy (TEM), which is able to generate nanometer‐scale micrographs of cellular degradation components in a fixed sample. Serial block face‐scanning electron microscopy is also used to visualize the 3D morphology of degradation machinery using the Amira software. In addition to TEM and 3D reconstruction, other imaging techniques are discussed, such as immunofluorescence and immunogold labeling, which can be used to classify cellular organelles, reliably and accurately. Results show how these methods may be used to accurately quantify cellular degradation machinery under various conditions, such as treatment with the endoplasmic reticulum stressor thapsigargin or ablation of the dynamin‐related protein 1. [ABSTRACT FROM AUTHOR]

The metabotropic glutamate receptor 1 (mGlu1) plays a pivotal role in synaptic transmission and neuronal plasticity. Despite the fact that several interacting proteins involved in the mGlu1 subcellular trafficking and intracellular transduction mechanisms have been identified, the protein network associated with this receptor in specific brain areas remains largely unknown. To identify novel mGlu1-associated protein complexes in the mouse cerebellum, we used an unbiased tissue-specific proteomic approach, namely co-immunoprecipitation followed by liquid chromatography/tandem mass spectrometry analysis. Many well-known protein complexes as well as novel interactors were identified, including G-proteins, Homer, δ2 glutamate receptor, 14-3-3 proteins, and Na/K-ATPases. A novel putative interactor, KCTD12, was further investigated. Reverse co-immunoprecipitation with anti-KCTD12 antibodies revealed mGlu1 in wild-type but not in KCTD12-knock-out homogenates. Freeze-fracture replica immunogold labeling co-localization experiments showed that KCTD12 and mGlu1 are present in the same nanodomain in Purkinje cell spines, although at a distance that suggests that this interaction is mediated through interposed proteins. Consistently, mGlu1 could not be co-immunoprecipitated with KCTD12 from a recombinant mammalian cell line co-expressing the two proteins. The possibility that this interaction was mediated via GABAB receptors was excluded by showing that mGlu1 and KCTD12 still co-immunoprecipitated from GABAB receptor knock-out tissue. In conclusion, this study identifies tissue-specific mGlu1-associated protein clusters including KCTD12 at Purkinje cell synapses. [ABSTRACT FROM AUTHOR]

Programmed cell death (PCD) in lateral root caps (LRCs) is crucial for maintaining root cap functionality. Endoplasmic reticulum (ER) bodies play important roles in plant immunity and PCD. However, the distribution of ER bodies and their communication with vacuoles in the LRC remain elusive. In this study, we investigated the ultrastructure of LRC cells of wild-type and transgenic Arabidopsis lines using an auto-acquisition transmission electron microscope (TEM) system and high-pressure freezing. Gigapixel-scale high-resolution TEM imaging of the transverse and longitudinal sections of roots followed by three-dimensional imaging identified sausage-shaped structures budding from the ER. These were subsequently identified as ER bodies using GFPh transgenic lines expressing green fluorescent protein (GFP) fused with an ER retention signal (HDEL). Immunogold labeling using an anti-GFP antibody detected GFP signals in the ER bodies and vacuoles. The fusion of ER bodies with vacuoles in LRC cells was identified using correlative light and electron microscopy. Imaging of the root tips of a GFPh transgenic line with a PYK10 promoter revealed the localization of PYK10, a member of the β-glucosidase family with an ER retention signal, in the ER bodies in the inner layer along with a fusion of ER bodies with vacuoles in the middle layer and collapse of vacuoles in the outer layer of the LRC. These findings suggest that ER bodies in LRC directly transport β-glucosidases to the vacuoles, and that a subsequent vacuolar collapse triggered by an unknown mechanism releases protective substances to the growing root tip to protect it from the invaders. [ABSTRACT FROM AUTHOR]

The Notch signalling pathway is one of the most conserved and well-characterised pathways involved in cell fate decisions and the development of many diseases, including cancer. Among them, it is worth noting the Notch4 receptor and its clinical application, which may have prognostic value in patients with colon adenocarcinoma. The study was performed on 129 colon adenocarcinomas. Immunohistochemical and fluorescence expression of Notch4 was performed using the Notch4 antibody. The associations between the IHC expression of Notch4 and clinical parameters were analysed using the Chi2 test or Chi2Yatesa test. The Kaplan–Meier analysis and the log-rank test were used to verify the relationship between the intensity of Notch4 expression and the 5-year survival rate of patients. Intracellular localisation of Notch4 was detected by the use of the immunogold labelling method and TEM. 101 (78.29%) samples had strong Notch4 protein expression, and 28 (21.71%) samples were characterised by low expression. The high expression of Notch4 was clearly correlated with the histological grade of the tumour (p < 0.001), PCNA immunohistochemical expression (p < 0.001), depth of invasion (p < 0.001) and angioinvasion (p < 0.001). We can conclude that high expression of Notch4 is correlated with poor prognosis of colon adenocarcinoma patients (log-rank, p < 0.001). [ABSTRACT FROM AUTHOR]

Synaptic plasticity involves proper establishment and rearrangement of structural and functional microdomains. Yet, visualization of the underlying lipid cues proved challenging. Applying a combination of rapid cryofixation, membrane freeze-fracturing, immunogold labeling and electron microscopy, we visualize and quantitatively determine the changes and the distribution of phosphatidylinositol-4,5-bisphosphate (PIP2) in the plasma membrane of dendritic spines and subareas thereof at ultra-high resolution. These efforts unravel distinct phases of PIP2 signals during induction of long-term depression (LTD). During the first minutes PIP2 rapidly increases in a PIP5K-dependent manner forming nanoclusters. PTEN contributes to a second phase of PIP2 accumulation. The transiently increased PIP2 signals are restricted to upper and middle spine heads. Finally, PLC-dependent PIP2 degradation provides timely termination of PIP2 cues during LTD induction. Together, this work unravels the spatial and temporal cues set by PIP2 during different phases after LTD induction and dissects the molecular mechanisms underlying the observed PIP2 dynamics. Ultrahigh-resolution views of PIP2 in freeze-fractured plasma membranes of dendritic spines during induction of long-term depression reveal spatial and temporal cues set by PIP2 and reveal the molecular mechanisms behind the observed PIP2 dynamics [ABSTRACT FROM AUTHOR]

