Osmoregulatory and immunological role of new canceled cells: Mitochondrial rich cells and its future perspective: A concise review.

Mitochondrial-rich cells (MRCs) are one of the most significant canceled type of epithelial cells. Morphologically these cells are totally different from other epithelial cells. These cells primarily implicated in sea-water and fresh-water adaptation, and acid-base regulation. However, in this review paper, we explored some of the most intriguing biological and immune-related functional developmental networks of MRCs. The main pinpoint, MRCs perform a dynamic osmoregulatory and immunological functional role in the gut and male reproductive system. The Na+/K+_ATPase (NKA) and Na+/K+/2Cl cotransporter (NKCC) are key acidifying proteins of MRCs for the ion-transporting function for intestinal homeostasis and maintenance of acidifying the luminal microenvironment in the male reproductive system. Further more importantly, MRCs play a novel immunological role through the exocrine secretion of nano-scale exosomes and multivesicular bodies (MVBs) pathway, which is very essential for sperm maturation, motility, acrosome reaction, and male sex hormones, and these an essential events to produce male gametes with optimal fertilizing ability. This effort is expected to promote the novel immunological role of MRCs, which might be essential for nano-scale exosome secretion.

Insights into the function of ESCRT and its role in enveloped virus infection.

The endosomal sorting complex required for transport (ESCRT) is an essential molecular machinery in eukaryotic cells that facilitates the invagination of endosomal membranes, leading to the formation of multivesicular bodies (MVBs). It participates in various cellular processes, including lipid bilayer remodeling, cytoplasmic separation, autophagy, membrane fission and re-modeling, plasma membrane repair, as well as the invasion, budding, and release of certain enveloped viruses. The ESCRT complex consists of five complexes, ESCRT-0 to ESCRT-III and VPS4, along with several accessory proteins. ESCRT-0 to ESCRT-II form soluble complexes that shuttle between the cytoplasm and membranes, mainly responsible for recruiting and transporting membrane proteins and viral particles, as well as recruiting ESCRT-III for membrane neck scission. ESCRT-III, a soluble monomer, directly participates in vesicle scission and release, while VPS4 hydrolyzes ATP to provide energy for ESCRT-III complex disassembly, enabling recycling. Studies have confirmed the hijacking of ESCRT complexes by enveloped viruses to facilitate their entry, replication, and budding. Recent research has focused on the interaction between various components of the ESCRT complex and different viruses. In this review, we discuss how different viruses hijack specific ESCRT regulatory proteins to impact the viral life cycle, aiming to explore commonalities in the interaction between viruses and the ESCRT system.

Enterovirus D68 capsid formation and stability requires acidic compartments.

IMPORTANCE: The respiratory picornavirus enterovirus D68 is a causative agent of acute flaccid myelitis, a childhood paralysis disease identified in the last decade. Poliovirus, another picornavirus associated with paralytic disease, is a fecal-oral virus that survives acidic environments when passing from host to host. Here, we follow up on our previous work showing a requirement for acidic intracellular compartments for maturation cleavage of poliovirus particles. Enterovirus D68 requires acidic vesicles for an earlier step, assembly, and maintenance of viral particles themselves. These data have strong implications for the use of acidification blocking treatments to combat enterovirus diseases.

Live-Imaging Detection of Multivesicular Body-Plasma Membrane Fusion and Exosome Release in Cultured Primary Neurons.

Exosomes represent a class of extracellular vesicles (EVs) derived from the endocytic pathway that is important for cell-cell communication and implicated in the spread of pathogenic protein aggregates associated with neurological diseases. Exosomes are released extracellularly when multivesicular bodies (also known as late endosomes) fuse with the plasma membrane (PM). An important breakthrough in exosome research is the ability to capture MVB-PM fusion and exosome release simultaneously in individual cells using live-imaging microscopy techniques. Specifically, researchers have created a construct fusing CD63, a tetraspanin enriched in exosomes, with the pH-sensitive reporter pHluorin whereby CD63-pHluorin fluorescence is quenched in the acidic MVB lumen and only fluoresces when released into the less acidic extracellular environment. Here, we describe a method using this CD63-pHluorin construct to visualize MVB-PM fusion/exosome secretion in primary neurons using total internal reflection fluorescence (TIRF) microscopy.

Cadmium-induced impairment of spermatozoa development by reducing exosomal-MVBs secretion: a novel pathway.

