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Abstract Melatonin supplementation during in vitro maturation (IVM) improves porcine oocyte maturation and embryonic development by exerting antioxidative effects. Nevertheless, the mechanism by which melatonin prevents polyspermy after in vitro fertilization (IVF) remains unclear. Here, we examined the effects of melatonin on cytoplasmic maturation and the incidence of polyspermic penetration in porcine oocytes. No statistically significant difference was observed in the rate of first polar body formation between the groups (Control, Melatonin, Melatonin + Luzindole, and Melatonin + 4-P-PDOT). Interestingly, melatonin supplementation significantly improved the cytoplasmic maturation of porcine oocytes by enhancing the normal distribution of organelles (Golgi apparatus, endoplasmic reticulum and mitochondria) and upregulating organelle-related gene expressions (P

The endoplasmic reticulum (ER), the largest cellular compartment, harbours the machinery for the biogenesis of secretory proteins and lipids, calcium storage/mobilisation, and detoxification. It is shaped as layered membranous sheets interconnected with a network of tubules extending throughout the cell. Understanding the influence of the ER morphology dynamics on molecular transport may offer clues to rationalising neuro-pathologies caused by ER morphogen mutations. It remains unclear, however, how the ER facilitates its intra-luminal mobility and homogenises its content. It has been recently proposed that intra-luminal transport may be enabled by active contractions of ER tubules. To surmount the barriers to empirical studies of the minuscule spatial and temporal scales relevant to ER nanofluidics, here we exploit the principles of viscous fluid dynamics to generate a theoretical physical model emulating in silico the content motion in actively contracting nanoscopic tubular networks. The computational model reveals the luminal particle speeds, and their impact in facilitating active transport, of the active contractile behaviour of the different ER components along various time–space parameters. The results of the model indicate that reproducing transport with velocities similar to those reported experimentally in single-particle tracking would require unrealistically high values of tubule contraction site length and rate. Considering further nanofluidic scenarios, we show that width contractions of the ER’s flat domains (perinuclear sheets) generate local flows with only a short-range effect on luminal transport. Only contractions of peripheral sheets can reproduce experimental measurements, provided they are able to contract fast enough.

Radial glia (RG) are the main progenitor cell type in the developing cortex. These cells are highly polarized, with a long basal process spanning the entire thickness of the cortex and acting as a support for neuronal migration. The RG cell terminates by an endfoot that contacts the pial (basal) surface. A shorter apical process also terminates with an endfoot that faces the ventricle, with a primary cilium protruding in the cerebrospinal fluid. These cell domains have particular subcellular compositions that are critical for the correct functioning of RG. When altered, this can affect proper development of the cortex, ultimately leading to cortical malformations, associated with different pathological outcomes. In this review, we focus on the current knowledge concerning the cell biology of these bipolar stem cells and discuss the role of their polarity in health and disease.

Yongyi Xie,1,* Nanhui Wu,1,* Suwei Tang,1,* Zhiyu Zhou,1 Jiashe Chen,1 Jie Li,1 Fei Wu,1 Mingyuan Xu,1 Xiaoxiang Xu,1 Yeqiang Liu,1 Xin Ma1,2 1Shanghai Skin Disease Hospital, Institute of Dermatology, School of Medicine, Tongji University, Shanghai, People’s Republic of China; 2Department of Dermatology, Yueyang Hospital of Integrated Traditional Chinese and Western Medicine, Shanghai University of Traditional Chinese Medicine, Shanghai, People’s Republic of China*These authors contributed equally to this workCorrespondence: Yeqiang Liu; Xin Ma, Shanghai Skin Disease Hospital, Institute of Dermatology, Baode Road 1278 Street, Shanghai, 200433, People’s Republic of China, Email 1500156@tongji.edu.cn; Nicole.ma@me.comAbstract: The endoplasmic reticulum (ER) is the main site of protein synthesis, transport, and modification. Its abnormal status has now emerged as an established cause of many pathological processes, such as tumors and autoimmune diseases. Recent studies also demonstrated that the defective functions of ER may lead to pigmentary diseases. Vitiligo is a depigmenting ailment skin disorder whose pathogenesis is now found to be associated with ER. However, the detailed mechanism is still unclear. In this review, we try to link the association between ER with its inter- and intra-organellar interactions in vitiligo pathogenesis and focus on the function, mechanism, and clinical potential of ER with vitiligo. Expand ER is found in melanocytes of vitiligo and ER stress (ERS) might be a bridge between oxidative stress and innate and adaptive immunity. Meanwhile, the tight association between ER and mitochondria or melanosomes in organelles levels, as well as genes and cytokines, is the new paradigm in the pathogenesis of vitiligo. This undoubtedly adds a new aspect to the understanding of vitiligo, facilitating the design of targeted therapies for vitiligo.Keywords: vitiligo, endoplasmic reticulum, organelles, cellular interactions, oxidative stress

Mucopolysaccharidoses (MPS) are a group of diseases caused by mutations in genes encoding lysosomal enzymes that catalyze reactions of glycosaminoglycan (GAG) degradation. As a result, GAGs accumulate in lysosomes, impairing the proper functioning of entire cells and tissues. There are 14 types/subtypes of MPS, which are differentiated by the kind(s) of accumulated GAG(s) and the type of a non-functional lysosomal enzyme. Some of these types (severe forms of MPS types I and II, MPS III, and MPS VII) are characterized by extensive central nervous system disorders. The aim of this work was to identify, using transcriptomic methods, organelle-related genes whose expression levels are changed in neuronopathic types of MPS compared to healthy cells while remaining unchanged in non-neuronopathic types of MPS. The study was conducted with fibroblast lines derived from patients with neuronopathic and non-neuronopathic types of MPS and control (healthy) fibroblasts. Transcriptomic analysis has identified genes related to cellular organelles whose expression is altered. Then, using fluorescence and electron microscopy, we assessed the morphology of selected structures. Our analyses indicated that the genes whose expression is affected in neuronopathic MPS are often associated with the structures or functions of the cell nucleus, endoplasmic reticulum, or Golgi apparatus. Electron microscopic studies confirmed disruptions in the structures of these organelles. Special attention was paid to up-regulated genes, such as PDIA3 and MFGE8, and down-regulated genes, such as ARL6IP6, ABHD5, PDE4DIP, YIPF5, and CLDN11. Of particular interest is also the GM130 (GOLGA2) gene, which encodes golgin A2, which revealed an increased expression in neuronopathic MPS types. We propose to consider the levels of mRNAs of these genes as candidates for biomarkers of neurodegeneration in MPS. These genes may also become potential targets for therapies under development for neurological disorders associated with MPS and candidates for markers of the effectiveness of these therapies. Although fibroblasts rather than nerve cells were used in this study, it is worth noting that potential genetic markers characteristic solely of neurons would be impractical in testing patients, contrary to somatic cells that can be relatively easily obtained from assessed persons.

We investigated whether the elimination of two major enzymes responsible for triacylglycerol synthesis altered the structure and physical state of organelle membranes under mild heat shock conditions in the fission yeast, Schizosaccharomyces pombe. Our study revealed that key intracellular membrane structures, lipid droplets, vacuoles, the mitochondrial network, and the cortical endoplasmic reticulum were all affected in mutant fission yeast cells under mild heat shock but not under normal growth conditions. We also obtained direct evidence that triacylglycerol-deficient cells were less capable than wild-type cells of adjusting their membrane physical properties during thermal stress. The production of thermoprotective molecules, such as HSP16 and trehalose, was reduced in the mutant strain. These findings suggest that an intact system of triacylglycerol metabolism significantly contributes to membrane protection during heat stress.