The two hallmarks of Alzheimer's disease (AD) are amyloid‐β (Aβ) plaques and neurofibrillary tangles marked by phosphorylated tau. Increasing evidence suggests that aggregating Aβ drives tau accumulation, a process that involves synaptic degeneration leading to cognitive impairment. Conversely, there is a realization that non‐fibrillar (oligomeric) forms of Aβ mediate toxicity in AD. Fibrillar (filamentous) aggregates of proteins across the spectrum of the primary and secondary tauopathies were the focus of recent structural studies with a filament structure‐based nosologic classification, but less emphasis was given to non‐filamentous co‐aggregates of insoluble proteins in the fractions derived from post‐mortem human brains. Here, we revisited sarkosyl‐soluble and ‐insoluble extracts to characterize tau and Aβ species by quantitative targeted mass spectrometric proteomics, biochemical assays, and electron microscopy. AD brain sarkosyl‐insoluble pellets were greatly enriched with Aβ42 at almost equimolar levels to N‐terminal truncated microtubule‐binding region (MTBR) isoforms of tau with multiple site‐specific post‐translational modifications (PTMs). MTBR R3 and R4 tau peptides were most abundant in the sarkosyl‐insoluble materials with a 10‐fold higher concentration than N‐terminal tau peptides. This indicates that the major proportion of the enriched tau was the aggregation‐prone N‐terminal and proline‐rich region (PRR) of truncated mixed 4R and 3R tau with more 4R than 3R isoforms. High concentration and occupancies of site‐specific phosphorylation pT181 (~22%) and pT217 (~16%) (key biomarkers of AD) along with other PTMs in the PRR and MTBR indicated a regional susceptibility of PTMs in aggregated tau. Immunogold labelling revealed that tau may exist in globular non‐filamentous form (N‐terminal intact tau) co‐localized with Aβ in the sarkosyl‐insoluble pellets along with tau filaments (N‐truncated MTBR tau). Our results suggest a model that Aβ and tau interact forming globular aggregates, from which filamentous tau and Aβ emerge. These characterizations contribute towards unravelling the sequence of events which lead to end‐stage AD changes. [ABSTRACT FROM AUTHOR]

Calcification starts with hydroxyapatite (HA) crystallization on cell membranous components, as with aortic valve interstitial cells (AVICs), wherein a cell-membrane-derived substance containing acidic phospholipids (PPM/PPLs) acts as major crystal nucleator. Since nucleic acid removal is recommended to prevent calcification in valve biosubstitutes derived from decellularized valve scaffolds, the involvement of ribosomal RNA (rRNA) and nuclear chromatin (NC) was here explored in three distinct contexts: (i) bovine AVIC pro-calcific cultures; (ii) porcine aortic valve leaflets that had undergone accelerated calcification after xenogeneic subdermal implantation; and (iii) human aortic valve leaflets affected by calcific stenosis. Ultrastructurally, shared AVIC degenerative patterns included (i) the melting of ribosomes with PPM/PPLs, and the same for apparently well-featured NC; (ii) selective precipitation of silver particles on all three components after adapted von Kossa reactions; and (iii) labelling by anti-rRNA immunogold particles. Shared features were also provided by parallel light microscopy. In conclusion, the present results indicate that rRNA and NC contribute to AVIC mineralization in vitro and in vivo, with their anionic charges enhancing the HA nucleation capacity exerted by PPM/PPL substrates, supporting the concept that nucleic acid removal is needed for valve pre-implantation treatments, besides better elucidating the modality of pro-calcific cell death. [ABSTRACT FROM AUTHOR]

Background: The brown planthopper (BPH) is one of the most destructive agricultural pests in Asia. RNA interference (RNAi)‐mediated pest management has been under development for years, and the selection of appropriate target genes is important for pest‐targeted RNAi. C‐type lectins (CTLs) are a class of genes that perform a variety of functions, such as the regulation of growth and development. Results: A CTL‐S protein named Nllet1, containing a single calcium ion (Ca2+)‐dependent carbohydrate‐binding domain (CRD) with a conserved triplet motif QPD was identified and functionally characterized in BPH. Expression profiles at both the transcriptional and translational levels show that Nllet1 accumulates during the serosal cuticle (SC) formation period. Immunofluorescence and immunogold labeling further demonstrated that Nllet1 is located in the serosal endocuticle (en‐SC). Maternal RNAi‐mediated silencing of Nllet1 disrupted the SC structure, accompanied by a loss of the outward barrier and 100% embryo mortality. Injection of 10 ng dsNllet1 or dsNllet1' per female adult BPH resulted in a total failure of egg hatching. Conclusion: Nllet1 is essential for SC formation and embryonic development in BPH, which helps us understand the important roles of CTL‐Ss. Additionally, BPH eggs show high sensitivity to the depletion of Nllet1. This study indicates that Nllet1 is a promising candidate gene that can be used to develop RNAi‐based control strategies at the BPH egg stage, and it can also be used as a target for developing novel ovicides. © 2022 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

