Shedding Of Vesicles Research Articles

Effect of Melatonin on Telocytes in the Seminal Vesicle of the Soay Ram: An Immunohistochemical, Ultrastructural and Morphometrical Study

Telocytes (TCs) are a special type of interstitial cell with characteristic cellular processes that are described in many organs. The current study aimed to investigate TCs in seminal vesicles of the Soay ram responding to melatonin treatment during the nonbreeding season by conventional immunohistochemical stains, and to detect the ultrastructural and morphometrical changes of TCs. TCs in the control group showed a broad range of staining affinity and also reacted positively to CD117/c-kit, CD34, desmin, S-100 protein, and progesterone and estrogen receptors alpha, while after melatonin treatment a strong reaction against these 6 antibodies was recorded. Electron microscopically, TCs in the control group were characterized by a small cell body with distinct long cytoplasmic extensions called telopodes (Tps). Tps had alternation of the thin segment (podomers) and dilated segments (podoms), in which the latter accommodate mitochondria, rough endoplasmic reticulum and caveolae. TCs and their Tps were interconnected by homo- and heterocellular junctions and form a wide network to communicate between different cell types. Tps showed close contact with immune cells, progenitor stem cells, smooth muscle cells and other interstitial cells. Melatonin caused a significant increase in the number of TCs, length of Tps, and number and diameter of secretory vesicles. Also, the melatonin-treated group showed exaggerated secretory activity in the form of a massive release of secretory vesicles from Tps. Moreover, Tps showed an increase in their contact with blood and lymphatic capillaries, nerve endings and Schwann cells. In addition, the shedding of secretory structures (exosomes, ectosomes, and multivesicular bodies) was greater from Tps, which were involved in paracrine signaling in the melatonin-treated group. The length and ramifications of Tps together with the intercellular junctions and the releasing of shed vesicles or exosomes assumed an essential role of TCs in intercellular signaling and coordination. On the basis of their distribution and morphology, we investigated whether the different locations of TCs could be associated with different roles.

Characterization of the ultrastructure in the uterovaginal junction of the hen

In poultry, the infundibulum is the place of fertilization, eggshell production, and sperm storage, while its uterovaginal junction (UVJ) is regarded as the most important site, which has abundant sperm storage tubules (SST). We examined the ultrastructure of the epithelium with relation to its unique secretory cytology in the UVJ of hens using transmission electron microscopy. The epithelium of the UVJ is lined with ciliated and secretory cells. Ciliated cells are characterized with light and dense secretory granules in supernuclear cytoplasm. Dense secretory granules in ciliated cells are larger in diameter (oneμm), surrounded with a transparent rim and concentric layers, whereas the dense granules in the ciliated cells of SST are smaller (0.52μm) in size and not surrounded by any transparent rim or layer. Ciliated cells also are involved in the shedding of exosomes and secretory vesicles in the lumen. Secretory exosomes are in close contact with cilia and directly release from the apical border into the lumen. Cell junctions are widely distributed between these cells. The secretory cells are associated with the release of secretions via apocrine blebs from microvilli and secretory vesicles, which protrude out from the surface of the epithelium. The dense secretory granules in these cells are smaller in size (0.6μm), absent of a transparent rim or layers, and are released into the lumen through secretory vesicles. The intracellular multivesicular body (MVB) also is observed in the supernuclear cytoplasm of secretory cells, which are related to the production of exosomes. In general, the apical protrusion of the epithelium in the form of apocrine secretions, the releasing of exosomes, the identification of intracellular MVB, and the release of dense granules give the epithelium a distinct morphology in the UVJ of the hen oviduct.