Abstract The oocyte cumulus complex is mainly composed of an oocyte, the perivitelline space, zona pellucida and numerous granulosa cells. The cumulus granulosa cells (cGCs) provide a particularly important microenvironment for oocyte development, regulating its growth, maturation and meiosis. In this study, we studied the internal structures and cell-to-cell connections of mouse cGCs using focused ion beam scanning electron microscopy (FIB-SEM). We reconstructed three-dimensional models to display characteristic connections between the oocyte and cGCs, and to illustrate various main organelles in cGCs together with their interaction relationship. A special form of cilium identified in granulosa cell was never reported in previous literature.

Prohibitins are the central regulatory element of cellular homeostasis, especially by modulating the response at different levels: Nucleus, mitochondria and membranes. Their localization and interaction with various proteins, homons, transcription and nuclear factors, and mtDNA indicate the globality and complexity of their pleiotropic properties, which remain to be investigated. A more detailed deciphering of cellular metabolism in relation to prohibitins under normal conditions and in various metabolic diseases will allow us to understand the precise role of prohibitins in the signaling cascades of PI3K/Akt, Raf/MAP/ERK, STAT3, p53, and others and to fathom their mutual influence. A valuable research perspective is to investigate the role of prohibitins in the molecular and cellular interactions between the two major players in the pathogenesis of obesity—adipocytes and macrophages - that form the basis of the meta-inflammatory response. Investigating the subtle intercellular communication and molecular cascades triggered in these cells will allow us to propose new therapeutic strategies to eliminate persistent inflammation, taking into account novel molecular genetic approaches to activate/inactivate prohibitins.

Background: Carotid arterial atherosclerotic stenosis is a well-recognized pathological basis of ischemic stroke; however, its underlying molecular mechanisms remain unknown. Vascular smooth muscle cells (VSMCs) play fundamental roles in the initiation and progression of atherosclerosis. Organelle dynamics have been reported to affect atherosclerosis development. However, the association between organelle dynamics and various cellular stresses in atherosclerotic progression remain ambiguous. Methods: In this study, we conducted transcriptomics and bioinformatics analyses of stable and vulnerable carotid plaques. Primary VSMCs were isolated from carotid plaques and subjected to histopathological staining to determine their expression profiles. Endoplasmic reticulum (ER), mitochondria, and lysosome dynamics were observed in primary VSMCs and VSMC cell lines using live-cell imaging. Moreover, the mechanisms underlying disordered organelle dynamics were investigated using comprehensive biological approaches. Results: ER whorls, a representative structural change under ER stress, are prominent dynamic reconstructions of VSMCs between vulnerable and stable plaques, followed by fragmented mitochondria and enlarged lysosomes, suggesting mitochondrial stress and lysosomal defects, respectively. Induction of mitochondrial stress alleviated ER stress and autophagy in an eukaryotic translation initiation factor (eIF)-2α-dependent manner. Furthermore, the effects of eIF2α on ER stress, mitochondrial stress, and lysosomal defects were validated using clinical samples. Conclusion: Our results indicate that morphological and functional changes in VSMC organelles, especially in ER whorls, can be used as reliable biomarkers for atherosclerotic progression. Moreover, eIF2α plays an important role in integrating multiple stress-signaling pathways to determine the behavior and fate of VSMCs.

Organic fluorescent probes have attracted attention for bioimaging due to their advantages, including high sensitivity, biocompatibility, and multi-functionality. However, some limitations related to low signal-to-background ratio and false positive and negative signals make them difficult for in situ target detection. Recently, organelle targeting self-assembled fluorescent probes have been studied to meet this demand. Most of the dye molecules suffer from a quenching effect, but, specifically, some dyes like Pyrene, Near-Infrared (NIR), Nitrobenzoxadiazole (NBD), Fluorescein isothiocyanate (FITC), Naphthalenediimides (NDI), and Aggregation induced emission (AIE) show unique characteristics when they undergo self-assembly or aggregation. Therefore, in this review, we classified the molecules according to the dye type and provided an overview of the organelle-targeting strategy with an emphasis on the construction of fluorescent nanostructures within complex cellular environments. Results demonstrated that fluorescent probes effectively target and localized inside the organelles (mitochondria, lysosome, and golgi body) and undergo self-assembly to form various nanostructures that possess bio-functionality with long retention time, organelles membrane disruption/ROS generation/enzyme activity suppression ability, and enhanced photodynamic properties for anticancer treatment. Furthermore, we systematically discussed the challenges that remain to be resolved for the high performance of these probes and mentioned some of the future directions for the design of molecules.

Abstract Naphthalimide derivatives are attracting increasing attention as fluorescent probes due to their excellent modifiability and photophysical properties. Their large, rigid coplanar structures and conjugated systems enable easy generation of fluorescence. The imide units and bromonaphthalene functionality offer facile and concise modification pathways in preparing amenable probes for different metal cations, anions, small molecules, and organelles. Therefore, naphthalimide probes have great potential for application in research fields, such as analytical chemistry, cell biology, and clinical diagnosis.

Abstract Background Plant cells usually sustain a stable membrane potential due to influx and/or efflux of charged ions across plasma membrane. With the growth and development of plants, different tissues and cells undergo systemic or local programmed decline. Whether the membrane potential of plasma membrane could report senescence signal of plant tissues and cells is unclear. Results We applied a maneuverable transmembrane potential (TMP) detection method with patch-clamp setup to examine the senescence signal of leaf tissue cells in situ over the whole life cycle in Arabidopsis thaliana. The data showed that the TMPs of plant tissues and cells were varied at different growth stages, and the change of TMP was higher at the vegetative growth stage than at the reproductive stage of plant growth. The distinct change of TMP was detectable between the normal and the senescent tissues and cells in several plant species. Moreover, diverse abiotic stimuli, such as heat stress, hyperpolarized the TMP in a short time, followed by depolarized membrane potential with the senescence occurring. We further examined the TMP of plant chloroplasts, which also indicates the senescence signal in organelles. Conclusions This convenient TMP detection method can report the senescence signal of plant tissues and cells, and can also indicate the potential of plant tolerance to environmental stress.