The accumulation of misfolded proteins is a hallmark of aging and many neurodegenerative diseases, making it important to understand how the cellular machinery recognizes and processes such proteins. A key question in this respect is whether misfolded proteins are handled in a similar way regardless of their genetic origin. To approach this question, we compared how three different misfolded proteins, guk1-7, gus1-3, and pro3-1, are handled by the cell. We show that all three are nontoxic, even though highly overexpressed, highlighting their usefulness in analyzing the cellular response to misfolding in the absence of severe stress. We found significant differences between the aggregation and disaggregation behavior of the misfolded proteins. Specifically, gus1-3 formed some aggregates that did not efficiently recruit the protein disaggregase Hsp104 and did not colocalize with the other misfolded reporter proteins. Strikingly, while all three misfolded proteins generally coaggregated and colocalized to specific sites in the cell, disaggregation was notably different; the rate of aggregate clearance of pro3-1 was faster than that of the other misfolded proteins, and its clearance rate was not hindered when pro3-1 colocalized with a slowly resolved misfolded protein. Finally, we observed using super-resolution light microscopy as well as immunogold labeling EM in which both showed an even distribution of the different misfolded proteins within an inclusion, suggesting that misfolding characteristics and remodeling, rather than spatial compartmentalization, allows for differential clearance of these misfolding reporters residing in the same inclusion. Taken together, our results highlight how properties of misfolded proteins can significantly affect processing. [ABSTRACT FROM AUTHOR]

• The large extracellular matrix protein hemicentin-1 from mouse and zebrafish physically binds to the basement membrane protein nidogen-2. • Hemincentin-1 and laminins compete for binding to the G3 domain of nidogen-2, with binding between hemicentin-1 and nidogen-2 occurring kinetically faster, but binding between laminin and nidogen-2 being thermodynamically more stable. Hemicentin-1 and nidogen-2 display partially overlapping localization in. • different mouse and zebrafish tissues. • Loss-of-function studies in zebrafish embryos indicate that hemicentin-1, nidogen-2 and laminin-5 are all required for proper epidermal-dermal junction formation. • Synergistic enhancement studies via combined partial loss-of-function conditions in zebrafish embryos point to a tight genetic interaction between hemicentin-1 and nidogen-2 as well as between laminin-5 and nidogen-2, but not between hemicentin-1 and laminin-5, to promote epidermal-dermal junction formation, in line with the identified differential physical binding properties. • A model is proposed according to which hemicentin-1 plays a temporary, chaperone-like role during early steps of basement membrane formation, allowing molecular nidogen-2 networking before its G3-mediated binding to laminins. Hemicentins are large proteins of the extracellular matrix that belong to the fibulin family and play pivotal roles during development and homeostasis of a variety of invertebrate and vertebrate tissues. However, bona fide interaction partners of hemicentins have not been described as yet. Here, applying surface plasmon resonance spectroscopy and co-immunoprecipitation, we identify the basement membrane protein nidogen-2 (NID2) as a binding partner of mouse and zebrafish hemicentin-1 (HMCN1), in line with the formerly described essential role of mouse HMCN1 in basement membrane integrity. We show that HMCN1 binds to the same protein domain of NID2 (G2) as formerly shown for laminins, but with an approximately 3.5-fold lower affinity and in a competitive manner. Furthermore, immunofluorescence and immunogold labeling revealed that HMCN1/Hmcn1 is localized close to basement membranes and in partial overlap with NID2/Nid2a in different tissues of mouse and zebrafish. Genetic knockout and antisense-mediated knockdown studies in zebrafish further show that loss of Nid2a leads to similar defects in fin fold morphogenesis as the loss of Laminin-α5 (Lama5) or Hmcn1. Finally, combined partial loss-of-function studies indicated that nid2a genetically interacts with both hmcn1 and lama5. Together, these findings suggest that despite their mutually exclusive physical binding, hemicentins, nidogens, and laminins tightly cooperate and support each other during formation, maintenance, and function of basement membranes to confer tissue linkage. [ABSTRACT FROM AUTHOR]

Extracellular vesicle‐bound DNA (evDNA) is an understudied extracellular vesicle (EV) cargo, particularly in cancer‐unrelated research. Although evDNA has been detected in urine, little is known about its characteristics, localization, and biomarker potential for kidney pathologies. To address this, we enriched EVs from urine of well‐characterized kidney transplant recipients undergoing allograft biopsy, characterized their evDNA and its association to allograft injury. The SEC‐based method enriched pure EVs from urine of kidney transplant recipients, regardless of the allograft injury. Urinary evDNA represented up to 29.2 ± 8% (mean ± SD) of cell‐free DNA (cfDNA) and correlated with cfDNA in several characteristics but was less fragmented (P < 0.001). Importantly, using DNase treatment and immunogold labelling TEM, we demonstrated that evDNA was bound to the surface of urinary EVs. Normalised evDNA yield (P = 0.042) and evDNA copy number (P = 0.027) significantly differed between patients with normal histology, rejection injury and non‐rejection injury, the later groups having significantly larger uEVs (mean diameter, P = 0.045) and more DNA bound per uEV. ddDNA is detectable in uEV samples of kidney allograft recipients, but its quantity is highly variable. In a proof‐of‐principle study, several evDNA characteristics correlated with clinical and histological parameters (P = 0.040), supporting that the potential of evDNA as a biomarker for kidney allograft injury should be further investigated. [ABSTRACT FROM AUTHOR]

Background: Zika virus (ZIKV), a member of the Flaviviridae family, has caused massive outbreaks of infection in tropical areas over the last decade and has now begun spreading to temperate countries. Little is currently known about the specific host factors involved in the intracellular life cycle of ZIKV. Flaviviridae viruses interact closely with host-cell lipid metabolism and associated secretory pathways. Another Flaviviridae, hepatitis C virus, is highly dependent on apolipoprotein E (ApoE) for the completion of its infectious cycle. We therefore investigated whether ZIKV also interacted with this protein. Methods: ZIKV infections were performed on both liver and microglia derived cell lines in order to proceed to colocalization analysis and immunoprecipitation assays of ApoE and Zika envelope glycoprotein (Zika E). Transmission electron microscopy combined to immunogold labeling was also performed on the infected cells and related supernatant to study the association of ApoE and Zika E protein in the virus-induced membrane rearrangements and secreted particles, respectively. Finally, the potential of neutralization of anti-ApoE antibodies on ZIKV particles was studied. Result: We demonstrated an interaction between ApoE and the Zika E protein. This specific interaction was observed in virus-induced host-cell membrane rearrangements, but also on newly formed intracellular particles. The partial neutralizing effect of anti-ApoE antibody and the immunogold labeling of the two proteins on secreted virions indicates that this interaction is conserved during ZIKV intracellular trafficking and release. Conclusions: These data suggest that another member of the Flaviviridae also interacts with ApoE, indicating that this could be a common mechanism for the viruses from this family. [ABSTRACT FROM AUTHOR]