Characterisation of plasmalemmal shedding of vesicles induced by the cholesterol/sphingomyelin binding protein, ostreolysin A-mCherry

Ostreolysin A (OlyA) is a 15-kDa protein that binds selectively to cholesterol/sphingomyelin membrane nanodomains. This binding induces the production of extracellular vesicles (EVs) that comprise both microvesicles with diameters between 100nm and 1μm, and larger vesicles of around 10-μm diameter in Madin-Darby canine kidney cells. In this study, we show that vesiculation of these cells by the fluorescent fusion protein OlyA-mCherry is not affected by temperature, is not mediated via intracellular Ca2+ signalling, and does not compromise cell viability and ultrastructure. Seventy-one proteins that are mostly of cytosolic and nuclear origin were detected in these shed EVs using mass spectroscopy. In the cells and EVs, 218 and 84 lipid species were identified, respectively, and the EVs were significantly enriched in lysophosphatidylcholines and cholesterol. Our collected data suggest that OlyA-mCherry binding to cholesterol/sphingomyelin membrane nanodomains induces specific lipid sorting into discrete patches, which promotes plasmalemmal blebbing and EV shedding from the cells. We hypothesize that these effects are accounted for by changes of local membrane curvature upon the OlyA-mCherry-plasmalemma interaction. We suggest that the shed EVs are a potentially interesting model for biophysical and biochemical studies of cell membranes, and larger vesicles could represent tools for non-invasive sampling of cytosolic proteins from cells and thus metabolic fingerprinting.

Extracellular vesicles are transferred from melanocytes to keratinocytes after UVA irradiation.

Ultraviolet (UV) irradiation induces skin pigmentation, which relies on the intercellular crosstalk of melanin between melanocytes to keratinocytes. However, studying the separate effects of UVA and UVB irradiation reveals differences in cellular response. Herein, we show an immediate shedding of extracellular vesicles (EVs) from the plasma membrane when exposing human melanocytes to UVA, but not UVB. The EV-shedding is preceded by UVA-induced plasma membrane damage, which is rapidly repaired by Ca2+-dependent lysosomal exocytosis. Using co-cultures of melanocytes and keratinocytes, we show that EVs are preferably endocytosed by keratinocytes. Importantly, EV-formation is prevented by the inhibition of exocytosis and increased lysosomal pH but is not affected by actin and microtubule inhibitors. Melanosome transfer from melanocytes to keratinocytes is equally stimulated by UVA and UVB and depends on a functional cytoskeleton. In conclusion, we show a novel cell response after UVA irradiation, resulting in transfer of lysosome-derived EVs from melanocytes to keratinocytes.

Concise Review: The Bystander Effect: Mesenchymal Stem Cell-Mediated Lung Repair.

Mesenchymal stem or stromal cells (MSCs), a heterogeneous subset of adult stem/progenitor cells, have surfaced as potential therapeutic units with significant clinical benefit for a wide spectrum of disease conditions, including those affecting the lung. Although MSCs carry both self-renewal and multilineage differentiation abilities, current dogma holds that MSCs mainly contribute to tissue regeneration and repair by modulating the host tissue via secreted cues. Thus, the therapeutic benefit of MSCs is thought to derive from so called bystander effects. The regenerative mechanisms employed by MSCs in the lung include modulation of the immune system as well as promotion of epithelial and endothelial repair. Apart from secreted factors, a number of recent findings suggest that MSCs engage in mitochondrial transfer and shedding of membrane vesicles as a means to enhance tissue repair following injury. Furthermore, it is becoming increasingly clear that MSCs are an integral component of epithelial lung stem cell niches. As such, MSCs play an important role in coupling information from the environment to stem and progenitor populations, such that homeostasis can be ensured even in the face of injury. It is the aim of this review to outline the major mechanisms by which MSCs contribute to lung regeneration, synthesizing recent preclinical findings with data from clinical trials and potential for future therapy. Stem Cells 2016;34:1437-1444.

Data supporting the shedding of larger extracellular vesicles by multidrug resistant tumour cells.

To date, there are no simple and minimally invasive methods to diagnose MDR. Extracellular vesicles (EVs) are shed by all cells, carry a specific cargo from the donor cells and are present in several body fluids, which means that they can potentially be easily collected from cancer patients and become the source of biomarkers to diagnose cancer. This data article contains a full list of the proteins identified in the EVs shed by an isogenic pair of chronic myeloid leukaemia cells (MDR cells and their drug-sensitive counterparts) by LC/MS/MS analysis, together with their GeneOntology analysis. In addition, it also contains data from protein content analysis and Dynamic light scattering count-rate events of the referred EVs as well as of the EVs shed from an isogenic pair of non-small cell lung cancer cells (MDR cells and their drug-sensitive counterparts). The interpretation of the data presented in this article and further extensive insights can be found in “Multidrug resistant tumour cells shed more microvesicles-like EVs and less exosomes than their drug-sensitive counterpart cells” [1].