Abstract Background Crops are under constant pressure due to global warming, which unfolds at a much faster pace than their ability to adapt through evolution. Agronomic traits are linked to cytoplasmic-nuclear genome interactions. It thus becomes important to understand the influence exerted by the organelles on gene expression under heat stress conditions and profit from the available genetic diversity. Maize (Zea mays) cytolines allow us to investigate how the gene expression changes under heat stress conditions in three different cytoplasmic environments, but each having the same nucleus. Analyzing retrograde signaling in such an experimental set-up has never been done before. Here, we quantified the response of three cytolines to heat stress as differentially expressed genes (DEGs), and studied gene expression patterns in the context of existing polymorphism in their organellar genomes. Results Our study unveils a plethora of new genes and GO terms that are differentially expressed or enriched, respectively, in response to heat stress. We report 19,600 DEGs as responding to heat stress (out of 30,331 analyzed), which significantly enrich 164 GO biological processes, 30 GO molecular functions, and 83 GO cell components. Our approach allowed for the discovery of a significant number of DEGs and GO terms that are not common in the three cytolines and could therefore be linked to retrograde signaling. Filtering for DEGs with a fold regulation > 2 (absolute values) that are exclusive to just one of the cytolines, we find a total of 391 up- and down-DEGs. Similarly, there are 19 GO terms with a fold enrichment > 2 that are cytoline-specific. Using GBS data we report contrasting differences in the number of DEGs and GO terms in each cytoline, which correlate with the genetic distances between the mitochondrial genomes (but not chloroplast) and the original nuclei of the cytolines, respectively. Conclusions The experimental design used here adds a new facet to the paradigm used to explain how gene expression changes in response to heat stress, capturing the influence exerted by different organelles upon one nucleus rather than investigating the response of several nuclei in their innate cytoplasmic environments.

The elimination of organelles to create the lens organelle-free zone (OFZ) is a macroautophagy/autophagy-dependent mechanism that occurs without causing cell death. This process is essential to the ability of the lens to focus incoming images on the retina, as the presence of these organelles would interfere with lens refraction of light and impair vision. Therefore, organelles are removed from differentiating lens fiber cells during development, their elimination beginning with the non-nuclear organelles in the central lens fiber cells, and expanding over time to the neighboring fiber cells in the lens cortex. The removal of fiber cell nuclei lags behind that of non-nuclear organelles, following the same spatiotemporal patterning. We find that removal of organelles to form the OFZ depends on the induction of autophagy through the inhibition of phosphoinositide 3-kinase (PI3K) signaling. Abbreviation OFZ– organelle-free zone

Migrasomes, a newly discovered organelle produced by migrating cells, are vesicles with membranous structure that form on the tips and intersections of retraction fibers (RFs). These structures are released into the extracellular environment or taken up by surrounding cells, mediating the release of cytoplasmic contents and intercellular communication. Retractosomes, a new type of small extracellular vesicles generated from broken-off RFs, are closely related to migrasomes in their physical location and origin, but were defined later. Despite their widespread existence in cells and biological organisms, little is known about the regulatory mechanisms underlying their formation and potential function. In this review, we provide an overview of the discovery, biogenesis, distribution, and functions of migrasomes and retractosomes, as well as their differences from exosomes.

Axons are processes of neurons, up to a metre long, that form the essential biological cables wiring nervous systems. They must survive, often far away from their cell bodies and up to a century in humans. This requires self-sufficient cell biology including structural proteins, organelles, and membrane trafficking, metabolic, signalling, translational, chaperone, and degradation machinery—all maintaining the homeostasis of energy, lipids, proteins, and signalling networks including reactive oxygen species and calcium. Axon maintenance also involves specialised cytoskeleton including the cortical actin-spectrin corset, and bundles of microtubules that provide the highways for motor-driven transport of components and organelles for virtually all the above-mentioned processes. Here, we aim to provide a conceptual overview of key aspects of axon biology and physiology, and the homeostatic networks they form. This homeostasis can be derailed, causing axonopathies through processes of ageing, trauma, poisoning, inflammation or genetic mutations. To illustrate which malfunctions of organelles or cell biological processes can lead to axonopathies, we focus on axonopathy-linked subcellular defects caused by genetic mutations. Based on these descriptions and backed up by our comprehensive data mining of genes linked to neural disorders, we describe the ‘dependency cycle of local axon homeostasis’ as an integrative model to explain why very different causes can trigger very similar axonopathies, providing new ideas that can drive the quest for strategies able to battle these devastating diseases.

The quality of oocytes determines their development competence, which will be rapidly lost if the oocytes are not fertilized at the proper time after ovulation. SIRT1, one of the sirtuin family members, has been proven to protect the quality of oocytes during postovulatory oocyte aging. However, evidence of the effect of SIRT1 on the activity of organelles including the mitochondria, the endoplasmic reticulum (ER), the Golgi apparatus, and the lysosomes in postovulatory aging oocyte is lacking. In this study, we investigated the distribution and function of organelles in postovulatory aged oocytes and discovered abnormalities. Luteolin, which is a natural flavonoid contained in vegetables and fruits, is an activator of SIRT1. When the oocytes were treated with luteolin, the abnormal distribution of mitochondria, ER, and Golgi complex were restored during postovulatory oocyte aging. The ER stress protein GRP78 and the lysosome protein LAMP1 increased, while the mitochondrial membrane potential and the Golgi complex protein GOLPH3 decreased in aged oocytes, and these were restored by luteolin treatment. EX-527, an inhibitor of SIRT1, disrupted the luteolin-mediated normal distribution and function of mitochondria, ER, Golgi apparatus, and lysosomes. In conclusion, we demonstrate that luteolin regulates the distribution and function of mitochondria, ER, Golgi apparatus, and lysosomes during postovulatory oocyte aging by activating SIRT1.

Abstract Background Recently, defects in mammalian oocytes maturation induced by environmental pollution results in the decreasing animal reproduction. Animal exposed to glyphosate is largely unavoidable because glyphosate is one of the most widely used herbicide worldwide due to its high-efficiency and broad-spectrum effects, which causes glyphosate an environmental contaminant found in soil, water and food. During the last few years, the growing and wider use of glyphosate has raised great concerns about its effects of reproductive toxicity. In this study, using porcine models, we investigated effects of glyphosate on organelle functions during oocyte meiosis. Results The results showed glyphosate exposure disrupted porcine oocyte maturation. Expression levels of cumulus expansion-related genes were interfered, further indicating the meiotic defects. The damaging effects were mediated by destruction of mitochondrial distribution and functions, which induced ROS accumulation and oxidative stress, also indicated by the decreased mRNA expression of related antioxidant enzyme genes. We also found an interference of endoplasmic reticulum (ER) distribution, disturbance of Ca2+ homeostasis, as well as fluctuation of ER stress, showing with the reduced ER stress-related mRNA or protein expression, which could indicate the dysfunction of ER for protein processing and signal transduction in glyphosate-exposed oocytes. Moreover, glyphosate exposure induced the disruption of lysosome function for autophagy, showing with the decrease of LAMP2 expression and autophagy-related genes mRNA expression. Additionally, our data showed the distribution of Golgi apparatus and the functions of ribosome were disturbed after glyphosate exposure, which might affect protein synthesis and transport. Conclusions Collectively, our study showed that exposed to glyphosate could affect animal reproduction by compromising the quality of oocytes through its wide toxic effects on organelle functions.

