卵泡液中细胞外囊泡及其携带的microRNA对卵泡发育的作用

细胞外囊泡(Extracellular vesicles,EVs)是指细胞分泌的双层膜转运囊泡。EVs能从细胞中摄取大分子物质,并将其转移至受体细胞。在这些大分子物质中,研究最多的就是microRNA (miRNA)。miRNA是一种参与基因表达调控的非编码RNA,已证实在哺乳动物卵泡液EVs中有不同的非编码RNA存在,EVs携带miRNA可以作为自分泌和旁分泌的替代机制,影响卵泡发育。文中系统介绍了EVs的种类、特征和分离鉴定方法,重点综述了EVs及携带的miRNA对卵泡发育的作用,包括早期卵泡发育、卵母细胞成熟、卵泡优势化以及对颗粒细胞功能的影响。同时对卵泡液中EVs及其携带的miRNA的未来研究进行了展望,为卵泡液中EVs及携带的miRNA功能的研究及应用提供了思路和方向。     

Cardioprotective role of extracellular vesicles: A highlight on exosome beneficial effects in cardiovascular diseases

Extracellular vesicles (EVs) are nano-sized vesicles, released from many cell types including cardiac cells, have recently emerged as intercellular communication tools in cell dynamics. EVs are an important mediator of signaling within cells that influencing the functional behavior of the target cells. In heart complex, cardiac cells can easily use EVs to transport bioactive molecules such as proteins, lipids, and RNAs to the regulation of neighboring cell function. Cross-talk between intracardiac cells plays pivotal roles in the heart homeostasis and in adaptive responses of the heart to stress. EVs were released by cardiomyocytes under baseline conditions, but stress condition such as hypoxia intensifies secretome capacity. EVs secreted by cardiac progenitor cells and cardiosphere-derived cells could be pinpointed as important mediators of cardioprotection and cardiogenesis. Furthermore, EVs from many different types of stem cells could potentially exert a therapeutic effect on the damaged heart. Recent evidence shows that cardiac-derived EVs are rich in microRNAs, suggesting a key role in the controlling of cellular processes. EVs harboring exosomes may be clinically useful in cell-free therapy approaches and potentially act as prognosis and diagnosis biomarkers of cardiovascular diseases.     

Movement of small RNAs in and between plants and fungi

RNA interference is a biological process whereby small RNAs inhibit gene expression through neutralizing targeted mRNA molecules. This process is conserved in eukaryotes. Here, recent work regarding the mechanisms of how small RNAs move within and between organisms is examined. Small RNAs can move locally and systemically in plants through plasmodesmata and phloem, respectively. In fungi, transportation of small RNAs may also be achieved by septal pores and vesicles. Recent evidence also supports bidirectional cross-kingdom communication of small RNAs between host plants and adapted fungal pathogens to affect the outcome of infection. We discuss several mechanisms for small RNA trafficking and describe evidence for transport through naked form, combined with RNA-binding proteins or enclosed by vesicles.     

Retroviral genomic RNAs are transported to the plasma membrane by endosomal vesicles

The viral genomes of alpha- and gamma-retroviruses follow an outbound route through the cytoplasm before assembling with the budding particle at the plasma membrane. We show here that murine leukemia virus (MLV) RNAs are transported on lysosomes and transferrin-positive endosomes. Transport on transferrin-positive vesicles requires both Gag and Env polyproteins. In the presence of Env, Gag is rerouted from lysosomes to transferrin-positive endosomes, and virion production becomes highly sensitive to drugs poisoning vesicular and endosomal traffic. Vesicular transport of the RNA does not require prior endocytosis, indicating that it is recruited directly from the cytosol. Viral prebudding complexes containing Env, Gag, and retroviral RNAs are thus formed on endosomes, and subsequently routed to the plasma membrane. This may allow retroviruses to hijack the endosomal machinery as part of their biosynthetic pathway. More generally, tethering to vesicles may provide an efficient mechanism for directed RNA transport.     