The Cutaneous Telocytes.

Telocytes (TCs) are interstitial cells found in stroma of many organs, including the skin dermis. Ultrastructurally, normal skin TCs recapitulates all the previously documented features in interstitum of other organs. Their (ultra)structural hallmark is the presence of particular shaped cellular prolongations (termed telopodes), along other features as cellular organelles representation and their distribution within cell body and its prolongations. Transmission electron microscopy (TEM) or high magnification light microscopy indicated that the particular shape of telopodes alternate characteristically thin segments (termed podomeres) and dilated segments (called podoms). A new and powerful technique, focused ion beam scanning electron microscopy (FIB-SEM), indicated that, ultrastructurally, telopodes could be either irregular ribbon-like structures, or uneven tubular-like structures. TEM images shown that podoms consists mitochondria, elements of endoplasmic reticulum and caveolae. Immunohisochemical studies on skin TCs revealed their positive expression for CD34 and PDGFRα, but for vimentin and c-kit, also. In normal dermis, TCs are involved in junctions, either homocellular (TCs-TCs), or heterocellular (TCs - other type of cells). The junctional attribute of TCs underlies their ability of forming a 3D network within dermis. Beyond the physical interactions, the connections between TCs and other cells could be also chemical, by paracrine secretion via shed vesicles as ultrastructural studies demonstrated. In normal dermis, TCs were found distributed in particular spatial relationships with other interstitial structures and/or cells: vascular structures, nerves, skin adnexa, stem cells and immune reactive cells.To date, the study of TCs was approached into two pathologic conditions: systemic sclerosis and psoriasis. In both diseases, the normal ultrastructure of TCs and also their distribution were shown to be altered. Moreover, the pattern of TCs ultrastructural changes differs in systemic sclerosis (cytoplasmic vacuolization, swollen mitochondria, lipofuscin bodies) from those appeared in psoriasis, characterized by important dystrophic changes (telopodes fragmentation, cytoplasmic disintegration, apoptotic nuclei, nuclear extrusions). Furthermore, in psoriasis, the lesional remission is (ultra)structurally displaying a recovery of dermal TCs at values similar to normal.Considering TCs ultrastructural features, their connections and spatiality in normal dermis and also their pathologic changes, TCs are credited with roles in skin homeostasis and/or pathogeny of dermatological disorders. In our opinion, further researches should be focused on identifying a specific marker for TCs and also on comprehending the pattern of their response in different dermatoses.

Secreted miR-34a in astrocytic shedding vesicles enhanced the vulnerability of dopaminergic neurons to neurotoxins by targeting Bcl-2.

MicroRNAs (miRNAs) are a class of noncoding RNAs that regulates target gene expression at posttranscriptional level, leading to further biological functions. We have demonstrated that microvesicles (MVs) can deliver miRNAs into target cells as a novel way of intercellular communication. It is reported that in central nervous system, glial cells release MVs, which modulate neuronal function in normal condition. To elucidate the potential role of glial MVs in disease, we evaluated the effects of secreted astrocytic MVs on stress condition. Our results demonstrated that after Lipopolysaccharide (LPS) stimulation, astrocytes released shedding vesicles (SVs) that enhanced vulnerability of dopaminergic neurons to neurotoxin. Further investigation showed that increased astrocytic miR-34a in SVs was involved in this progress via targeting anti-apoptotic protein Bcl-2 in dopaminergic neurons. We also found that inhibition of astrocytic miR-34a after LPS stimulation can postpone dopaminergic neuron loss under neurotoxin stress. These data revealed a novel mechanism underlying astrocyte-neuron interaction in disease.Electronic supplementary materialThe online version of this article (doi:10.1007/s13238-015-0168-y) contains supplementary material, which is available to authorized users.