Cellular membranes are formed from different lipids in various amounts and proportions depending on the subcellular localization. The lipid composition of membranes is sensitive to changes in the cellular environment, and its alterations are linked to several diseases. Lipids not only form lipid-lipid interactions but also interact with other biomolecules, including proteins.Molecular dynamics (MD) simulations are a powerful tool to study the properties of cellular membranes and membrane-protein interactions on different timescales and resolutions. Over the last few years, software and hardware for biomolecular simulations have been optimized to routinely run long simulations of large and complex biological systems. On the other hand, high-throughput techniques based on lipidomics provide accurate estimates of the composition of cellular membranes at the level of subcellular compartments. Lipidomic data can be analyzed to design biologically relevant models of membranes for MD simulations. Similar applications easily result in a massive amount of simulation data where the bottleneck becomes the analysis of the data. In this context, we developed LipidDyn, a Python-based pipeline to streamline the analyses of MD simulations of membranes of different compositions. Once the simulations are collected, LipidDyn provides average properties and time series for several membrane properties such as area per lipid, thickness, order parameters, diffusion motions, lipid density, and lipid enrichment/depletion. The calculations exploit parallelization, and the pipeline includes graphical outputs in a publication-ready form. We applied LipidDyn to different case studies to illustrate its potential, including membranes from cellular compartments and transmembrane protein domains. LipidDyn is available free of charge under the GNU General Public License from https://github.com/ELELAB/LipidDyn.

Eukaryotes use various mechanisms to maintain cell division stability during sporogenesis, and in particular during meiosis to achieve production of haploid spores. In addition to establishing even chromosome segregation in meiosis I and II, it is crucial for meiotic cells to guarantee balanced partitioning of organelles to the daughter cells, to properly inherit cellular functions. In plants, cytological studies in model systems have yielded insights into the meiotic behaviour of different organelles, i.e., clearly revealing a distinct organization at different stages throughout meiosis indicating for an active regulatory mechanism determining their subcellular dynamics. However, how, and why plant meiocytes organize synchronicity of these elements and whether this is conserved across all plant genera is still not fully elucidated. It is generally accepted that the highly programmed intracellular behaviour of organelles during meiosis serves to guarantee balanced cytoplasmic inheritance. However, recent studies also indicate that it contributes to the regulation of key meiotic processes, like the organization of cell polarity and spindle orientation, thus exhibiting different functionalities than those characterized in mitotic cell division. In this review paper, we will outline the current knowledge on organelle dynamics in plant meiosis and discuss the putative strategies that the plant cell uses to mediate this programmed spatio-temporal organization in order to safeguard balanced separation of organelles. Particular attention is thereby given to putative molecular mechanisms that underlie this dynamic organelle organization taken into account existing variations in the meiotic cell division program across different plant types. Furthermore, we will elaborate on the structural role of organelles in plant meiosis and discuss on organelle-based cellular mechanisms that contribute to the organization and molecular coordination of key meiotic processes, including spindle positioning, chromosome segregation and cell division. Overall, this review summarizes all relevant insights on the dynamic behaviour and inheritance of organelles during plant meiosis, and discusses on their functional role in the structural and molecular regulation of meiotic cell division.

Mice and mouse platelets are major experimental models for hemostasis and thrombosis; however, important physiological data from this model has received little to no quantitative, 3D ultrastructural analysis. We used state-of-the-art, serial block imaging scanning electron microscopy (SBF-SEM, nominal Z-step size was 35 nm) to image resting platelets from C57BL/6 mice. α-Granules were identified morphologically and rendered in 3D space. The quantitative analysis revealed that mouse α-granules typically had a variable, elongated, rod shape, different from the round/ovoid shape of human α–granules. This variation in length was confirmed qualitatively by higher-resolution, focused ion beam (FIB) SEM at a nominal 5 nm Z-step size. The unexpected α-granule shape raises novel questions regarding α-granule biogenesis and dynamics. Does the variation arise at the level of the megakaryocyte and α-granule biogenesis or from differences in α-granule dynamics and organelle fusion/fission events within circulating platelets? Further quantitative analysis revealed that the two major organelles in circulating platelets, α-granules and mitochondria, displayed a stronger linear relationship between organelle number/volume and platelet size, i.e., a scaling in number and volume to platelet size, than found in human platelets suggestive of a tighter mechanistic regulation of their inclusion during platelet biogenesis. In conclusion, the overall spatial arrangement of organelles within mouse platelets was similar to that of resting human platelets, with mouse α-granules clustered closely together with little space for interdigitation of other organelles.

The article discloses a modern definition of the immune response. The phylogenesis of immunity constitutes a single system and is an integral part of the development of multicellular microorganisms. The emergence of multicellular organisms contributed to the formation of a separate community of cells that would be responsible for maintaining homeostasis of the internal environment of the body. The interaction of these cells was a prototype of modern immunity, and the immune response became a form of regulation of the constancy of the internal environment. The role of the immune response in the body is as follows: the search and elimination of foreign particles, both exogenously penetrating (pathogens of infectious diseases) and endogenously formed (cells infected with viruses, tumor cells). The role of the innate and acquired immune response is determined. Particular emphasis is placed on the pathogen recognizing receptors, on their various types. The concept of a pathogen of recognizing receptors is disclosed, their interaction and activation is shown for various types of pathogens. Modern perceptions of interleukins and transcription factors are characterized.

Abstract Background Polyamidoamine (PAMAM) dendrimer applications have extended from tumor cells to multidrug-resistant tumor cells. However, their transportation in multidrug-resistant tumor cells remains unclear. Herein, we investigated the exocytosis rule and mechanism of PAMAM dendrimers in multidrug-resistant tumor cells. Results Using a multidrug-resistant human breast cancer cell model (MCF-7/ADR), we performed systematic analyses of the cellular exocytosis dynamics, pathways and mechanisms of three PAMAM dendrimers with different surface charges: positively charged PAMAM-NH2, neutral PAMAM-OH and negatively charged PAMAM-COOH. The experimental data indicated that in MCF-7/ADR cells, the exocytosis rate was the highest for PAMAM-NH2 and the lowest for PAMAM-OH. Three intracellular transportation processes and P-glycoprotein (P-gp) participated in PAMAM-NH2 exocytosis in MCF-7/ADR cells. Two intracellular transportation processes, P-gp and multidrug resistance (MDR)-associated protein participated in PAMAM-COOH exocytosis. P-gp and MDR-associated protein participated in PAMAM-OH exocytosis. Intracellular transportation processes, rather than P-gp and MDR-associated protein, played major roles in PAMAM dendrimer exocytosis. PAMAM-NH2 could enter MCF-7/ADR cells by forming nanoscale membrane holes, but this portion of PAMAM-NH2 was eliminated by P-gp. Compared with PAMAM-OH and PAMAM-COOH, positively charged PAMAM-NH2 was preferentially attracted to the mitochondria and cell nuclei. Major vault protein (MVP) promoted exocytosis of PAMAM-NH2 from the nucleus but had no effect on the exocytosis of PAMAM-OH or PAMAM-COOH. Conclusions Positive charges on the surface of PAMAM dendrimer promote its exocytosis in MCF-7/ADR cells. Three intracellular transportation processes, attraction to the mitochondria and cell nucleus, as well as nuclear efflux generated by MVP led to the highest exocytosis observed for PAMAM-NH2. Our findings provide theoretical guidance to design a surface-charged tumor-targeting drug delivery system with highly efficient transfection in multidrug-resistant tumor cells. Especially, to provide more DNA to the nucleus and enhance DNA transfection efficiency in multidrug-resistant tumor cells using PAMAM-NH2, siRNA-MVP or an inhibitor should be codelivered to decrease MVP-mediated nuclear efflux. Graphical abstract

Ferroptosis is fairly different from other types of cell-death in biochemical processes, morphological changes and genetics as a special programmed cell-death. Here we summarize the current literatures on ferroptosis, including the cascade reaction of key material metabolism in the process, dysfunction of organelles, the relationship between different organelles and the way positive and negative key regulatory factors to affect ferroptosis in the epigenetic level. Based on material metabolism or epigenetic regulation, it is obvious that the regulatory network of ferroptosis is interrelated and complex.

