Observation of Vacuoles in Arabidopsis Developing Pollen Grains with Whole-Cell Electron Tomography.

Arabidopsis thaliana developing pollen grains serve as an excellent system for studying vacuole dynamics. Here, we present a methodological approach that utilizes the serial tomography package in Etomo software from IMOD to generate whole-cell tomograms on A. thaliana developing pollens for visualizing vacuoles on the whole-cell scale. In order to understand the vacuole dynamics along with the pollen maturation, we also introduce a sampling method aimed at harvesting the pollen grains at various stages, marked by the vegetative nucleus or generative cell. The cryo-fixation/freeze-substitution technique can then be applied to preserve the fine structures of the pollen grains and facilitate detailed ultrastructure examination. Through this method, large-volume whole-cell electron tomograms regarding vacuolar morphologies and ultrastructural changes during pollen development and maturation have been obtained. Overall, the method presented here provides valuable insights into the dynamic nature of vacuoles in Arabidopsis developing pollen.

Whole-Cell Electron Tomography Analysis of Vacuole Biogenesis.

Vacuoles in plant cells are the most prominent organelles that harbor distinctive features, including lytic function, storage of proteins and sugars, balance of cell volume, and defense responses. Despite their dominant size and functional versatility, the nature and biogenesis of vacuoles in plants per se remain elusive and several models have been proposed. Recently, we used the whole-cell 3D electron tomography (ET) technique to study vacuole formation and distribution at nanometer resolution and demonstrated that small vacuoles are derived from multivesicular body maturation and fusion. Good sample preparation is a critical step to get high-quality electron tomography images. In this chapter, we provide detailed sample preparation methods for high-resolution ET in Arabidopsis thaliana root cells, including high-pressure freezing, subsequent freeze-substitution fixation, embedding, and serial sectioning.

High-resolution live cell imaging to define ultrastructural and dynamic features of the halotolerant yeast Debaryomyces hansenii.

Although some budding yeasts have proved tractable and intensely studied models, others are more recalcitrant. Debaryomyces hansenii, an important yeast species in food and biotechnological industries with curious physiological characteristics, has proved difficult to manipulate genetically and remains poorly defined. To remedy this, we have combined live cell fluorescent dyes with high-resolution imaging techniques to define the sub-cellular features of D. hansenii, such as the mitochondria, nuclei, vacuoles and the cell wall. Using these tools, we define biological processes like the cell cycle, organelle inheritance and various membrane trafficking pathways of D. hansenii for the first time. Beyond this, reagents designed to study Saccharomyces cerevisiae proteins were used to access proteomic information about D. hansenii. Finally, we optimised the use of label-free holotomography to image yeast, defining the physical parameters and visualising sub-cellular features like membranes and vacuoles. Not only does this work shed light on D. hansenii but this combinatorial approach serves as a template for how other cell biological systems, which are not amenable to standard genetic procedures, can be studied.

Accurate automated segmentation of autophagic bodies in yeast vacuoles using cellpose 2.0.

Segmenting autophagic bodies in yeast TEM images is a key technique for measuring changes in autophagosome size and number in order to better understand macroautophagy/autophagy. Manual segmentation of these images can be very time consuming, particularly because hundreds of images are needed for accurate measurements. Here we describe a validated Cellpose 2.0 model that can segment these images with accuracy comparable to that of human experts. This model can be used for fully automated segmentation, eliminating the need for manual body outlining, or for model-assisted segmentation, which allows human oversight but is still five times as fast as the current manual method. The model is specific to segmentation of autophagic bodies in yeast TEM images, but researchers working in other systems can use a similar process to generate their own Cellpose 2.0 models to attempt automated segmentations. Our model and instructions for its use are presented here for the autophagy community.Abbreviations: AB, autophagic body; AvP, average precision; GUI, graphical user interface; IoU, intersection over union; MVB, multivesicular body; ROI, region of interest; TEM, transmission electron microscopy; WT,wild type.

Tau filaments with the chronic traumatic encephalopathy fold in a case of vacuolar tauopathy with VCP mutation D395G.