Simple Summary: The desert locust, Schistocerca gregaria, can form gigantic swarms of millions of individuals that devastate the vegetation of invaded landscapes. Locust food search, reproduction, and aggregation behaviors are triggered and controlled by complex olfactory signals. Insects detect odorants through different types of olfactory sensilla on the antenna that house olfactory sensory neurons and associated support cells, both of which express the proteins required for olfactory signaling. Among these proteins, two members of the CD36 lipid transporter/receptor family, named sensory neuron membrane proteins 1 and 2 (SNMP1 and SNMP2), are indicated to be of vital importance. Towards a better understanding of the role of the two SNMPs in the olfactory system of S. gregaria, we have analysed their antennal topography and subcellular localization using specific antibodies. The results indicate sensilla type- and cell type-specific distribution patterns of the SNMP proteins. SNMP1 was located in the receptive dendrites of subpopulations of olfactory sensory neurons as well as in the microvilli of associated support cells, suggesting a dual function of this protein, both in olfactory signal detection and in sensillum lymph maintenance, respectively. In contrast, SNMP2 was found solely in support cells and their microvilli membranes, suggesting a role limited to sensillum lymph recovery processes. Insect olfactory sensilla house olfactory sensory neurons (OSNs) and supports cells (SCs). The olfactory sensory processes require, besides the odorant receptors (ORs), insect-specific members of the CD36 family, named sensory neuron membrane proteins (SNMPs). While SNMP1 is considered to act as a coreceptor in the OR-mediated detection of pheromones, SNMP2 was found to be expressed in SCs; however, its function is unknown. For the desert locust, Schistocerca gregaria, we previously visualized mRNA for SNMP1 in OSNs and SNMP2 mRNA in cells associated with OSN clusters. Towards an understanding of their functional implication, it is imperative to explore the cellular and the subcellular localization the SNMP proteins. Therefore, we have generated polyclonal antibodies against SNMP1 and SNMP2 and used fluorescence immunohistochemistry (FIHC) to visualize the SNMP proteins. We found SNMP1 in the somata and respective dendrites of all OSNs in trichoid sensilla and in subsets of OSNs in basiconic sensilla. Notably, SNMP1 was also detected in SCs of these sensilla types. In contrast, SNMP2 protein was only visualized in SCs of basiconic and coeloconic sensilla, but not of trichoid sensilla. Exploring the subcellular localization by electron microscopy using anti-SNMP1-ab and anti-SNMP2-ab revealed an immunogold labelling of SC microvilli bordering the sensillum lymph. Together our findings suggest a dual role of SNMP1 in the antenna of S. gregaria, in some OSN subpopulations in odor detection as well as in functions of some SCs, whereas the role of SNMP2 is limited to the functions of support cells. [ABSTRACT FROM AUTHOR]

Simple Summary: Infections of white spot syndrome virus could be a lethal disease in shrimp culture systems. The 3D structures of the virus-associated structures were obtained through electron tomography. The relationships between the mature and immature particles were connected by the localizations of viral proteins. Our study provided new structural information on the virus through 3D, which extends the limited 2D knowledge of pathogen assembling in the white spot disease, further contributing to the development of a strategy for therapy or prevention. White spot syndrome virus (WSSV) has been reported to cause severe economic loss in the shrimp industry. With WSSV being a large virus still under investigation, the 3D structure of its assembly remains unclear. The current study was planned to clarify the 3D structures of WSSV infections in the cell nucleus of red swamp crayfish (Procambarus clarkii). The samples from various tissues were prepared on the seventh day post-infection. The serial sections of the intestinal tissue were obtained for electron tomography after the ultrastructural screening. After 3D reconstruction, the WSSV-associated structures were further visualized, and the expressions of viral proteins were confirmed with immuno-gold labeling. While the pairs of sheet-like structures with unknown functions were observed in the nucleus, the immature virions could be recognized by the core units of nucleocapsids on a piece of the envelope. The maturation of the particle could include the elongation of core units and the filling of empty nucleocapsids with electron-dense materials. Our observations may bring to light a possible order of WSSV maturation in the cell nucleus of the crayfish, while more investigations remain necessary to visualize the detailed viral–host interactions. [ABSTRACT FROM AUTHOR]

Bacterial decay in compression wood (CW) tracheids of waterlogged archaeological wood (WAW) was investigated using light microscopy, confocal laser scanning microscopy, transmission electron microscopy (TEM), and TEM immunogold labeling. Erosion bacteria were identified as the main degraders, and the extent of cell wall degradation differed depending on the severity of CW tracheids (mild vs. severe). Mild CW tracheids showed preferential decay in the inner S2 layer, with the locally degraded and/or fragmented S3 layer remaining. In contrast, severe CW tracheids revealed gradual degradation of the cell wall from the erosion progressing from exposed faces of the cell wall as decay progressed. The overall decay was more extensive in mild than in severe CW tracheids, and degradation of the highly lignified outer S2 layer (S2L) was only detected in mild CW tracheids. TEM immunogold labeling of 1,4-β-galactan, homogalacturonan (HG), heteroxylan, and heteromannan epitopes showed that there was no preferential degradation of pectins and hemicelluloses by action of diffusible enzymes and/or agents through the un-decayed cell wall during bacterial decay, in both mild and severe CW tracheids. Inter-CW tracheid bordered pit membranes showed higher decay resistance than CW tracheid walls. Degradation of HG and heteromannan epitopes was suppressed in pit membranes. [ABSTRACT FROM AUTHOR]