Cadmium is a heavy environmental pollutant that presents a high risk to male-fertility and targets the different cellular and steroidogenic supporting germ cells networks during spermatogenesis. However, the mechanism accounting for its toxicity in multivesicular bodies (MVBs) biogenesis, and exosomal secretion associated with spermatozoa remains obscure. In the current study, the light and electron microscopy revealed that, the Sertoli cells perform a dynamic role with secretion of well-developed early endosomes (Ee) and MVBs pathway associated with spermatozoa during spermatogenesis. In addition, some apical blebs containing nano-scale exosomes located on the cell surface and after fragmentation nano-scale exosomes were directly linked with spermatozoa in the luminal compartment of seminiferous tubules, indicating normal spermatogenesis. Controversially, the cadmium treated group showed limited and deformed spermatozoa with damaging acromion process and mid-peace, and the cytoplasmic vacuolization of spermatids. After cadmium treatment, there is very limited biogenesis of MVBs inside the cytoplasm of Sertoli cells, and no obvious secretions of nano-scale exosomes interacted with spermatozoa. Interestingly, the cadmium treated group demonstrated relatively higher formation of autophagosomes and autolysosome, and the autophagosomes were enveloped by MVBs that later formed the amphisome which degraded by lysosomes, indicating the hypo-spermatogenesis. Moreover, cadmium declined the exosomal protein cluster of differentiation (CD63) and increased the autophagy-related proteins microtubule-associated light chain (LC3), sequestosome 1 (P62) and lysosomal-associated membrane protein 2 (LAMP2) expression level were confirmed by Western blotting. These results provide rich information regarding how cadmium is capable of triggering impaired spermatozoa development during spermatogenesis by reduction of MVBs pathway through high activation of autophagic pathway. This study explores the toxicant effect of cadmium on nano-scale exosomes secretion interacting with spermatozoa.

Interplay between exosomes and autophagy machinery in pain management: State of the art.

Despite recent progress regarding inexpensive medical approaches, many individuals suffer from moderate to severe pain globally. The discovery and advent of exosomes, as biological nano-sized vesicles, has revolutionized current knowledge about underlying mechanisms associated with several pathological conditions. Indeed, these particles are touted as biological bio-shuttles with the potential to carry specific signaling biomolecules to cells in proximity and remote sites, maintaining cell-to-cell communication in a paracrine manner. A piece of evidence points to an intricate relationship between exosome biogenesis and autophagy signaling pathways at different molecular levels. A close collaboration of autophagic response with exosome release can affect the body's hemostasis and physiology of different cell types. This review is a preliminary attempt to highlight the possible interface of autophagy flux and exosome biogenesis on pain management with a special focus on neuropathic pain. It is thought that this review article will help us to understand the interplay of autophagic response and exosome biogenesis in the management of pain under pathological conditions. The application of therapies targeting autophagy pathway and exosome abscission can be an alternative strategy in the regulation of pain.

Targeting Membrane Trafficking as a Strategy for Cancer Treatment.

Membrane trafficking is emerging as an attractive therapeutic strategy for cancer. Recent reports have found a connection between Wnt signaling, receptor-mediated endocytosis, V-ATPase, lysosomal activity, and macropinocytosis through the canonical Wnt pathway. In macropinocytic cells, a massive internalization of the plasma membrane can lead to the loss of cell-surface cadherins, integrins, and other antigens that mediate cell-cell adhesion, favoring an invasive phenotype. V-ATPase is a key regulator in maintaining proper membrane trafficking, homeostasis, and the earliest developmental decisions in the Xenopus vertebrate development model system. Here, we review how the interference of membrane trafficking with membrane trafficking inhibitors might be clinically relevant in humans.

The Incredible Potential of Exosomes as Biomarkers in the Diagnosis of Colorectal Cancer.

Colorectal cancer (CRC) is common cancer that is one of the leading causes of cancerrelated deaths around the world. The creation of new biomarkers for this disease is an important public health strategy for lowering the disease's mortality rate. According to new research, exosomes may be important sources of biomarkers in CRC. Exosomes are nanometer-sized membrane vesicles (30-200 nm) secreted by normal and cancer cells that transport RNA and proteins between cells and are thought to help with intercellular communication. Exosomes have been linked to CRC initiation and progression, and some differentially expressed RNAs and proteins in exosomes have been identified as potential cancer detection candidates. As a result, studying the relationship between exosomes and CRC may aid in the development of new biomarkers for the disease. This article discusses the importance of exosomes as biomarkers in the diagnosis of CRC, as well as their use in the treatment of CRC metastasis, chemoresistance, and recrudescence. The benefits and drawbacks of using exosomes as tumour markers are also discussed.