Platelet microparticles in transfusion

Platelets are active participants in maintaining haemostasis. Upon activation, platelet shed vesicles from their cytoplasmic membrane known as platelet microparticles. These platelet microparticles and their unique cargo are thought to participate in intercellular communication. Platelet transfusion is frequently required for patients with low platelet counts or platelet disorders to restore normal functions. Unfortunately, adverse transfusion reactions are most commonly associated with platelet transfusion than any other type of labile blood products. In this review, we raise the potential implications of platelet microparticles in adverse transfusion reactions.

Crosstalk between red blood cells and the immune system and its impact on atherosclerosis.

Atherosclerosis is a chronic multifactorial disease of the arterial wall characterized by inflammation, oxidative stress, and immune system activation. Evidence exists on a pathogenic role of oxidized red blood cells (RBCs) accumulated in the lesion after intraplaque hemorrhage. This review reports current knowledge on the impact of oxidative stress in RBC modifications with the surface appearance of senescent signals characterized by reduced expression of CD47 and glycophorin A and higher externalization of phosphatidylserine. The review summarizes findings indicating that oxidized, senescent, or stored RBCs, due to surface antigen modification and release of prooxidant and proinflammatory molecules, exert an impaired modulatory activity on innate and adaptive immune cells and how this activity contributes to atherosclerotic disease. In particular RBCs from patients with atherosclerosis, unlike those from healthy subjects, fail to control lipopolysaccharide-induced DC maturation and T lymphocyte apoptosis. Stored RBCs, accompanied by shedding of extracellular vesicles, stimulate peripheral blood mononuclear cells to release proinflammatory cytokines, augment mitogen-driven T cell proliferation, and polarize macrophages toward the proinflammatory M1 activation pathway. Collectively, literature data suggest that the crosstalk between RBCs with immune cells represents a novel mechanism by which oxidative stress can contribute to atherosclerotic disease progression and may be exploited for therapeutic interventions.

Differential Proteomic Analysis of Syncytiotrophoblast Extracellular Vesicles from Early-Onset Severe Preeclampsia, using 8-Plex iTRAQ Labeling Coupled with 2D Nano LC-MS/MS

Aims: Previous studies have revealed that the increased shedding of syncytiotrophoblast extracellular vesicles (STBM) may lead to preeclampsia (PE). We aimed to identify the proteins carried by STBM and their potential pathological roles in early-onset severe PE. Methods: In this study, we performed a differential proteomic analysis of STBM from early-onset severe PE patients, using iTRAQ isobaric tags and 2D nano LC-MS/MS. STBM were generated by the in vitro explant culture method, and then verified by electron microscopy and western blot analysis. Results: A total of 18 533 unique peptides and 3 317 proteins were identified, 3 292 proteins were quantified. We identified 194 differentially expressed proteins in STBM from early-onset severe PE patients, 122 proteins were up-regulated and 72 proteins were down-regulated. Further bioinformatics analysis revealed that mitochondrion, transmembrane transport and transmembrane transporter activity were the most abundant categories in gene ontology (GO) annotation. Glycolysis/ gluconeogenesis, citrate cycle, fatty acid elongation, steroid hormone biosynthesis and oxidative phosphorylation were the five significantly represented pathways. Four differentially expressed proteins (siglec-6, calnexin, CD63 and S100-A8) related to inflammation, coagulation or immunoregulation were independently verified using western blot. Conclusions: The identification of key proteins carried by STBM may serve not only as a basis for better understanding and further exploring the etiology and pathogenesis of PE, but also as potential biomarkers and in providing targets for future therapy in PE, especially in early-onset severe PE(sPE).