Abstract Immunogold labeling allows localization of proteins at the electron microscopy (EM) level of resolution, and quantification of signals. The present paper summarizes methodological issues and experiences gained from studies on the distribution of synaptic and other neuron-specific proteins in cell cultures and brain tissues via a pre-embedding method. An optimal protocol includes careful determination of a fixation condition for any particular antibody, a well-planned tissue processing procedure, and a strict evaluation of the credibility of the labeling. Here, tips and caveats on different steps of the sample preparation protocol are illustrated with examples. A good starting condition for EM-compatible fixation and permeabilization is 4% paraformaldehyde in PBS for 30 min at room temperature, followed by 30 min incubation with 0.1% saponin. An optimal condition can then be readjusted for each particular antibody. Each lot of the secondary antibody (conjugated with a 1.4 nm small gold particle) needs to be evaluated against known standards for labeling efficiency. Silver enhancement is required to make the small gold visible, and quality of the silver-enhanced signals can be affected by subsequent steps of osmium tetroxide treatment, uranyl acetate en bloc staining, and by detergent or ethanol used to clean the diamond knife for cutting thin sections. Most importantly, verification of signals requires understanding of the protein of interest in order to validate for correct localization of antibodies at expected epitopes on particular organelles, and quantification of signals needs to take into consideration the penetration gradient of reagents and clumping of secondary antibodies.

The drug efflux transporter P-glycoprotein (Pgp; ABCB1) plays an important role in drug absorption, disposition, and elimination. There is an ongoing debate whether, in addition to its localization at the plasma membrane, Pgp may also be expressed at the limiting membrane of endolysosomes (ELs), mediating active EL drug sequestration. If true, this would be an important mechanism to prevent drugs from reaching their intracellular targets. However, direct evidence demonstrating the functional expression of Pgp at the limiting membrane of ELs is lacking. This prompted us to perform a biochemical and ultrastructural study on the intracellular localization of Pgp in native rat liver. For this purpose, we established an improved subcellular fractionation procedure for the enrichment of ELs and employed different biochemical and ultrastructural methods to characterize the Pgp localization and function in the enriched EL fractions. Whereas the biochemical methods seemed to indicate that Pgp is functionally expressed at EL limiting membranes, transmission electron microscopy (TEM) indicated that this only occurs rarely, if at all. Instead, Pgp was found in the limiting membrane of early endosomes and intraluminal vesicles. In additional TEM experiments, using a Pgp-overexpressing brain microvessel endothelial cell line (hCMEC/D3-MDR1-EGFP), we examined whether Pgp is expressed at the limiting membrane of ELs when cells are exposed to high levels of the Pgp substrate doxorubicin. Pgp was seen in early endosomes but only rarely in endolysosomes, whereas Pgp immunogold labeling was detected in large autophagosomes. In summary, our data demonstrate the importance of combining biochemical and ultrastructural methods to investigate the relationship between Pgp localization and function. [ABSTRACT FROM AUTHOR]

Key message: The article concerns the association between callose synthase and cytoskeleton by biochemical and ultrastructural analyses in the pollen tube. Results confirmed this association and immunogold labeling showed a colocalization. Callose is a cell wall polysaccharide involved in fundamental biological processes, from plant development to the response to abiotic and biotic stress. To gain insight into the deposition pattern of callose, it is important to know how the enzyme callose synthase is regulated through the interaction with the vesicle-cytoskeletal system. Actin filaments likely determine the long-range distribution of callose synthase through transport vesicles but the spatial/biochemical relationships between callose synthase and microtubules are poorly understood, although experimental evidence supports the association between callose synthase and tubulin. In this manuscript, we further investigated the association between callose synthase and microtubules through biochemical and ultrastructural analyses in the pollen tube model system, where callose is an essential component of the cell wall. Results by native 2-D electrophoresis, isolation of callose synthase complex and far-western blot confirmed that callose synthase is associated with tubulin and can therefore interface with cortical microtubules. In contrast, actin and sucrose synthase were not permanently associated with callose synthase. Immunogold labeling showed colocalization between the enzyme and microtubules, occasionally mediated by vesicles. Overall, the data indicate that pollen tube callose synthase exerts its activity in cooperation with the microtubular cytoskeleton. [ABSTRACT FROM AUTHOR]

Nano secondary ion mass spectrometry (nanoSIMS) imaging is a rapidly growing field in biological sciences, which enables investigators to describe the chemical composition of cells and tissues with high resolution. One of the major challenges of nanoSIMS is to identify specific molecules or organelles, as these are not immediately recognizable in nanoSIMS and need to be revealed by SIMS-compatible probes. Few laboratories have generated such probes, and none are commercially available. To address this, we performed a systematic study of probes initially developed for electron microscopy. Relying on nanoscale SIMS, we found that antibodies coupled to 6 nm gold particles are surprisingly efficient in terms of labeling specificity while offering a reliable detection threshold. These tools enabled accurate visualization and sample analysis and were easily employed in correlating SIMS with other imaging approaches, such as fluorescence microscopy. We conclude that antibodies conjugated to moderately sized gold particles are promising tools for SIMS imaging. [ABSTRACT FROM AUTHOR]