Dominantly inherited mutation D395G in the gene encoding valosin-containing protein causes vacuolar tauopathy, a type of behavioural-variant frontotemporal dementia, with marked vacuolation and abundant filamentous tau inclusions made of all six brain isoforms. Here we report that tau inclusions were concentrated in layers II/III of the frontotemporal cortex in a case of vacuolar tauopathy. By electron cryomicroscopy, tau filaments had the chronic traumatic encephalopathy (CTE) fold. Tau inclusions of vacuolar tauopathy share this cortical location and the tau fold with CTE, subacute sclerosing panencephalitis and amyotrophic lateral sclerosis/parkinsonism-dementia complex, which are believed to be environmentally induced. Vacuolar tauopathy is the first inherited disease with the CTE tau fold.

Regional differences in the ultrastructure of mucosal macrophages in the rat large intestine.

We previously clarified the histological characteristics of macrophages in the rat small intestine using serial block-face scanning electron microscopy (SBF-SEM). However, the regional differences in the characteristics of macrophages throughout the large intestine remain unknown. Here, we performed a pilot study to explore the regional differences in the ultrastructure of mucosal macrophages in the large intestine by using SBF-SEM analysis. SBF-SEM analysis conducted on the luminal side of the cecum and descending colon revealed macrophages as amorphous cells possessing abundant lysosomes and vacuoles. Macrophages in the cecum exhibited a higher abundance of lysosomes and a lower abundance of vacuoles than those in the descending colon. Macrophages with many intraepithelial cellular processes were observed beneath the intestinal superficial epithelium in the descending colon. Moreover, macrophages in contact with nerve fibers were more prevalent in the cecum than in the descending colon, and a subset of them surrounded a nerve bundle only in the cecum. In conclusion, the present pilot study suggested that the quantity of some organelles (lysosomes and vacuoles) in macrophages differed between the cecum and the descending colon and that there were some region-specific subsets of macrophages like nerve-associated macrophages in the cecum.

Morphological analysis for two types of viral particles in vacuoles of SARS-CoV-2-infected cells.

In this study, we analyzed the morphological structure of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in human cells. We identified the two types of viral particles present within the vacuoles of infected cells. Using transmission electron microscopy, we observed that SARS-CoV-2 particles exhibited both low- and high-electron-density structures, which was further confirmed through three-dimensional reconstruction using electron tomography. The budding of these particles was exclusively observed within these vacuoles. Intriguingly, viral particles with low-electron-density structures were confined to vacuoles, whereas those with high-electron-density structures were found in vacuoles and on the cell membrane surface of infected cells. Notably, high-electron-density particles found within vacuoles exhibited the same morphology as those outside the infected cells. This observation suggests that the two types of viral particles identified in this study had different maturation status. Our findings provide valuable insights into the molecular details of SARS-CoV-2 particles, contributing to our understanding of the virus.

Intracellular sphingolipid sorting drives membrane phase separation in the yeast vacuole.

The yeast vacuole membrane can phase separate into ordered and disordered domains, a phenomenon that is required for micro-lipophagy under nutrient limitation. Despite its importance as a biophysical model and physiological significance, it is not yet resolved if specific lipidome changes drive vacuole phase separation. Here we report that the metabolism of sphingolipids (SLs) and their sorting into the vacuole membrane can control this process. We first developed a vacuole isolation method to identify lipidome changes during the onset of phase separation in early stationary stage cells. We found that early stationary stage vacuoles are defined by an increased abundance of putative raft components, including 40% higher ergosterol content and a nearly 3-fold enrichment in complex SLs (CSLs). These changes were not found in the corresponding whole cell lipidomes, indicating that lipid sorting is associated with domain formation. Several facets of SL composition-headgroup stoichiometry, longer chain lengths, and increased hydroxylations-were also markers of phase-separated vacuole lipidomes. To test SL function in vacuole phase separation, we carried out a systematic genetic dissection of their biosynthetic pathway. The abundance of CSLs controlled the extent of domain formation and associated micro-lipophagy processes, while their headgroup composition altered domain morphology. These results suggest that lipid trafficking can drive membrane phase separation in vivo and identify SLs as key mediators of this process in yeast.

Subsarcolemmal and cytoplasmic p62 positivity and rimmed vacuoles are distinctive for PLIN4-myopathy.

PLIN4-myopathy is a recently identified autosomal dominant muscular disorder caused by the coding 99 bp repeat expansion in PLIN4, presenting with distal or proximal weakness. Here, we report one family and one sporadic case of adult-onset PLIN4-associated limb-girdle weakness, whose diagnoses were achieved by a comprehensive genetic analysis workup. We provided additional evidence that the combination of subsarcolemmal/cytoplasmic ubiquitin/p62 positive deposits and rimmed vacuoles could serve as a strong indicator of PLIN4-myopathy. Moreover, we found novel myopathological features that were ultrastructural subsarcolemmal filamentous materials and membrane-bound granulofilamentous inclusions formed by the co-deposition of disrupted lipid droplets and p62 protein aggregates.