The Role of Extracellular Vesicles in Senescence

Cells can communicate in a variety of ways, such as by contacting each other or by secreting certain factors. Recently, extracellular vesicles (EVs) have been proposed to be mediators of cell communication. EVs are small vesicles with a lipid bilayer membrane that are secreted by cells and contain DNA, RNAs, lipids, and proteins. These EVs are secreted from various cell types and can migrate and be internalized by recipient cells that are the same or different than those that secrete them. EVs harboring various components are involved in regulating gene expression in recipient cells. These EVs may also play important roles in the senescence of cells and the accumulation of senescent cells in the body. Studies on the function of EVs in senescent cells and the mechanisms through which nonsenescent and senescent cells communicate through EVs are being actively conducted. Here, we summarize studies suggesting that EVs secreted from senescent cells can promote the senescence of other cells and that EVs secreted from nonsenescent cells can rejuvenate senescent cells. In addition, we discuss the functional components (proteins, RNAs, and other molecules) enclosed in EVs that enter recipient cells.     

Endosomal transport of septin mRNA and protein indicates local translation on endosomes and is required for correct septin filamentation

Endosomes transport lipids and proteins over long distances by shuttling along microtubules. They also carry mRNAs on their surface, but the precise molecular function of this trafficking process is unknown. By live cell imaging of polarized fungal hyphae, we show microtubule-dependent transport of septin mRNA and encoded septin protein on the same shuttling endosomes. Consistent with the hypothesis that septin mRNA is translated on endosomes, the accumulation of septin protein on endosomes requires the recruitment of septin mRNA. Furthermore, ribosomal proteins co-localise with shuttling endosomes, but only if mRNA is present. Importantly, endosomal trafficking is essential for an efficient delivery of septin protein to filaments at growth poles, a process necessary to establish unipolar growth. Thus, we propose that local mRNA translation loads endosomes with septins for assembly and efficient delivery to septin filaments.     

Extracellular vesicles from neurons promote neural induction of stem cells through cyclin D1

Extracellular vesicles (EVs) are thought to mediate the transport of proteins and RNAs involved in intercellular communication. Here, we show dynamic changes in the buoyant density and abundance of EVs that are secreted by PC12 cells stimulated with nerve growth factor (NGF), N2A cells treated with retinoic acid to induce neural differentiation, and mouse embryonic stem cells (mESCs) differentiated into neuronal cells. EVs secreted from in vitro differentiated cells promote neural induction of mESCs. Cyclin D1 enriched within the EVs derived from differentiated neuronal cells contributes to this induction. EVs purified from cells overexpressing cyclin D1 are more potent in neural induction of mESC cells. Depletion of cyclin D1 from the EVs reduced the neural induction effect. Our results suggest that EVs regulate neural development through sorting of cyclin D1.     

Potential Transfer of Polyglutamine and CAG-Repeat RNA in Extracellular Vesicles in Huntington’s Disease: Background and Evaluation in Cell Culture

In Huntington’s disease (HD) the imperfect expanded CAG repeat in the first exon of the HTT gene leads to the generation of a polyglutamine (polyQ) protein, which has some neuronal toxicity, potentially mollified by formation of aggregates. Accumulated research, reviewed here, implicates both the polyQ protein and the expanded repeat RNA in causing toxicity leading to neurodegeneration in HD. Different theories have emerged as to how the neurodegeneration spreads throughout the brain, with one possibility being the transport of toxic protein and RNA in extracellular vesicles (EVs). Most cell types in the brain release EVs and these have been shown to contain neurodegenerative proteins in the case of prion protein and amyloid-beta peptide. In this study, we used a model culture system with an overexpression of HTT-exon 1 polyQ-GFP constructs in human 293T cells and found that the EVs did incorporate both the polyQ-GFP protein and expanded repeat RNA. Striatal mouse neural cells were able to take up these EVs with a consequent increase in the green fluorescent protein (GFP) and polyQ-GFP RNAs, but with no evidence of uptake of polyQ-GFP protein or any apparent toxicity, at least over a relatively short period of exposure. A differentiated striatal cell line expressing endogenous levels of Hdh mRNA containing the expanded repeat incorporated more of this mRNA into EVs as compared to similar cells expressing this mRNA with a normal repeat length. These findings support the potential of EVs to deliver toxic expanded trinucleotide repeat RNAs from one cell to another, but further work will be needed to evaluate potential EV and cell-type specificity of transfer and effects of long-term exposure. It seems likely that expanded HD-associated repeat RNA may appear in biofluids and may have use as biomarkers of disease state and response to therapy.     