Coupling size exclusion chromatography to ultracentrifugation improves detection of exosomal proteins from human plasma by LC-MS.

OBJECTIVES: Exosomes are small lipid bilayer vesicles that are defined by their endocytic origin and size range of 30-140 nm. They are constantly produced by different cell types, by both healthy and abnormal cells, and can be isolated from almost all body fluids.Little information exists in isolating exosomes from plasma due to the complexity of its content and the presence of contaminating plasma proteins. DESIGN AND METHODS: We carried-out liquid chromatography-mass spectrometry (LC-MS/MS) analyses of plasma-derived vesicles from 4 healthy donors obtained by 2 coupled methodologies: Ultracentrifugation (UC) coupled with size-exclusion chromatography (SEC) to isolate and subsequently enrich exosomes.We compared the proteins detected by UC alone and UC coupled with SEC. RESULTS: In the coupled UC + SEC methodology we found 52.25% more proteins enriched in exosomes as CD9, Annexins, YWHAZ (14-3-3 family) and others, than by using UC alone. There is also a reduction of 98.8% of contaminating plasma proteins by coupling UC and SEC in comparison to using UC alone. CONCLUSIONS: We conclude that exosomes can be successfully isolated from plasma using a very simple combination of standard methods, which could largely improve the proteomics profiling of plasma exosomes.

A Diverse Membrane Interaction Network for Plant Multivesicular Bodies: Roles in Proteins Vacuolar Delivery and Unconventional Secretion.

Vesicle trafficking between the membrane-bound organelles in plant cells plays crucial roles in the precise transportation of various materials, and thus supports cell proliferation and cellular polarization. Conventionally, plant prevacuolar compartments (PVCs), identified as multivesicular bodies (MVBs), play important roles in both the secretory pathway as intermediate compartments and the endocytic pathway as late endosomes. In recent years, the PVC/MVBs have been proposed to play important roles in both protein vacuolar delivery and unconventional secretion, but several important questions on the new regulators and environmental cues that coordinate the PVC/MVB-organelle membrane interactions and their biological significances remain. In this review, we first summarize the identity and nature of the plant PVC/MVBs, and then we present an update on our current understanding on the interaction of PVC/MVBs with other organelles in the plant endomembrane system with focus on the vacuole, autophagosome, and plasma membrane (PM) in plant development and stress responses. Finally, we raise some open questions and present future perspectives in the study of PVC/MVB-organelle interactions and associated biological functions.

Vacuole Biogenesis in Plants: How Many Vacuoles, How Many Models?

Vacuoles are the largest membrane-bounded organelles and have essential roles in plant growth and development, but several important questions on the biogenesis and dynamics of lytic vacuoles (LVs) remain. Here, we summarize and discuss recent research and models of vacuole formation, and propose, with testable hypotheses, that besides inherited vacuoles, plant cells can also synthesize LVs de novo from multiple organelles and routes in response to growth and development or external factors. Therefore, LVs may be further classified into different subgroups and/or populations with different pH, cargos, and functions, among which multivesicular body (MVB)-derived small vacuoles are the main source for central vacuole formation in arabidopsis root cortical cells.

Isolation and characterization of exosomes released from mosquito cells infected with dengue virus.

Exosomes are endocytic origin small-membrane vesicles secreted to the extracellular space by most cell types. Exosomes released from virus infected-cells can mediate the cell-to-cell communication to promote or modulate viral transmission. Dengue virus (DENV) is an arbovirus transmitted by Aedes mosquitoes bite to humans. Interestingly, the role of exosomes during the DENV infection in mammalian cells has already been described. However, little is known about exosomes derived from infected mosquito cells. Thus, the exosomes released from DENV-infected C6/36 cells were isolated, purified and analyzed using an antibody against the tetraspanin CD9 from human that showed cross-reactivity with the homologs to human CD9 found in Aedes albopictus (AalCD9). The exosomes from DENV infected cells were larger than the exosomes secreted from uninfected cells, contained virus-like particles, and they were able to infect naïve C6/36 cells, suggesting that exosomes are playing a role in virus dissemination.

In vivo multivesicular bodies and their exosomes in the absorptive cells of the zebrafish (Danio Rerio) gut.