Lysosomes are acidic, membrane-bound organelles that play essential roles in cellular quality control, metabolism, and signaling. The lysosomes of a cell are commonly depicted as vesicular organelles. Yet, lysosomes in fact show a high degree of ultrastructural heterogeneity. In some biological contexts, lysosome membranes naturally transform into tubular, non-vesicular morphologies. Though the purpose and regulation of tubular lysosomes has been historically understudied, emerging evidence suggests that tubular lysosomes may carry out unique activities, both degradative and non-degradative, that are critical to cell behavior, function, and viability. Here, we discuss recent advances in understanding the biological significance of tubular lysosomes in cellular physiology, and we highlight a growing number of examples that indicate the centrality of this special class of lysosomes to health and disease.

Aflatoxin B1 (AFB1) is a secondary metabolite produced by the fungus Aspergillus, which is ubiquitous in moldy grain products. Aflatoxin B1 has been reported to possess hepatotoxicity, renal toxicity, and reproductive toxicity. Previous studies have shown that AFB1 is toxic to mammalian oocytes. However, the potential toxicity of AFB1 on the organelles of mouse oocytes is unknown. In this study, we found that exposure to AFB1 significantly reduced mouse oocyte development capacity. Further analysis showed that the endoplasmic reticulum (ER) failed to accumulate around the spindle, and scattered in the cytoplasm under AFB1 exposure. Similar to the ER, the Golgi apparatus showed a uniform localization pattern following AFB1 treatment. In addition, we found that AFB1 exposure caused the condensation of lysosomes in the cytoplasm, presenting as a clustered or spindle peripheral-localization pattern, which indicated that protein modification, transport, and degradation were affected. Mitochondrial distribution was also altered by AFB1 treatment. In summary, our study showed that AFB1 exposure had toxic effects on the distribution of mouse oocyte organelles, which further led to a decline in oocyte quality.

The Gravity Force to which living beings are subjected on Earth rules the functionality of most biological processes in many tissues. It has been reported that a situation of Microgravity (such as that occurring in space) causes negative effects on living beings. Astronauts returning from space shuttle missions or from the International Space Station have been diagnosed with various health problems, such as bone demineralization, muscle atrophy, cardiovascular deconditioning, and vestibular and sensory imbalance, including impaired visual acuity, altered metabolic and nutritional status, and immune system dysregulation. Microgravity has profound effects also on reproductive functions. Female astronauts, in fact, suppress their cycles during space travels, and effects at the cellular level in the early embryo development and on female gamete maturation have also been observed. The opportunities to use space flights to study the effects of gravity variations are limited because of the high costs and lack of repeatability of the experiments. For these reasons, the use of microgravity simulators for studying, at the cellular level, the effects, such as those, obtained during/after a spatial trip, are developed to confirm that these models can be used in the study of body responses under conditions different from those found in a unitary Gravity environment (1 g). In view of this, this study aimed to investigate in vitro the effects of simulated microgravity on the ultrastructural features of human metaphase II oocytes using a Random Positioning Machine (RPM). We demonstrated for the first time, by Transmission Electron Microscopy analysis, that microgravity might compromise oocyte quality by affecting not only the localization of mitochondria and cortical granules due to a possible alteration of the cytoskeleton but also the function of mitochondria and endoplasmic reticulum since in RPM oocytes we observed a switch in the morphology of smooth endoplasmic reticulum (SER) and associated mitochondria from mitochondria-SER aggregates to mitochondria–vesicle complexes. We concluded that microgravity might negatively affect oocyte quality by interfering in vitro with the normal sequence of morphodynamic events essential for acquiring and maintaining a proper competence to fertilization in human oocytes.

As a vital parameter in living cells and tissues, the micro-environment is crucial for the living organisms. Significantly, organelles require proper micro-environment to achieve normal physiological processes, and the micro-environment in organelles can reflect the state of organelles in living cells. Moreover, some abnormal micro-environments in organelles are closely related to organelle dysfunction and disease development. So, visualizing and monitoring the variation of micro-environments in organelles is helpful for physiologists and pathologists to study the mechanisms of the relative diseases. Recently, a large variety of fluorescent probes was developed to study the micro-environments in living cells and tissues. However, the systematic and comprehensive reviews on the organelle micro-environment in living cells and tissues have rarely been published, which may hinder the research progress in the field of organic fluorescent probes. In this review, we will summarize the organic fluorescent probes for monitoring the microenvironment, such as viscosity, pH values, polarity, and temperature. Further, diverse organelles (mitochondria, lysosome, endoplasmic reticulum, cell membrane) about microenvironments will be displayed. In this process, the fluorescent probes about the “off-on” and ratiometric category (the diverse fluorescence emission) will be discussed. Moreover, the molecular designing, chemical synthesis, fluorescent mechanism, and the bio-applications of these organic fluorescent probes in cells and tissues will also be discussed. Significantly, the merits and defects of current microenvironment-sensitive probes are outlined and discussed, and the development tendency and challenges for this kind of probe are presented. In brief, this review mainly summarizes some typical examples and highlights the progress of organic fluorescent probes for monitoring micro-environments in living cells and tissues in recent research. We anticipate that this review will deepen the understanding of microenvironment in cells and tissues and facilitate the studies and development of physiology and pathology.

Organelles within eukaryotic cells are not isolated static compartments, instead being morphologically diverse and highly dynamic in order to respond to cellular needs and carry out their diverse and cooperative functions. One phenomenon exemplifying this plasticity, and increasingly gaining attention, is the extension and retraction of thin tubules from organelle membranes. While these protrusions have been observed in morphological studies for decades, their formation, properties and functions are only beginning to be understood. In this review, we provide an overview of what is known and still to be discovered about organelle membrane protrusions in mammalian cells, focusing on the best-characterised examples of these membrane extensions arising from peroxisomes (ubiquitous organelles involved in lipid metabolism and reactive oxygen species homeostasis) and mitochondria. We summarise the current knowledge on the diversity of peroxisomal/mitochondrial membrane extensions, as well as the molecular mechanisms by which they extend and retract, necessitating dynamic membrane remodelling, pulling forces and lipid flow. We also propose broad cellular functions for these membrane extensions in inter-organelle communication, organelle biogenesis, metabolism and protection, and finally present a mathematical model that suggests that extending protrusions is the most efficient way for an organelle to explore its surroundings.