Cancerous epithelial cell lines shed extracellular vesicles with a bimodal size distribution that is sensitive to glutamine inhibition

Extracellular shed vesicles (ESVs) facilitate a unique mode of cell–cell communication wherein vesicle uptake can induce a change in the recipient cellʼs state. Despite the intensity of ESV research, currently reported data represent the bulk characterization of concentrated vesicle samples with little attention paid to heterogeneity. ESV populations likely represent diversity in mechanisms of formation, cargo and size. To better understand ESV subpopulations and the signaling cascades implicated in their formation, we characterize ESV size distributions to identify subpopulations in normal and cancerous epithelial cells. We have discovered that cancer cells exhibit bimodal ESV distributions, one small-diameter and another large-diameter population, suggesting that two mechanisms may govern ESV formation, an exosome population and a cancer-specific microvesicle population. Altered glutamine metabolism in cancer is thought to fuel cancer growth but may also support metastatic niche formation through microvesicle production. We describe the role of a glutaminase inhibitor, compound 968, in ESV production. We have discovered that inhibiting glutamine metabolism significantly impairs large-diameter microvesicle production in cancer cells.

Horse adipose-derived mesenchymal stromal cells constitutively produce membrane vesicles: a morphological study.

Mesenchymal stromal cells (MSCs) are multipotent somatic cells that can differentiate into a variety of mature cell types. Over recent years, their biological in vitro and in vivo properties have elicited great expectations in the field of regenerative medicine, immunotherapy and tumour treatment. An increasing number of experimental observations suggest that their biological effects are probably related to a paracrine mechanism via the release of trophic factors and cytokines as well as through the production of membrane vesicles (MVs). These are nanometric membrane-bound structures, comprising shedding vesicles (SV) and exosomes (Ex), that enclose and transfer signalling molecules to target cells. We hypothesized that MVs may be implicated in the biological effects of MSCs from horse adipose tissue (E-AdMSCs), a type of MSC that has been extensively studied in recent years for its remarkable efficacy in tissue regeneration. By means of electron microscopy, we ascertained, for the first time, that equine adipose-derived MSCs constitutively produce MVs (E-Ad-MSCs). The analysis of MVs separated by ultracentrifugation allowed us to describe their general morphological features. Through the examination of cell monolayers by TEM, additionally, we distinguished the different pathways of SV and Ex formation, demonstrating that both fractions are produced by E-AdMSC. The accurate description of MV heterogeneous morphological characteristics led us to emphasize the possible implications of the relationship between different morphologies versus different functions. The data presented in this paper has an additional value, as they can be noteworthy for horses as well as for other mammalian species, including humans.

Microfluidic isolation of cancer-cell-derived microvesicles from hetergeneous extracellular shed vesicle populations.

Extracellular shed vesicles, including exosomes and microvesicles, are disseminated throughout the body and represent an important conduit of cell communication. Cancer-cell-derived microvesicles have potential as a cancer biomarker as they help shape the tumor microenvironment to promote the growth of the primary tumor and prime the metastatic niche. It is likely that, in cancer cell cultures, the two constituent extracellular shed vesicle subpopulations, observed in dynamic light scattering, represent an exosome population and a cancer-cell-specific microvesicle population and that extracellular shed vesicle size provides information about provenance and cargo. We have designed and implemented a novel microfluidic technology that separates microvesicles, as a function of diameter, from heterogeneous populations of cancer-cell-derived extracellular shed vesicles. We measured cargo carried by the microvesicle subpopulation processed through this microfluidic platform. Such analyses could enable future investigations to more accurately and reliably determine provenance, functional activity, and mechanisms of transformation in cancer.

Abstract 1658: Enhanced shedding of extracellular vesicles from amoeboid prostate cancer cells: Potential effects on the tumor microenvironment