Intercellular communication of gut epithelial cells is critical to gut mucosal homeostasis. Exosomes are important intercellular mediators in communication between cell to cell. Although many literature focus on the immunologic roles in the gut by the exosomes, the biological process of exosomes in the absorptive cells remains unknown. Uncovering the distribution, classification and formation process of multivesicular bodies (MVBs) and their exosomes in the absorptive cells of the zebrafish gut, is urgently needed to establish a platform for immunological research of fish gut exosomes. The expression levels of CD63 and TSG101 were different among the three segments of the gut, and they were enriched at the apex of the mid gut villi. The characteristics of MVBs and their exosomes in the absorptive cells were further revealed by transmission electron microscopy (TEM). Early endosomes (ee) were mainly present in the apical and basal cytoplasm of absorptive cells. Late endosomes (le) were mostly distributed with the supranuclear part of these cells. "Heterogeneous" MVBs were detected underlying the apical membranes of absorptive cells. Many exosomes with some MVB-like structures occurred in the lumen, indicating that the release process was mainly through apical secretion. Various MVBs with exosomes and the endosome-heterogeneous MVB-exosome complex existed widely in the mid gut absorptive cells, concluding that zebrafish as a potential model for in vivo MVBs and their exosomes research. All the results were summarized in a schematic diagram illustrating the morphological characteristics of gut MVBs and their exosomes in zebrafish.

Exosome Release Is Regulated by mTORC1.

Exosomes are small membrane-bound vesicles released into extracellular spaces by many types of cells. These nanovesicles carry proteins, mRNA, and miRNA, and are involved in cell waste management and intercellular communication. In the present study, it is shown that exosome release, which leads to net loss of cellular membrane and protein content, is negatively regulated by mechanistic target of rapamycin complex 1 (mTORC1). It is found that in cells and animal models exosome release is inhibited by sustained activation of mTORC1, leading to intracellular accumulation of CD63-positive exosome precursors. Inhibition of mTORC1 by rapamycin or nutrient and growth factor deprivation stimulates exosome release, which occurs concomitantly with autophagy. The drug-stimulated release is blocked by siRNA-mediated downregulation of small GTPase Rab27A. Analysis of the cargo content in exosomes released from rapamycin-treated cells reveals that inhibition of mTORC1 does not significantly alter its majority protein and miRNA profiles. These observations demonstrate that exosome release, like autophagy, is negatively regulated by mTORC1 in response to changes in nutrient and growth factor conditions.

In vivo dynamic distribution of multivesicular bodies and exosomes in spleen of DTMUV infected duck.

Exosomes are vesicles secreted by the multivesicular bodies (MVBs), which have been shown to mediate immunity regulation and virus transmission. In this study, the dynamic distribution and function of the MVBs and their exosomes was investigated through morphological characterization and molecular analyses in duck spleens infected with duck Tembusu virus (DTMUV) at different times post infection (1hpi, 2hpi, 12hpi, 24hpi). CD63, the marker of MVBs and exosomes, was distributed in the sheathed capillaries and the periellipsoidal lymphatic sheaths (PELS) of the white pulp. The numbers of MVBs and their exosomes were dramatically increased at 2 hpi, and with the increasing infection time, the numbers of MVBs and their exosomes were gradually decreased. DTMUV proteins were associated with exosomes according to double label immunofluorescence results. Ultrastructural characterization by transmission electron microscopy revealed four developing stages of MVBs containing exosomes were detected in high endothelial cells of the sheathed capillaries, lymphocytes and the ellipsoid-associated macrophages in PELS. Free exosomes were observed in the extracellular matrix and the blood vessels. Genes and proteins related to the endocytosis pathway were obviously up-regulated at 2 hpi as confirmed by RT-qPCR and western blotting. We speculated that DTMUV mediates host invasion through the endocytosis pathway by utilizing MVBs and their exosomes. The in vivo distribution pattern of MVBs and their exosomes in DTMUV infected spleens is shown for the first time in this study. This report could lay the foundations for understanding the infection mechanism of DTMUV.

Biogenesis and Function of Multivesicular Bodies in Plant Immunity.

Multivesicular bodies (MVBs) are specialized endosomes that contain intraluminal vesicles generated from invagination and budding of the limiting membrane. In the endocytic pathway, MVBs are late endosomes whose content can be degraded through fusion with lysosomes/vacuoles or released into the extracellular space after fusion with the plasma membrane (PM). The proteins retained on the limiting membrane of MVBs are translocated to the membrane of lysosomes/vacuoles or delivered back to the PM. It has been long suspected that MVBs might fuse with the PM to form paramural bodies in plant cells, possibly leading to release of building blocks for deposition of papillae and antimicrobial molecules against invading pathogens. Over the past decade or so, major progress has been made in establishing the critical roles of MVBs and associated membrane trafficking in pathogen recognition, defense signaling, and deployment of defense-related molecules during plant immune responses. Regulatory proteins and signaling pathways associated with induced biogenesis and trafficking of MVBs during plant immune responses have also been identified and characterized. Recent successful isolation of plant extracellular vesicles and proteomic profiling of their content have provided additional support for the roles of MVBs in plant-pathogen interactions. In this review, we summarize the important progress and discuss how MVBs, particularly through routing of cellular components to different destinations, contribute to the complex network of plant immune system.