A bacterium that is not a microbe.

A new discovery challenges the prevailing view of the boundaries of bacterial cell size.

Mitochondria shed their outer membrane in response to infection-induced stress.

The outer mitochondrial membrane (OMM) is essential for cellular homeostasis. Yet little is known of the mechanisms that remodel it during natural stresses. We found that large "SPOTs" (structures positive for OMM) emerge during Toxoplasma gondii infection in mammalian cells. SPOTs mediated the depletion of the OMM proteins mitofusin 1 and 2, which restrict parasite growth. The formation of SPOTs depended on the parasite effector TgMAF1 and the host mitochondrial import receptor TOM70, which is required for optimal parasite proliferation. TOM70 enabled TgMAF1 to interact with the host OMM translocase SAM50. The ablation of SAM50 or the overexpression of an OMM-targeted protein promoted OMM remodeling independently of infection. Thus, Toxoplasma hijacks the formation of SPOTs, a cellular response to OMM stress, to promote its growth.

In-Depth Characterization of the Chikungunya Virus Replication Cycle.

The chikungunya virus has spread globally with a remarkably high attack rate. Infection causes arthralgic sequelae that can last for years. Nevertheless, there are no specific drugs or vaccines to contain the virus. Understanding the biology of the virus, such as its replication cycle, is a powerful tool to identify new drugs and comprehend virus-host interactions. Even though the chikungunya virus has been known for a long time (it was first described in 1952), many aspects of the replication cycle remain unclear. Furthermore, part of the cycle is based on observations of other alphaviruses. In this study, we used electron and scanning microscopy, as well as biological assays, to analyze and investigate the stages of the chikungunya virus replication cycle. Based on our data, we found infection cellular activities other than those usually described for the chikungunya virus replication cycle, i.e., we show particles enveloping intracellularly without budding in a membrane-delimited morphogenesis area, and we also observed virion release by membrane protrusions. Our work provides novel details regarding the biology of chikungunya virus and fills gaps in our knowledge of its replication cycle. These findings may contribute to a better understanding of virus-host interactions and support the development of antivirals. IMPORTANCE The understanding of virus biology is essential to containing virus dissemination, and exploring the virus replication cycle is a powerful tool to do this. There are many points in the biology of the chikungunya virus that need to be clarified, especially regarding its replication cycle. Our incomplete understanding of chikungunya virus infection stages is based on studies with other alphaviruses. We systematized the chikungunya virus replication cycle using microscopic imaging in the order of infection stages, as follows: entry, replication, protein synthesis, assembly/morphogenesis, and release. The imaging evidence shows novel points in the replication cycle of enveloping without budding, as well as particle release by cell membrane protrusion.

Label-free imaging and evaluation of characteristic properties of asthma-derived eosinophils using optical diffraction tomography.

Optical diffraction tomography (ODT), an emerging imaging technique that does not require fluorescent staining, can measure the three-dimensional distribution of the refractive index (RI) of organelles. In this study, we used ODT to characterize the pathological characteristics of human eosinophils derived from asthma patients presenting with eosinophilia. In addition to morphological information about organelles appearing in eosinophils, including the cytoplasm, nucleus, and vacuole, we succeeded in imaging specific granules and quantifying the RI values of the granules. Interestingly, ODT analysis showed that the RI (i.e., molecular density) of granules was significantly different between eosinophils from asthma patients and healthy individuals without eosinophilia, and that vacuoles were frequently found in the cells of asthma patients. Our results suggest that the physicochemical properties of eosinophils derived from patients with asthma can be quantitatively distinguished from those of healthy individuals. The method will provide insight into efficient evaluation of the characteristics of eosinophils at the organelle level for various diseases with eosinophilia.

Atg39 selectively captures inner nuclear membrane into lumenal vesicles for delivery to the autophagosome.