Viscoelasticity and its alteration in time and space has turned out to act as a key element in fundamental biological processes in living systems, such as morphogenesis and motility. Based on experimental and theoretical findings it can be proposed that viscoelasticity of cells, spheroids and tissues seems to be a collective characteristic that demands macromolecular, intracellular component and intercellular interactions. A major challenge is to couple the alterations in the macroscopic structural or material characteristics of cells, spheroids and tissues, such as cell and tissue phase transitions, to the microscopic interferences of their elements. Therefore, the biophysical technologies need to be improved, advanced and connected to classical biological assays. In this review, the viscoelastic nature of cytoskeletal, extracellular and cellular networks is presented and discussed. Viscoelasticity is conceptualized as a major contributor to cell migration and invasion and it is discussed whether it can serve as a biomarker for the cells’ migratory capacity in several biological contexts. It can be hypothesized that the statistical mechanics of intra- and extracellular networks may be applied in the future as a powerful tool to explore quantitatively the biomechanical foundation of viscoelasticity over a broad range of time and length scales. Finally, the importance of the cellular viscoelasticity is illustrated in identifying and characterizing multiple disorders, such as cancer, tissue injuries, acute or chronic inflammations or fibrotic diseases.

Heat stress (HS) has been known to cause reproductive failure in animals, especially in summer. HS severely affects the developmental potential of oocytes and leads to low fertility rates. Previous studies have reported that HS compromises embryo development in bovine oocytes, and reduces ovarian development in mice, thereby impairing reproductive function in animals. However, the effect of high temperature (HT) on the organelles of porcine oocytes is unknown. In this study, we reported that exposure to HT for 24 h (41°C) significantly decreased meiotic maturation in porcine oocytes (p < 0.05). Further experiments on organelles found that HT induced mitochondrial dysfunction, increased abnormal mitochondrial distribution, and decreased mitochondrial membrane potential (MMP). We also found that HT induced abnormal endoplasmic reticulum (ER) distribution and higher expression of glucose regulatory protein 78 (GRP78), suggesting that HT exposure induces ER stress. Our results also indicated that exposure to HT induced abnormal distribution and dysfunction of the Golgi apparatus, which resulted from a decrease in the expression of the vesicle transporter, Ras-related protein Rab-11A (RAB11A). In addition, we found that HT exposure led to lysosomal damage by increasing the expression of lysosome-associated membrane protein 2 (LAMP2) and microtubule-associated protein 1A/1B-light chain 3 (LC3). In summary, our study revealed that HT exposure disrupts organelle dynamics, which further leads to the failure of meiotic maturation in porcine oocytes.

Protein fluorescence reporting systems are of crucial importance to in-depth life science research, providing systematic labeling tools for visualization of microscopic biological activities in vivo and revolutionizing basic research. Cotton somatic cell regeneration efficiency is low, causing difficulty in cotton transformation. It is conducive to screening transgenic somatic embryo using the fluorescence reporting system. However, available fluorescence labeling systems in cotton are currently limited. To optimize the fluorescence reporting system of cotton with an expanded range of available fluorescent proteins, we selected 11 fluorescent proteins covering red, green, yellow, and cyan fluorescence colors and expressed them in cotton. Besides mRuby2 and G3GFP, the other nine fluorescent proteins (mCherry, tdTomato, sfGFP, Clover, EYFP, YPet, mVenus, mCerulean, and ECFP) were stably and intensely expressed in transgenic callus and embryo, and inherited in different cotton organs derive from the screened embryo. In addition, transgenic cotton expressing tdTomato appears pink under white light, not only for callus and embryo tissues but also various organs of mature plants, providing a visual marker in the cotton genetic transformation process, accelerating the evaluation of transgenic events. Further, we constructed transgenic cotton expressing mCherry-labeled organelle markers in vivo that cover seven specific subcellular compartments: plasma membrane, endoplasmic reticulum, tonoplast, mitochondrion, plastid, Golgi apparatus, and peroxisome. We also provide a simple and highly efficient strategy to quickly determine the subcellular localization of uncharacterized proteins in cotton cells using organelle markers. Lastly, we built the first cotton stomatal fluorescence reporting system using stomata-specific expression promoters (ProKST1, ProGbSLSP, and ProGC1) to drive Clover expression. The optimized fluorescence labeling system for transgenic somatic embryo screening and functional gene labeling in this study offers the potential to accelerating somatic cell regeneration efficiency and the in vivo monitoring of diverse cellular processes in cotton.

Opioids are the most widely used analgesics and therefore have often been the focus of pharmacological research. Macrophages are the most plastic cells in the hematopoietic system. They show great functional diversity in various organism tissues and are an important consideration for the study of phagocytosis, cellular immunity, and molecular immunology. The expression of opioid receptors in macrophages indicates that opioid drugs act on macrophages and regulate their functions. This article reviewed the collection of research on effects of opioids on macrophage function. Studies show that opioids, both endogenous and exogenous, can affect the function of macrophages, effecting their proliferation, chemotaxis, transport, phagocytosis, expression of cytokines and chemokine receptors, synthesis and secretion of cytokines, polarization, and apoptosis. Many of these effects are closely associated with mitochondrial function and functions of other organelles in macrophages. Therefore, in depth research into effects of opioids on macrophage organelles may lead to some interesting new discoveries. In view of the important role of macrophages in HIV infection and tumor progression, this review also discusses effects of opioids on macrophages in these two pathological conditions.

Peroxisomes are ubiquitous, single membrane-bound organelles that play a crucial role in lipid metabolism and human health. While peroxisome number is maintained by the division of existing peroxisomes, nascent peroxisomes can be generated from the endoplasmic reticulum (ER) membrane in yeasts. During formation and proliferation, peroxisomes maintain membrane contacts with the ER. In addition to the ER, contacts between peroxisomes and other organelles such as lipid droplets, mitochondria, vacuole, and plasma membrane have been reported. These membrane contact sites (MCS) are dynamic and important for cellular function. This review focuses on the recent developments in peroxisome biogenesis and the functional importance of peroxisomal MCS in yeasts.

Cells prepare for fluctuations in nutrient availability by storing energy in the form of neutral lipids in organelles called Lipid Droplets (LDs). Upon starvation, fatty acids (FAs) released from LDs are trafficked to different cellular compartments to be utilized for membrane biogenesis or as a source of energy. Despite the biochemical pathways being known in detail, the spatio-temporal regulation of FA synthesis, storage, release, and breakdown is not completely understood. Recent studies suggest that FA trafficking and metabolism are facilitated by inter-organelle contact sites that form between LDs and other cellular compartments such as the Endoplasmic Reticulum (ER), mitochondria, peroxisomes, and lysosomes. LD-LD contact sites are also sites where FAs are transferred in a directional manner to support LD growth and expansion. As the storage site of neutral lipids, LDs play a central role in FA homeostasis. In this mini review, we highlight the role of LD contact sites with other organelles in FA trafficking, channeling, and metabolism and discuss the implications for these pathways on cellular lipid and energy homeostasis.

Abstract Ferroptosis, a novel form of regulated cell death, is different from other types of cell death in morphology, genetics and biochemistry. Increasing evidence indicates that ferroptosis has significant implications on cell death linked to cardiomyopathy, tumorigenesis, and cerebral hemorrhage to name a few. Here we summarize current literature on ferroptosis, including organelle dysfunction, signaling transduction pathways, metabolic reprogramming and epigenetic regulators in cancer progression. With regard to organelles, mitochondria-induced cysteine starvation, endoplasmic reticulum-related oxidative stress, lysosome dysfunction and golgi stress-related lipid peroxidation all contribute to induction of ferroptosis. Understanding the underlying mechanism in ferroptosis could provide insight into the treatment of various intractable diseases including cancers.