Abstract The gene encoding the cytoskeletal regulator DIAPH3 is lost at high frequency in metastatic prostate cancer, and silencing of DIAPH3 evokes a transition to an amoeboid phenotype in multiple tumor cell backgrounds. This amoeboid transformation is accompanied by increases in plasma membrane deformations (blebbing), cell migration, invasion, and metastasis. DIAPH3 silencing also promotes the shedding of atypically large (>1μm) extracellular vesicles (EV) containing bioactive cargo. Whether loss of DIAPH3 also stimulates the release of bioactive nano-sized EV (exosomes) is not established. Here we examined the mechanism of release and potential biological functions of EV shed from DIAPH3-silenced and other prostate cancer cells. We observed that stimulation of LNCaP cells with the prostate stroma-derived growth factor heparin-binding EGF-like growth factor (HB-EGF), combined with p38MAPK inhibition caused exosome shedding, a process mediated by ERK1/2 hyperactivation. DIAPH3 silencing in DU145 cells also increased rates of exosome production. EV isolated from DIAPH3-silenced cells activated AKT1 and androgen signaling, increased proliferation of recipient tumor cells, and suppressed proliferation of human macrophages and peripheral blood mononuclear cells (PBMC). DU145-derived EV contained miR-125a, which suppressed AKT1 expression and proliferation in recipient human peripheral blood mononuclear cells and macrophages. Our findings suggest that EV across a wide size range (∼70nm to >1um) are produced by amoeboid prostate cancer cells as a result of DIAPH3 loss and/or growth factor stimulation. These EV may condition the tumor microenvironment through multiple mechanisms, including promoting the growth of cancer cells and suppression of tumor-infiltrating immune cells. Citation Format: Jayoung Kim, Samantha Morley, Minh Le, Denis Bedoret, Dale Umetsu, Dolores Di Vizio, Michael Freeman. Enhanced shedding of extracellular vesicles from amoeboid prostate cancer cells: Potential effects on the tumor microenvironment. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1658. doi:10.1158/1538-7445.AM2014-1658

Distinct susceptibilities of corneal Pseudomonas aeruginosa clinical isolates to neutrophil extracellular trap-mediated immunity.

Ocular bacterial keratitis, often associated with Pseudomonas aeruginosa bacterial infection, commonly occurs in contact lens wearers and may lead to vision impairment. In this study, we analyzed the contribution of neutrophil extracellular traps (NETs) to the mediation of protection during ocular keratitis. Both invasive and cytotoxic P. aeruginosa clinical isolates induced NET release by neutrophils. NETs carried the characteristic histone proteins, elastase, lysozyme, myeloperoxidase, and metabolic enzymes. While the invasive P. aeruginosa strains PAO1 (serogroup O5) and 6294 (serogroup O6) were trapped by NETs, the cytotoxic P. aeruginosa strains 6077, 6206 (serogroup O11), and PA14 (serogroup 010) were less sensitive to NET capture. The mechanism of escape by the cytotoxic strains from adhesion to NETs involved the shedding of outer membrane vesicles (OMVs) that outcompeted the cytotoxic P. aeruginosa strains for NET binding. When ocular infection was caused by an invasive strain in vivo, NETs were released at the ocular surface to capture bacteria, limiting their spread. Treatment with MNase I had a dose-dependent effect, with low doses of MNase speeding up bacterial clearance and high doses of MNase having toxic consequences. Cumulatively, our data suggest that NET-mediated immunity is a two-step process. Initially, pathogens attach to NET fragments; subsequently, upon nuclease activity, active serine proteases, which proteolytically degrade NET-associated proteins and promote DNase activity, are released. Therefore, a balance between NET production and NET degradation is needed to achieve maximal NET immunity.

Distinct sensitivity of the corneal P. aeruginosa clinical isolates to neutrophil extracellular trap-mediated immunity (INM8P.449)

Abstract Ocular bacterial keratitis is a frequent sight-threatening condition usually associated with P. aeruginosa infection. In this study we analyzed the contribution of the neutrophil extracellular traps (NETs) in mediating protection during ocular keratitis. Both, invasive and cytotoxic, P. aeruginosa clinical isolates induced NET release by neutrophils. NETs shared a common protein signature regardless of the inducing stimuli and were decorated with histones, elastase, lysozyme, myeloperoxidase, and metabolic enzymes. While the cytotoxic P. aeruginosa strains 6077 and 6206 strains (serogroup O11) and PA14 (serogroup 010) were resistant to NET capture, the invasive P. aeruginosa strains PAO1 (serogroup O5), 6294 (serogroup O6) were trapped by NETs. The mechanism of escape of the cytotoxic strains from adhesion to NETs depended on shedding of outer membrane vesicles (OMV) that competed with P. aeruginosa for NET binding. Unlike cytotoxic strains, NETs adherent invasive strains were weakly sensitive to NET-mediated killing. When ocular infection was caused by invasive strain, NETs were released at the ocular surface to capture bacteria, limiting it’s spread. Treatment with DNase I facilitated the clearance of NET-captured bacteria, decreasing ocular bacterial burden and disease pathology. Cumulatively, these data suggest that current therapeutic strategies might utilize DNAse I to promote recovery from acute keratitis.