Omics insights into extracellular vesicles in embryo implantation and their therapeutic utility

Implantation success relies on intricate interplay between the developing embryo and the maternal endometrium. Extracellular vesicles (EVs) represent an important player of this intercellular signalling through delivery of functional cargo (proteins and RNAs) that reprogram the target cells protein and RNA landscape. Functionally, the signalling reciprocity of endometrial and embryo EVs regulates the site of implantation, preimplantation embryo development and hatching, antioxidative activity, embryo attachment, trophoblast invasion, arterial remodelling, and immune tolerance. Omics technologies including mass spectrometry have been instrumental in dissecting EV cargo that regulate these processes as well as molecular changes in embryo and endometrium to facilitate implantation. This has also led to discovery of potential cargo in EVs in human uterine fluid (UF) and embryo spent media (ESM) of diagnostic and therapeutic value in implantation success, fertility, and pregnancy outcome. This review discusses the contribution of EVs in functional hallmarks of embryo implantation, and how the integration of various omics technologies is enabling design of EV-based diagnostic and therapeutic platforms in reproductive medicine.     

Ticket to a bubble ride: Cargo sorting into exosomes and extracellular vesicles

Extracellular vesicles (EVs) are released by cells into the extracellular milieu to facilitate intercellular communication in both physiological and pathological condition. EVs contain selective repertoires of proteins, RNAs, lipids and metabolites that moderate signalling pathways in the recipient cells. The enrichment of a particular set of proteins or RNAs within the EVs highlights the existence of specific sorting mechanisms that orchestrate the selective packaging of the cargo. The molecular machinery of cargo sorting has remained obscure over the years and functional studies are required to understand this complex mechanism. In this article, we offer a brief overview of the molecular mechanisms that are known to regulate sorting of various molecules into EVs. We also discuss how different pathways of biogenesis alter the exosomal cargo as well and the implications of the cellular state on the content of the EVs. Understanding the sorting of exosomal cargo could further be exploited in clinical settings for targeted drug delivery and to block disease progression.     

Anti‐human CD9 antibody Fab fragment impairs the internalization of extracellular vesicles and the nuclear transfer of their cargo proteins

The intercellular communication mediated by extracellular vesicles (EVs) has gained international interest during the last decade. Interfering with the mechanisms regulating this cellular process might find application particularly in oncology where cancer cell‐derived EVs play a role in tumour microenvironment transformation. Although several mechanisms were ascribed to explain the internalization of EVs, little is our knowledge about the fate of their cargos, which are crucial to mediate their function. We recently demonstrated a new intracellular pathway in which a fraction of endocytosed EV‐associated proteins is transported into the nucleoplasm of the host cell via a subpopulation of late endosomes penetrating into the nucleoplasmic reticulum. Silencing tetraspanin CD9 both in EVs and recipient cells strongly decreased the endocytosis of EVs and abolished the nuclear transfer of their cargos. Here, we investigated whether monovalent Fab fragments derived from 5H9 anti‐CD9 monoclonal antibody (referred hereafter as CD9 Fab) interfered with these cellular processes. To monitor the intracellular transport of proteins, we used fluorescent EVs containing CD9‐green fluorescent protein fusion protein and various melanoma cell lines and bone marrow‐derived mesenchymal stromal cells as recipient cells. Interestingly, CD9 Fab considerably reduced EV uptake and the nuclear transfer of their proteins in all examined cells. In contrast, the divalent CD9 antibody stimulated both events. By impeding intercellular communication in the tumour microenvironment, CD9 Fab‐mediated inhibition of EV uptake, combined with direct targeting of cancerous cells could lead to the development of novel anti‐melanoma therapeutic strategies.     