Integral membrane proteins such as ion channels, transporters, and receptors shape cell activity and mediate cell-to-cell communication in the brain. The distribution, quantity, and clustering arrangement of those proteins contribute to the physiological properties of the cell; therefore, precise quantification of their state can be used to gain insight into cellular function. Using a highly sensitive immunoelectron microscopy technique called sodium dodecyl sulfate-digested freeze-fracture replica immunogold labeling (SDS-FRL), multiple membrane proteins can be tagged with different sizes of immunogold particles at once and visualized two-dimensionally. For quantification, gold particles in the images must be annotated, and then different mathematical and statistical methods must be applied to characterize the distribution states of proteins of interest. To perform such analyses in a user-friendly manner, we developed a program with a simple graphical user interface called Gold In-and-Out (GIO), which integrates several classical and novel analysis methods for immunogold labeled replicas into one self-contained package. GIO takes an input of particle coordinates, then allows users to implement analysis methods such as nearest neighbor distance (NND) and particle clustering. The program not only performs the selected analysis but also automatically compares the results of the real distribution to a random distribution of the same number of particles on the membrane region of interest. In addition to classical approaches for analyzing protein distribution, GIO includes new tools to analyze the positional bias of a target protein relative to a morphological landmark such as dendritic spines, and can also be applied for synaptic protein analysis. Gold Rippler provides a normalized metric of particle density that is resistant to differences in labeling efficiency among samples, while Gold Star is useful for quantifying distances between a protein and landmark. This package aims to help standardize analysis methods for subcellular and synaptic protein localization with a user-friendly interface while increasing the efficiency of these time-consuming analyses. [ABSTRACT FROM AUTHOR]

The parasitic protozoan Giardia intestinalis, the causative agent of giardiasis, presents a stable and elaborated cytoskeleton, which shapes and supports several intracellular structures, including the ventral disc, the median body, the funis, and four pairs of flagella. Giardia trophozoite is the motile form that inhabits the host small intestine and attaches to epithelial cells, leading to infection. The ventral disc is considered one important element of adhesion to the intestinal cells. It is adjacent to the plasma membrane in the ventral region of the cell and consists of a spiral layer of microtubules and microribbons. In this work, we studied the organization of the cytoskeleton in the ventral disc of G. intestinalis trophozoites using high-resolution scanning electron microscopy or helium ion microscopy in plasma membrane-extracted cells. Here, we show novel morphological details about the arrangement of cross-bridges in different regions of the ventral disc. Results showed that the disc is a non-uniformly organized structure that presents specific domains, such as the margin and the ventral groove region. High-resolution scanning electron microscopy allowed observation of the labeling pattern for several anti-tubulin antibodies using secondary gold particle-labeled antibodies. Labeling in the region of the emergence of the microtubules and supernumerary microtubules using an anti-acetylated tubulin antibody was observed. Ultrastructural analysis and immunogold labeling for gamma-tubulin suggest that disc microtubules originate from a region bounded by the bands of the banded collar and merge with microtubules formed at the perinuclear region. Actin-like filaments and microtubules of the disc are associated, showing an interconnection between elements of the cytoskeleton of the trophozoite. [ABSTRACT FROM AUTHOR]

Invasive aspergillosis and scedosporiosis are life-threatening fungal infections with similar clinical manifestations in immunocompromised patients. Contrarily, Scedosporium apiospermum is susceptible to some azole derivative but often resistant to amphotericin B. Histopathological examination alone cannot diagnose these two fungal species. Pathogenesis studies could contribute to explore candidate protein markers for new diagnosis and treatment methods leading to a decrease in mortality. In the present study, proteomics was conducted to identify significantly altered proteins in A549 cells infected with or without Aspergillus fumigatus and S. apiospermum as measured at initial invasion. Protein validation was performed with immunogold labelling alongside immunohistochemical techniques in infected A549 cells and lungs from murine models. Further, cytokine production was measured, using the Bio-Plex-Multiplex immunoassay. The cytoskeletal proteins HSPA9, PA2G4, VAT1, PSMA2, PEX1, PTGES3, KRT1, KRT9, CLIP1 and CLEC20A were mainly changed during A. fumigatus infection, while the immunologically activated proteins WNT7A, GAPDH and ANXA2 were principally altered during S. apiospermum infection. These proteins are involved in fungal internalisation and structural destruction leading to pulmonary disorders. Interleukin (IL)-21, IL-1α, IL-22, IL-2, IL-8, IL-12, IL-17A, interferon-γ and tumour necrosis factor-α were upregulated in both aspergillosis and scedosporiosis, although more predominately in the latter, in accordance with chitin synthase-1 and matrix metalloproteinase levels. Our results demonstrated that during invasion, A. fumigatus primarily altered host cellular integrity, whereas S. apiospermum chiefly induced and extensively modulated host immune responses. [ABSTRACT FROM AUTHOR]

Small-conductance calcium-activated potassium (SK) channels are crucial for learning and memory. However, many aspects of their spatial organization in neurons are still unknown. In this study, we have taken a novel approach to answering these questions combining a pre-embedding immunogold labeling with an automated dual-beam electron microscope that integrates focused ion beam milling and scanning electron microscopy (FIB/SEM) to gather 3D map ultrastructural and biomolecular information simultaneously. Using this new approach, we evaluated the number and variability in the density of extrasynaptic SK2 channels in 3D reconstructions from six dendritic segments of excitatory neurons and six inhibitory neurons present in the stratum radiatum of the CA1 region of the mouse. SK2 immunoparticles were observed throughout the surface of hippocampal neurons, either scattered or clustered, as well as at intracellular sites. Quantitative volumetric evaluations revealed that the extrasynaptic SK2 channel density in spines was seven times higher than in dendritic shafts and thirty-five times higher than in interneurons. Spines showed a heterogeneous population of SK2 expression, some spines having a high SK2 content, others having a low content and others lacking SK2 channels. SK2 immunonegative spines were significantly smaller than those immunopositive. These results show that SK2 channel density differs between excitatory and inhibitory neurons and demonstrates a large variability in the density of SK2 channels in spines. Furthermore, we demonstrated that SK2 expression was associated with excitatory synapses, but not with inhibitory synapses in CA1 pyramidal cells. Consequently, regulation of excitability and synaptic plasticity by SK2 channels is expected to be neuron class- and target-specific. These data show that immunogold FIB/SEM represent a new powerful EM tool to correlate structure and function of ion channels with nanoscale resolution. [ABSTRACT FROM AUTHOR]