Mechanisms that turn over components of the nucleus and inner nuclear membrane (INM) remain to be fully defined. We explore how components of the INM are selected by a cytosolic autophagy apparatus through a transmembrane nuclear envelope-localized cargo adaptor, Atg39. A split-GFP reporter showed that Atg39 localizes to the outer nuclear membrane (ONM) and thus targets the INM across the nuclear envelope lumen. Consistent with this, sequence elements that confer both nuclear envelope localization and a membrane remodeling activity are mapped to the Atg39 lumenal domain; these lumenal motifs are required for the autophagy-mediated degradation of integral INM proteins. Interestingly, correlative light and electron microscopy shows that the overexpression of Atg39 leads to the expansion of the ONM and the enclosure of a network of INM-derived vesicles in the nuclear envelope lumen. Thus, we propose an outside-in model of nucleophagy where INM is delivered into vesicles in the nuclear envelope lumen, which can be targeted by the autophagosome.

Lactoferrin Protects against Methamphetamine Toxicity by Modulating Autophagy and Mitochondrial Status.

Lactoferrin (LF) was used at first as a vehicle to deliver non-soluble active compounds to the body, including the central nervous system (CNS). Nonetheless, it soon became evident that, apart from acting as a vehicle, LF itself owns active effects in the CNS. In the present study, the effects of LF are assessed both in baseline conditions, as well as to counteract methamphetamine (METH)-induced neurodegeneration by assessing cell viability, cell phenotype, mitochondrial status, and specific autophagy steps. In detail, cell integrity in baseline conditions and following METH administration was carried out by using H&E staining, Trypan blue, Fluoro Jade B, and WST-1. Western blot and immuno-fluorescence were used to assess the expression of the neurofilament marker ßIII-tubulin. Mitochondria were stained using Mito Tracker Red and Green and were further detailed and quantified by using transmission electron microscopy. Autophagy markers were analyzed through immuno-fluorescence and electron microscopy. LF counteracts METH-induced degeneration. In detail, LF significantly attenuates the amount of cell loss and mitochondrial alterations produced by METH; and mitigates the dissipation of autophagy-related proteins from the autophagy compartment, which is massively induced by METH. These findings indicate a protective role of LF in the molecular mechanisms of neurodegeneration.

Plasma Membrane Fluidity: An Environment Thermal Detector in Plants.

The lipid matrix in cell membranes is a dynamic, bidimensional array of amphipathic molecules exhibiting mesomorphism, which contributes to the membrane fluidity changes in response to temperature fluctuation. As sessile organisms, plants must rapidly and accurately respond to environmental thermal variations. However, mechanisms underlying temperature perception in plants are poorly understood. We studied the thermal plasticity of membrane fluidity using three fluorescent probes across a temperature range of -5 to 41 °C in isolated microsomal fraction (MF), vacuolar membrane (VM), and plasma membrane (PM) vesicles from Arabidopsis plants. Results showed that PM were highly fluid and exhibited more phase transitions and hysteresis, while VM and MF lacked such attributes. These findings suggest that PM is an important cell hub with the capacity to rapidly undergo fluidity modifications in response to small changes of temperatures in ranges spanning those experienced in natural habitats. PM fluidity behaves as an ideal temperature detector: it is always present, covers the whole cell, responds quickly and with sensitivity to temperature variations, functions with a cell free-energy cost, and it is physically connected with potential thermal signal transducers to elicit a cell response. It is an optimal alternative for temperature detection selected for the plant kingdom.

LPS-induced autophagy in human dental pulp cells is associated with p38.

Lipopolysaccharides (LPS), which are components of the cell wall of Gram-negative bacteria, are among the important factors that induce inflammation, including pulpitis. Autophagy in human dental pulp cells (hDPCs) acts as a protective mechanism that promotes cell survival under adverse conditions through different signaling pathways. In this study, we examined whether LPS increases autophagy in hDPCs and investigated the role of mitogen-activated protein kinases signaling and nuclear factor κB (NF-κB) in this process. We found that stimulation of hDPCs with 0.1 µg/mL LPS increased the protein and mRNA levels of autophagy markers, beclin1 and microtubule associated protein light chain 3II (LC3II). In addition, acridine orange staining and transmission electron microscopy demonstrated the induction of autophagy upon the treatment of LPS. Furthermore, LPS affected phosphorylation of p38, extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase (JNK), and the nuclear translocation of NF-κB. While p38 inhibitor suppressed the LPS-induced increase in protein levels of beclin1 and LC3-II. Our results suggest that LPS induced autophagy in hDPCs and affected the phosphorylation of p38, ERK, and JNK, as well as the nuclear translocation of NF-κB. Phosphorylation of p38 may be involved in LPS-induced autophagy in hDPCs.