Imaging extracellular vesicles: current and emerging methods

Extracellular vesicles (EVs) are lipid bilayer-enclosed nanoparticles released by cells. They range from 30?nm to several micrometers in diameter, and ferry biological cargos such as proteins, lipids, RNAs and DNAs for local and distant intercellular communications. EVs have since been found to play a role in development, as well as in diseases including cancers. To elucidate the roles of EVs, researchers have established different methods to visualize and study their spatiotemporal properties. However, since EV are nanometer-sized, imaging them demands a full understanding of each labeling strategy to ensure accurate monitoring. This review covers current and emerging strategies for EV imaging for prospective studies.     

Protein Composition Reflects Extracellular Vesicle Heterogeneity

Extracellular vesicles (EVs) are membrane‐enclosed particles that are released by virtually all cells from all living organisms. EVs shuttle biologically active cargo including protein, RNA, and DNA between cells. When shed by cancer cells, they function as potent intercellular messangers with important functional consequences. Cells produce a diverse spectrum of EVs, spanning from small vesicles of 40–150 nm in diameter, to large vesicles up to 10 μm in diameter. While this diversity was initially considered to be purely based on size, it is becoming evident that different classes of EVs, and different populations within one EV class may harbor distinct molecular cargo and play specific functions. Furthermore, there are considerable cell type‐dependent differences in the cargo and function of shed EVs. This review focuses on the most recent proteomic studies that have attempted to capture the EV heterogeneity by directly comparing the protein composition of different EV classes and EV populations derived from the same cell source. Recent studies comparing protein composition of the same EV class(es) derived from different cell types are also summarized. Emerging approaches to study EV heterogeneity and their important implications for future studies are also discussed.     

Viral miRNAs exploiting the endosomal-exosomal pathway for intercellular cross-talk and immune evasion

The class of persistent gamma-herpesviruses has developed a variety of strategies that exploit host-cell regulatory pathways to ensure a long-lasting, well-balanced infection of their host. However when these pathways are deregulated, an otherwise harmless infection can lead to disease including cancer. We recently demonstrated that the human herpes virus 4 (HHV4) also known as Epstein-Barr virus (EBV), encodes for small regulatory non-coding microRNAs (miRNAs) that can be transferred from an infected cell to uninfected neighboring cells. Upon arrival these miRNAs are functional in the recipient cell, in that they are able to down regulate specific target genes. These secreted miRNAs are transported to recipient cells via small nano-sized vesicles (known as exosomes) that are of endosomal origin, formed as intraluminal vesicles (ILV) inside multivesicular bodies (MVB). One question that needs to be addressed is how viral miRNAs are sorted into these exosomes. Mature miRNAs, including those of viral origin, are loaded into RNA-induced silencing complexes (RISC) for gene silencing via blocking mRNA translation and/or initiating mRNA decay. Recent insights indicate that cytoplasmic RNA granules rich in RISC complexes are closely associated with endosomes. In fact, selective components of RISC, including GW182 and Argonaut proteins, miRNAs and mRNAs are present in exosomes. Thus miRNA function, mRNA stability and exosome-mediated intercellular communication converge at the level of endosomes. Since endosomes can be considered as key intracellular cross-roads that regulate communication of cells with their exterior, including neighboring cells, it is perhaps not surprising that viruses have found means to exploit this pathway to their benefit. Little is known however, how and if (micro) RNA species are specifically sorted into ILVs and what (micro)RNA-binding proteins are involved. Here we discuss recent developments relating to intracellular trafficking and function of miRNA-containing protein complexes that EBV may exploit for promoting or restricting miRNAs sorting into exosomes for intercellular regulatory functions. This article is part of a Special Issue entitled: MicroRNAs in viral gene regulation.