Abstract Calcium/calmodulin-dependent protein kinase II (CaMKII), an abundant protein in neurons, is involved in synaptic plasticity and learning. CaMKII associates with multiple proteins located at or near the postsynaptic density (PSD), and CaMKII is known to translocate from cytoplasm to PSD under excitatory conditions. The present study examined the laminar distribution of CaMKII at the PSD by immunogold labeling in dissociated hippocampal cultures under low calcium (EGTA or APV), control, and stimulated (depolarization with high K+ or NMDA) conditions. The patterns of CaMKII distribution are classified with particular reference to the two layers of the PSD: (1) the PSD core, a layer within ~ 30–40 nm to the postsynaptic membrane, and (2) the PSD pallium, a deeper layer beyond the PSD core, ~ 100–120 nm from the postsynaptic membrane. Under low calcium conditions, a subpopulation (40%) of synapses stood out with no CaMKII labeling at the PSD, indicating that localization of CaMKII at the PSD is sensitive to calcium levels. Under control conditions, the majority (~ 60–70%) of synapses had label for CaMKII dispersed evenly in the spine, including the PSD and the nearby cytoplasm. Upon stimulation, the majority (60–75%) of synapses had label for CaMKII concentrated at the PSD, delineating the PSD pallium from the cytoplasm. Median distance of label for CaMKII to postsynaptic membrane was higher in low calcium samples (68–77 nm), than in control (59–63 nm) and stimulated samples (49–53 nm). Thus, upon stimulation, not only more CaMKII translocated to the PSD, but they also were closer to the postsynaptic membrane. Additionally, there were two relatively infrequent labeling patterns that may represent intermediate stages of CaMKII distribution between basal and stimulated conditions: (1) one type showed label preferentially localized near the PSD core where CaMKII may be binding to NR2B, an NMDA receptor concentrated at the PSD core, and (2) the second type showed label preferentially in the PSD pallium, where CaMKII may be binding to Shank, a PSD scaffold protein located in the PSD pallium. Both of these distribution patterns may portray the initial stages of CaMKII translocation upon synaptic activation. In addition to binding to PSD proteins, the concentrated CaMKII labeling at the PSD under heightened excitatory conditions could also be formed by self-clustering of CaMKII molecules recruited to the PSD. Most importantly, these accumulated CaMKII molecules do not extend beyond the border of the PSD pallium, and are likely held in the pallium by binding to Shank under these conditions.

Alzheimer's disease (AD) is a devastating neurodegenerative disorder characterized by initial memory impairments that progress to dementia. In this sense, synaptic dysfunction and loss have been established as the pathological features that best correlate with the typical early cognitive decline in this disease. At the histopathological level, post mortem AD brains typically exhibit intraneuronal neurofibrillary tangles (NFTs) along with the accumulation of amyloid-beta (Abeta) peptides in the form of extracellular deposits. Specifically, the oligomeric soluble forms of Abeta are considered the most synaptotoxic species. In addition, neuritic plaques are Abeta deposits surrounded by activated microglia and astroglia cells together with abnormal swellings of neuronal processes named dystrophic neurites. These periplaque aberrant neurites are mostly presynaptic elements and represent the first pathological indicator of synaptic dysfunction. In terms of losing synaptic proteins, the hippocampus is one of the brain regions most affected in AD patients. In this work, we report an early decline in spatial memory, along with hippocampal synaptic changes, in an amyloidogenic APP/PS1 transgenic model. Quantitative electron microscopy revealed a spatial synaptotoxic pattern around neuritic plaques with significant loss of periplaque synaptic terminals, showing rising synapse loss close to the border, especially in larger plaques. Moreover, dystrophic presynapses were filled with autophagic vesicles in detriment of the presynaptic vesicular density, probably interfering with synaptic function at very early synaptopathological disease stages. Electron immunogold labeling showed that the periphery of amyloid plaques, and the associated dystrophic neurites, was enriched in Abeta oligomers supporting an extracellular location of the synaptotoxins. Finally, the incubation of primary neurons with soluble fractions derived from 6-month-old APP/PS1 hippocampus induced significant loss of synaptic proteins, but not neuronal death. Indeed, this preclinical transgenic model could serve to investigate therapies targeted at initial stages of synaptic dysfunction relevant to the prodromal and early AD. [ABSTRACT FROM AUTHOR]

There are approximately 38 million people globally living with Human immunodeficiency virus 1 (HIV-1) and given the tremendous success of combination antiretroviral therapy (cART) this has dramatically reduced mortality and morbidity with prevention benefits. However, HIV-1 persists during cART within the human body and re-appears upon cART interruption. This HIV-1 reservoir remains a barrier to cure with cellular sites of viral persistence not fully understood. In this study we provide evidence corroborating a recently published article in STM demonstrating the role of platelets as a novel cellular disseminator of HIV-1 particles in the setting of viral suppression. Using classical transmission electron microscopy with and without immunogold labeling, we visualize HIV-1 in both platelets and monocytes in cART suppressed HIV donors. Our study suggests that due to the close proximity of platelets and monocytes an alternative life cycle of HIV-1 cycling within monocytes and platelets without the need of active replication under cART occurs. Our findings are supported by the lack of detectable HIV-1 particles in platelets derived from HIV uninfected donors or the 'Berlin' patient suggesting that platelets may serve as an underappreciated hidden bearer for HIV-1 and should be considered in HIV remission studies and trials. [ABSTRACT FROM AUTHOR]