Plant PIEZO homologs modulate vacuole morphology during tip growth.

In animals, PIEZOs are plasma membrane-localized cation channels involved in diverse mechanosensory processes. We investigated PIEZO function in tip-growing cells in the moss Physcomitrium patens and the flowering plant Arabidopsis thaliana PpPIEZO1 and PpPIEZO2 redundantly contribute to the normal growth, size, and cytoplasmic calcium oscillations of caulonemal cells. Both PpPIEZO1 and PpPIEZO2 localized to vacuolar membranes. Loss-of-function, gain-of-function, and overexpression mutants revealed that moss PIEZO homologs promote increased complexity of vacuolar membranes through tubulation, internalization, and/or fission. Arabidopsis PIEZO1 also localized to the tonoplast and is required for vacuole tubulation in the tips of pollen tubes. We propose that in plant cells the tonoplast has more freedom of movement than the plasma membrane, making it a more effective location for mechanosensory proteins.

Norepinephrine Protects against Methamphetamine Toxicity through ß2-Adrenergic Receptors Promoting LC3 Compartmentalization.

Norepinephrine (NE) neurons and extracellular NE exert some protective effects against a variety of insults, including methamphetamine (Meth)-induced cell damage. The intimate mechanism of protection remains difficult to be analyzed in vivo. In fact, this may occur directly on target neurons or as the indirect consequence of NE-induced alterations in the activity of trans-synaptic loops. Therefore, to elude neuronal networks, which may contribute to these effects in vivo, the present study investigates whether NE still protects when directly applied to Meth-treated PC12 cells. Meth was selected based on its detrimental effects along various specific brain areas. The study shows that NE directly protects in vitro against Meth-induced cell damage. The present study indicates that such an effect fully depends on the activation of plasma membrane ß2-adrenergic receptors (ARs). Evidence indicates that ß2-ARs activation restores autophagy, which is impaired by Meth administration. This occurs via restoration of the autophagy flux and, as assessed by ultrastructural morphometry, by preventing the dissipation of microtubule-associated protein 1 light chain 3 (LC3) from autophagy vacuoles to the cytosol, which is produced instead during Meth toxicity. These findings may have an impact in a variety of degenerative conditions characterized by NE deficiency along with autophagy impairment.

Testosterone synthesis in testicular Leydig cells after long-term exposure to a static electric field (SEF).

China's clean energy and resources are mainly located in the west and north while electric load center is concentrated in the middle and east. Thus, these resources and energy need to be converted into electrical energy in situ and transported to electric load center through ultra-high voltage direct current (UHVDC) transmissions. China has built 25,000 km UHVDC transmission lines of 800 kV and 1100 kV, near which the impact of electric field on health has attracted public attention. Previous studies showed that time-varying electromagnetic field exposure could disturb testosterone secretion. To study the effect of non-time-varying electric field caused by direct current transmission lines on testosterone synthesis, male ICR mice were continually (24 h/d) exposed to static electric field of 56.3 ± 1.4 kV/m. Results showed that on the 3rd day of exposure and on the 7th day after ceasing the exposure of 28 d, serum testosterone level and testicular oxidative stress indicators didn't change significantly. On the 28th day of exposure, serum testosterone levels, testicular glutathione peroxidase (GSH-Px) activity, the mRNA and protein levels of testicular StAR, PBR, CYP11A1 decreased significantly, and testicular malondialdehyde (MDA) content increased significantly. Meanwhile, electron-dense edges and vacuolation appeared in lipid droplets of Leydig cells. The gap between inner mitochondrial membrane (IMM) and outer mitochondrial membrane (OMM) enlarged, which would cause the swelling of mitochondria, the rupture and deficiency of mitochondrial membranes. Analysis showed that testicular oxidative stress could induce the damage of mitochondrial structure in Leydig cells, which would decrease the rate of cholesterol transport from cytoplasm to mitochondria. Since cholesterol is the necessary precursor of testosterone synthesis, testosterone synthesis was inhibited. The decrease of the mRNA and protein expression levels of StAR and PBR in testes could diminish the cholesterol transported from OMM to IMM. The decrease of the mRNA and protein expression levels of CYP11A1 could reduce the pregnenolone required in testosterone synthesis and inhibit testosterone synthesis consequently.