Abstract Background Giardia duodenalis causes giardiasis in humans, particularly in developing countries. Despite the availability of treatments, resistance to some of the commercial anti-Giardia drugs has been reported in addition to their harmful side effects. Therefore, novel treatments for giardiasis are required. In this study, we aimed to assess the in vitro activity of crude extracts of Ageratum conyzoides against G. duodenalis trophozoites. Methods Plants were classified into three groups based on their flower colors: white (W), purple (P), and white–purple (W–P). Plants were separately cut into leaf (L) and flower (F) parts. Changes in internal organelle morphology of trophozoites following exposure to crude extracts were assessed using transmission electron microscopy (TEM). In subsequent experiments, efficacy of the most active essential oils from crude extracts [half maximal inhibitory concentrations (IC50) ≤ 100 μg/mL] against G. duodenalis trophozoites was tested. In vitro anti-Giardia assays using essential oils were performed in the same way as those performed using crude extracts. Results LW–P and FP extracts showed high activity (IC50 ≤ 100 μg/mL) against G. duodenalis trophozoites, with IC50 ± SD values of 45.67 ± 0.51 and 96.00 ± 0.46 μg/mL, respectively. In subsequent experiments, IC50 ± SD values of LW–P and FP essential oils were 35.00 ± 0.50 and 89.33 ± 0.41 μg/mL, respectively. TEM revealed the degeneration of flagella and ventral discs of G. duodenalis trophozoites following exposure to crude extracts. Conclusion Crude LW–P and FP extracts of A. conyzoides showed the highest activity against G. duodenalis. Exposure to crude extract induced changes in the flagella and ventral discs of G. duodenalis trophozoites, which play important roles in attachment to the surface of mucosal cells. Our results suggest that the tested extracts warrant further research in terms of their efficacy and safety as giardiasis treatment.

Transcription factor EB (TFEB) is a member of the microphthalmia-associated transcription factor/transcription factor E (MiTF/TFE) family and critically involved in the maintenance of structural integrity and functional balance of multiple cells. In this review, we described the effects of post-transcriptional modifications, including phosphorylation, acetylation, SUMOylation, and ubiquitination, on the subcellular localization and activation of TFEB. The activated TFEB enters into the nucleus and induces the expressions of targeted genes. We then presented the role of TFEB in the biosynthesis of multiple organelles, completion of lysosome-autophagy pathway, metabolism regulation, immune, and inflammatory responses. This review compiles existing knowledge in the understanding of TFEB regulation and function, covering its essential role in response to cellular stress. We further elaborated the involvement of TFEB dysregulation in the pathophysiological process of various diseases, such as the catabolic hyperactivity in tumors, the accumulation of abnormal aggregates in neurodegenerative diseases, and the aberrant host responses in inflammatory diseases. In this review, multiple drugs have also been introduced, which enable regulating the translocation and activation of TFEB, showing beneficial effects in mitigating various disease models. Therefore, TFEB might serve as a potential therapeutic target for human diseases. The limitation of this review is that the mechanism of TFEB-related human diseases mainly focuses on its association with lysosome and autophagy, which needs deep description of other mechanism in diseases progression after getting more advanced information.

Podophyllotoxin (POD) is one of the most characterized lignans that is commonly found in podophyllum, and its preparations and derivatives are widely used in clinical treatment due to strong antitumor and antivirus activities. POD has been reported for its neurotoxicity, liver toxicity, and potential reproductive toxicity. In the present study, we investigated the effects of POD on the organelles of mouse oocytes during meiosis. Our results showed that exposure to POD significantly reduced the developmental competence of mouse oocytes. Further analysis revealed that the endoplasmic reticulum (ER) failed to accumulate to the spindle periphery, suggesting that POD exposure might affect protein synthesis during oocyte meiotic maturation. Similarly, abnormal Golgi apparatus distribution was found after POD exposure, which could be confirmed by the aberrant localization of Rab11a-related vesicles, indicating that POD induced vesicle-based protein transport disorder. We also found the aberrant accumulation of lysosomes in the cytoplasm of POD-exposed oocytes, which implied that POD might lead to aberrant protein degradation. Moreover, the perinuclear distribution of mitochondria was also significantly disturbed, indicating the mitochondrial dysfunction after POD exposure. In all, our study illustrated that exposure to POD might disrupt protein synthesis, transport, degradation, and ATP production by its effects on the distribution and functions of organelles during mouse oocyte meiotic maturation.

The diatom Phaeodactylum tricornutum is a marine unicellular microalga that exists under three main morphotypes: oval, fusiform, and triradiate. Previous works have demonstrated that the oval morphotype of P. tricornutum Pt3 strain presents specific metabolic features. Here, we compared the cellular organization of the main morphotypes of the diatom P. tricornutum Pt3 strain through transmission electron and advanced light microscopies. The three morphotypes share similarities including spectral characteristics of the plastid, the location of the nucleus, the organization of mitochondria around the plastid as well as the existence of both a F-actin cortex, and an intracellular network of F-actin. In contrast, compared to fusiform and triradiate cells, oval cells spontaneously release proteins more rapidly. In addition, comparison of whole transcriptomes of oval versus fusiform or triradiate cells revealed numerous differential expression of positive and negative regulators belonging to the complex dynamic secretory machinery. This study highlights the specificities occurring within the oval morphotype underlying that the oval cells secrete proteins more rapidly.

Bisphenol A (BPA) is one of the ubiquitous environmental endocrine disruptors (EEDs). Previous studies have shown that the reproduction toxicity of BPA could cause severe effects on the mammal oocytes and disturb the quality of mature oocytes. However, the toxic effects of BPA on the organelles of mouse oocytes have not been reported. In this study, to investigate whether BPA can be toxic to the organelles, we used different concentrations of BPA (50, 100, and 200 μM) to culture mouse oocytes in vitro. The results showed that 100 μM BPA exposure could significantly decrease the developmental capacity of oocytes. Then, we used the immunofluorescence staining, confocal microscopy, and western blotting to investigate the toxic effects of BPA on the organelles. The results revealed that mitochondrial dysfunction is manifested by abnormal distribution and decreased mitochondrial membrane potential. Moreover, the endoplasmic reticulum (ER) is abnormally distributed which is accompanied by ER stress showing increased expression of GRP78. For the Golgi apparatus, BPA-exposed dose not disorder the Golgi apparatus distribution but caused abnormal structure of Golgi apparatus, which is manifested by the decrease of GM130 protein expression. Moreover, we also found that BPA-exposed led to the damage of lysosome, which were shown by the increase of LAMP2 protein expression. Collectively, our findings demonstrated that the exposure of BPA could damage the normal function of the organelles, which may explain the reduced maturation quality of oocytes.

Organelles are important subsystems of cells. The damage and inactivation of organelles are closely related to the occurrence of diseases. Organelles’ functional activity can be observed by fluorescence molecular tools. Nowadays, a series of aggregation-induced emission (AIE) bioprobes with organelles-targeting ability have emerged, showing great potential in visualizing the interactions between probes and different organelles. Among them, AIE luminogen (AIEgen)-based peptide bioprobes have attracted more and more attention from researchers due to their good biocompatibility and photostability and abundant diversity. In this review, we summarize the progress of AIEgen-peptide bioprobes in targeting organelles, including the cell membrane, nucleus, mitochondria, lysosomes and endoplasmic reticulum, in recent years. The structural characteristics and biological applications of these bioprobes are discussed, and the development prospect of this field is forecasted. It is hoped that this review will provide guidance for the development of AIEgen-peptide bioprobes at the organelles level and provide a reference for related biomedical research.