Amyloidosis comprises a spectrum of disorders characterized by the extracellular deposition of amorphous material, originating from an abnormal serum protein. The typing of amyloid into its many variants represents a pivotal step for a correct patient management. Several methods are currently used, including mass spectrometry, immunofluorescence, immunohistochemistry, and immunogold labeling. The aim of the present study was to investigate the accuracy and reliability of immunohistochemistry by means of a recently developed amyloid antibody panel applicable on fixed paraffin-embedded tissues in an automated platform. Patients with clinically and pathologically proven amyloidosis were divided into two cohorts: a pilot one, which included selected amyloidosis cases from 2009 to 2018, and a retrospective one (comprising all consecutive amyloidosis cases analyzed between November 2018 and May 2020). The above-referred panel of antibodies for amyloid classification was tested in all cases using an automated immunohistochemistry platform. When fresh-frozen material was available, immunofluorescence was also performed. Among 130 patients, a total of 143 samples from different organs was investigated. They corresponded to 51 patients from the pilot cohort and 79 ones from the retrospective cohort. In 82 cases (63%), fresh-frozen tissue was tested by immunofluorescence, serving to define amyloid subtype only in 30 of them (36.6%). On the contrary, the automated immunohistochemistry procedure using the above-referred new antibodies allowed to establish the amyloid type in all 130 cases (100%). These included: ALλ (n = 60, 46.2%), ATTR (n = 29, 22.3%), AA (n = 19, 14.6%), ALκ (n = 18, 13.8%), ALys (n = 2, 1.5%), and Aβ2M amyloidosis (n = 2, 1.5%). The present immunohistochemistry antibody panel represents a sensitive, reliable, fast, and low-cost method for amyloid typing. Since immunohistochemistry is available in most pathology laboratories, it may become the new gold standard for amyloidosis classification, either used alone or combined with mass spectrometry in selected cases. [ABSTRACT FROM AUTHOR]

SARS-CoV-2 infection can cause fatal inflammatory lung pathology, including thrombosis and increased pulmonary vascular permeability leading to edema and hemorrhage. In addition to the lung, cytokine storm-induced inflammatory cascade also affects other organs. SARS-CoV-2 infection-related vascular inflammation is characterized by endotheliopathy in the lung and other organs. Whether SARS-CoV-2 causes endotheliopathy by directly infecting endothelial cells is not known and is the focus of the present study. We observed 1) the co-localization of SARS-CoV-2 with the endothelial cell marker CD31 in the lungs of SARS-CoV-2-infected mice expressing hACE2 in the lung by intranasal delivery of adenovirus 5-hACE2 (Ad5-hACE2 mice) and non-human primates at both the protein and RNA levels, and 2) SARS-CoV-2 proteins in endothelial cells by immunogold labeling and electron microscopic analysis. We also detected the co-localization of SARS-CoV-2 with CD31 in autopsied lung tissue obtained from patients who died from severe COVID-19. Comparative analysis of RNA sequencing data of the lungs of infected Ad5-hACE2 and Ad5-empty (control) mice revealed upregulated KRAS signaling pathway, a well-known pathway for cellular activation and dysfunction. Further, we showed that SARS-CoV-2 directly infects mature mouse aortic endothelial cells (AoECs) that were activated by performing an aortic sprouting assay prior to exposure to SARS-CoV-2. This was demonstrated by co-localization of SARS-CoV-2 and CD34 by immunostaining and detection of viral particles in electron microscopic studies. Moreover, the activated AoECs became positive for ACE-2 but not quiescent AoECs. Together, our results indicate that in addition to pneumocytes, SARS-CoV-2 also directly infects mature vascular endothelial cells in vivo and ex vivo , which may contribute to cardiovascular complications in SARS-CoV-2 infection, including multipleorgan failure. [ABSTRACT FROM AUTHOR]

• Recombinant FimH of V. parahaemolyticus (r Vp FimH) was purified to near homogeneity. • The r Vp FimH generated T-cell memory and mixed immune response in BALB/c mice. • Agglutination positive anti-r Vp FimH antiserum cross-reacted with Vp FimH. • Immunization with r Vp FimH conferred 100 % protection against bacterial challenge. Vibrio parahaemolyticus causes vibriosis in wide range of marine organisms, and is responsible for food borne illnesses in humans through consumption of contaminated uncooked/partially cooked seafood. Continued and widespread antibiotics usage to increase the productivity has led to antibiotics resistance development. This has necessitated the need to develop alternative methods to control its infection. Use of safe and effective vaccines against the virulence factors not only protects from infection, it also minimizes antibiotic usage. The colonization of V. parahaemolyticus in the host and disease development requires several adhesins present on the cell surface, and thereby make them attractive vaccine candidates. V. parahaemolyticus produces extracellular type 1 fimbriae that have been shown to play a role in adhesion, biofilm formation and virulence. FimH is one of the minor components of the type 1 fimbriae occurring on its very tip. Being present on the cell surface, it is highly immunogenic, and can be targeted as a potential vaccine candidate. The present study describes the immunogenic and vaccine potential of recombinant V. parahaemolyticus FimH (r Vp FimH) expressed in E. coli. Immunization of BALB/c mice with the r Vp FimH elicited a strong mixed immune response, T-cell memory (evidenced by antibody isotyping, cytokine profiling and T-cell proliferation assay), and agglutination positive antibodies. FACS analysis and immunogold labeling showed that the polyclonal anti-r Vp FimH antibodies were able to recognize the FimH on V. parahaemolyticus cells. In vivo challenge of the r Vp FimH-immunized mice with 2×LD 50 dose of live bacteria showed one hundred percent survival. Thus, our findings clearly demonstrate the potential of FimH as an effective vaccine candidate against V. parahaemolyticus. [ABSTRACT FROM AUTHOR]