Unconventional Trafficking of Mammalian Phospholipase D3 to Lysosomes.

Variants in the phospholipase D3 (PLD3) gene have genetically been linked to late-onset Alzheimer's disease. We present a detailed biochemical analysis of PLD3 and reveal its endogenous localization in endosomes and lysosomes. PLD3 reaches lysosomes as a type II transmembrane protein via a (for mammalian cells) uncommon intracellular biosynthetic route that depends on the ESCRT (endosomal sorting complex required for transport) machinery. PLD3 is sorted into intraluminal vesicles of multivesicular endosomes, and ESCRT-dependent sorting correlates with ubiquitination. In multivesicular endosomes, PLD3 is subjected to proteolytic cleavage, yielding a stable glycosylated luminal polypeptide and a rapidly degraded N-terminal membrane-bound fragment. This pathway closely resembles the delivery route of carboxypeptidase S to the yeast vacuole. Our experiments reveal a biosynthetic route of PLD3 involving proteolytic processing and ESCRT-dependent sorting for its delivery to lysosomes in mammalian cells.

The Multivesicular Body and Autophagosome Pathways in Plants.

In eukaryotic cells, the endomembrane system consists of multiple membrane-bound organelles, which play essential roles in the precise transportation of various cargo proteins. In plant cells, vacuoles are regarded as the terminus of catabolic pathways whereas the selection and transport of vacuolar cargoes are mainly mediated by two types of organelles, multivesicular bodies (MVBs) also termed prevacuolar compartments (PVCs) and autophagosomes. MVBs are single-membrane bound organelles with intraluminal vesicles and mediate the transport between the trans-Golgi network (TGN) and vacuoles, while autophagosomes are double-membrane bound organelles, which mediate cargo delivery to the vacuole for degradation and recycling during autophagy. Great progress has been achieved recently in identification and characterization of the conserved and plant-unique regulators involved in the MVB and autophagosome pathways. In this review, we present an update on the current knowledge of these key regulators and pay special attention to their conserved protein domains. In addition, we discuss the possible interplay between the MVB and autophagosome pathways in regulating vacuolar degradation in plants.

Current knowledge on exosome biogenesis and release.

Exosomes are nanosized membrane vesicles released by fusion of an organelle of the endocytic pathway, the multivesicular body, with the plasma membrane. This process was discovered more than 30 years ago, and during these years, exosomes have gone from being considered as cellular waste disposal to mediate a novel mechanism of cell-to-cell communication. The exponential interest in exosomes experienced during recent years is due to their important roles in health and disease and to their potential clinical application in therapy and diagnosis. However, important aspects of the biology of exosomes remain unknown. To explore the use of exosomes in the clinic, it is essential that the basic molecular mechanisms behind the transport and function of these vesicles are better understood. We have here summarized what is presently known about how exosomes are formed and released by cells. Moreover, other cellular processes related to exosome biogenesis and release, such as autophagy and lysosomal exocytosis are presented. Finally, methodological aspects related to exosome release studies are discussed.

The emerging role of exosome-derived non-coding RNAs in cancer biology.

Exosomes are a new means of intercellular information exchange that have aroused great research interest. Long neglected in research, exosomes were deemed nonfunctional cellular components to be discarded. However, it has been gradually revealed that exosomes are an important tool for the exchange of intercellular information and material. Exosomes contain specific repertoires of non-coding RNAs (ncRNAs, including microRNA and lncRNA), indicating that a specific RNA sorting mechanism may exist. Correspondingly, intracellular multivesicular bodies (MVBs) are produced after fusion with the cell membrane to release exosomes rather than inducing autophagy, which reveals that there may be a specific regulatory mechanism for MVB secretion. Cells can trigger cancer-related disorders after the recognition and uptake of circulating exosomal ncRNAs, providing indications for early tumor biopsy and treatment. The use of exosomes as a biological carrier in targeted therapy has been demonstrated. However, there may be a specific, unknown switch for loading drugs. This review focuses on the mechanisms of exosome biogenesis, release, and uptake. We also review the promotion of tumor development by exosomal ncRNAs including chemotherapy resistance, metastasis and the prospective use of exosomes in cancer diagnosis and treatment.