Unraveling the fine structure of the brain is important to provide a better understanding of its normal and abnormal functioning. Application of high-resolution electron microscopic techniques gives us an unprecedented opportunity to discern details of the brain parenchyma at nanoscale resolution, although identifying different cell types and their unique features in two-dimensional, or three-dimensional images, remains a challenge even to experts in the field. This article provides insights into how to identify the different cell types in the central nervous system, based on nuclear and cytoplasmic features, amongst other unique characteristics. From the basic distinction between neurons and their supporting cells, the glia, to differences in their subcellular compartments, organelles and their interactions, ultrastructural analyses can provide unique insights into the changes in brain function during aging and disease conditions, such as stroke, neurodegeneration, infection and trauma. Brain parenchyma is composed of a dense mixture of neuronal and glial cell bodies, together with their intertwined processes. Intracellular components that vary between cells, and can become altered with aging or disease, relate to the cytoplasmic and nucleoplasmic density, nuclear heterochromatin pattern, mitochondria, endoplasmic reticulum and Golgi complex, lysosomes, neurosecretory vesicles, and cytoskeletal elements (actin, intermediate filaments, and microtubules). Applying immunolabeling techniques to visualize membrane-bound or intracellular proteins in neurons and glial cells gives an even better appreciation of the subtle differences unique to these cells across contexts of health and disease. Together, our observations reveal how simple ultrastructural features can be used to identify specific changes in cell types, their health status, and functional relationships in the brain.

ABSTRACT Mitogen-activated protein kinases (MAPKs) are a conserved family of protein kinases that regulate signal transduction, proliferation, and development throughout eukaryotes. The apicomplexan parasite Toxoplasma gondii expresses three MAPKs. Two of these, extracellular signal-regulated kinase 7 (ERK7) and MAPKL1, have been implicated in the regulation of conoid biogenesis and centrosome duplication, respectively. The third kinase, MAPK2, is specific to and conserved throughout the Alveolata, although its function is unknown. We used the auxin-inducible degron system to determine phenotypes associated with MAPK2 loss of function in Toxoplasma. We observed that parasites lacking MAPK2 failed to duplicate their centrosomes and therefore did not initiate daughter cell budding, which ultimately led to parasite death. MAPK2-deficient parasites initiated but did not complete DNA replication and arrested prior to mitosis. Surprisingly, the parasites continued to grow and replicate their Golgi apparatus, mitochondria, and apicoplasts. We found that the failure in centrosome duplication is distinct from the phenotype caused by the depletion of MAPKL1. As we did not observe MAPK2 localization at the centrosome at any point in the cell cycle, our data suggest that MAPK2 regulates a process at a distal site that is required for the completion of centrosome duplication and the initiation of parasite mitosis. IMPORTANCE Toxoplasma gondii is a ubiquitous intracellular protozoan parasite that can cause severe and fatal disease in immunocompromised patients and the developing fetus. Rapid parasite replication is critical for establishing a productive infection. Here, we demonstrate that a Toxoplasma protein kinase called MAPK2 is conserved throughout the Alveolata and essential for parasite replication. We found that parasites lacking MAPK2 protein were defective in the initiation of daughter cell budding and were rendered inviable. Specifically, T. gondii MAPK2 (TgMAPK2) appears to be required for centrosome replication at the basal end of the nucleus, and its loss causes arrest early in parasite division. MAPK2 is unique to the Alveolata and not found in metazoa and likely is a critical component of an essential parasite-specific signaling network.

Dinoflagellates are major components of phytoplankton that play critical roles in many microbial food webs, many of them being hosts of countless intracellular parasites. The phototrophic dinoflagellate Scrippsiella acuminata (Dinophyceae) can be infected by the microeukaryotic parasitoids Amoebophrya spp. (Syndiniales), some of which primarily target and digest the host nucleus. Early digestion of the nucleus at the beginning of the infection is expected to greatly impact the host metabolism, inducing the knockout of the organellar machineries that highly depend upon nuclear gene expression, such as the mitochondrial OXPHOS pathway and the plastid photosynthetic carbon fixation. However, previous studies have reported that chloroplasts remain functional in swimming host cells infected by Amoebophrya. We report here a multi-approach monitoring study of S. acuminata organelles over a complete infection cycle by nucleus-targeting Amoebophrya sp. strain A120. Our results show sustained and efficient photosystem II activity as a hallmark of functional chloroplast throughout the infection period despite the complete digestion of the host nucleus. We also report the importance played by light on parasite production, i.e., the amount of host biomass converted to parasite infective propagules. Using a differential gene expression analysis, we observed an apparent increase of all 3 mitochondrial and 9 out of the 11 plastidial genes involved in the electron transport chains (ETC) of the respiration pathways during the first stages of the infection. The longer resilience of organellar genes compared to those encoded by the nucleus suggests that both mitochondria and chloroplasts remain functional throughout most of the infection. This extended organelle functionality, along with higher parasite production under light conditions, suggests that host bioenergetic organelles likely benefit the parasite Amoebophrya sp. A120 and improve its fitness during the intracellular infective stage.

Abstract This study aimed to evaluate the humoral immune response in pigs immunized intranasally and intramuscularly with recombinant Toxoplasma gondii rROP2 protein in combination with the adjuvant Iscomatrix. Twelve mixed breed pigs divided into three groups (n=4) were used, G1 received recombinant ROP2 proteins (200 µg/dose) plus Iscomatrix, G2 received PBS plus Iscomatrix, and G3 as the control group. The intranasal (IN) and intramuscular (IM) routes were used. Animals were challenged orally with VEG strain oocysts and treated on day three after challenge. Fever, anorexia, and prostration were the clinical signs observed in all animals. All the G1 animals produced antibodies above the cut-off on the day of the challenge, while the G2 and G3 remained below the cut-off. Better partial protection against parasitemia and cyst tissue formation was observed in G1 than G3. The protection factors against tissue cyst formation were 40.0% and 6.1% for G1 and G2, respectively, compared to G3. In conclusion, there were not systemic antibody responses in pigs with IN immunization with rROP2+Iscomatrix; however, after IM immunization, those animals produced higher titers than animal controls. We associated these results with partial protection obtained against parasitemia and tissue cysts formation.

The design principles dictating the spatio-temporal organisation of eukaryotic cells, and in particular the mechanisms controlling the self-organisation and dynamics of membrane-bound organelles such as the Golgi apparatus, remain elusive. Although this organelle was discovered 120 years ago, such basic questions as whether vesicular transport through the Golgi occurs in an anterograde (from entry to exit) or retrograde fashion are still strongly debated. Here, we address these issues by studying a quantitative model of organelle dynamics that includes: de-novo compartment generation, inter-compartment vesicular exchange, and biochemical conversion of membrane components. We show that anterograde or retrograde vesicular transports are asymptotic behaviors of a much richer dynamical system. Indeed, the structure and composition of cellular compartments and the directionality of vesicular exchange are intimately linked. They are emergent properties that can be tuned by varying the relative rates of vesicle budding, fusion and biochemical conversion.