Enhanced shedding of extracellular vesicles from amoeboid prostate cancer cells

The gene encoding the cytoskeletal regulator DIAPH3 is lost at high frequency in metastatic prostate cancer, and DIAPH3 silencing evokes a transition to an amoeboid tumor phenotype in multiple cell backgrounds. This amoeboid transformation is accompanied by increased tumor cell migration, invasion, and metastasis. DIAPH3 silencing also promotes the formation of atypically large (>1 μm) membrane blebs that can be shed as extracellular vesicles (EV) containing bioactive cargo. Whether loss of DIAPH3 also stimulates the release of nano-sized EV (e.g., exosomes) is not established. Here we examined the mechanism of release and potential biological functions of EV shed from DIAPH3-silenced and other prostate cancer cells. We observed that stimulation of LNCaP cells with the prostate stroma-derived growth factor heparin-binding EGF-like growth factor (HB-EGF), combined with p38MAPK inhibition caused EV shedding, a process mediated by ERK1/2 hyperactivation. DIAPH3 silencing in DU145 cells also increased rates of EV production. EV isolated from DIAPH3-silenced cells activated AKT1 and androgen signaling, increased proliferation of recipient tumor cells, and suppressed proliferation of human macrophages and peripheral blood mononuclear cells. DU145 EV contained miR-125a, which suppressed AKT1 expression and proliferation in recipient human peripheral blood mononuclear cells and macrophages. Our findings suggest that EV produced as a result of DIAPH3 loss or growth factor stimulation may condition the tumor microenvironment through multiple mechanisms, including the proliferation of cancer cells and suppression of tumor-infiltrating immune cells.

Ciliary and Flagellar Membrane Vesicle (Ectosome) Purification

Eukaryotic cilia/flagella are ideal organelles for the analysis of membrane trafficking, membrane assembly, and the functions of a variety of signal transduction molecules. Cilia are peninsular organelles and the membrane lipids, membrane proteins, and microtubular-associated components are selectively transported into cilia through the region formed by the basal body/transition region and tightly associated ciliary membrane. Cilia can be isolated from many organisms without disrupting cells and many will rapidly regenerate cilia (with the ciliary membrane lipids and proteins) to replace those that are released. Despite their ease of isolation, we have relatively little understanding of the mechanisms that regulate lipid and protein transport into ciliary membranes (Pazour and Bloodgood, 2008; Bloodgood, 2009; Bloodgood, 2012).Chlamydomonas flagella shed membrane vesicles, also called ectosomes (Wood et al., 2013) from flagellar tips and these vesicles can be purified from the culture medium without damaging or deflagellating cells (McLean et al., 1974; Bergman et al., 1975; Snell, 1976; Kalshoven et al., 1990). Based on a comparison of biotinylated proteins on the shed vesicles with biotinylated proteins isolated from purified flagella and cell bodies, the ectosomes contain most, but not all, flagellar surface proteins and none of the major cell body proteins (Dentler, 2013). Although ectosomes have only been purified from Chlamydomonas cells, preliminary evidence indicates that similar vesicles are released from Tetrahymena cilia (Dentler, unpublished). Flagellar (and ciliary) membranes or membrane proteins also can be released from purified flagella/cilia. Most membrane proteins can be solubilized by extracting purified cilia with nonionic detergent [Triton X-100 or X-114 or Nonidet P-40 (NP-40)] and pelleting the microtubules (axonemes). However, not all membranes are released by detergent (Dentler, 1980) and the supernatant also contains all of the flagellar proteins that are not attached to the microtubules. Intact membrane vesicles can be released from flagella by agitation of flagella, often with low concentrations of nonionic detergents or freeze-thawing (Witman et al., 1972; Snell, 1976; Dentler, 1980; Dentler, 1995; Bloodgood and May, 1982; Pasquale and Goodenough, 1987; Iomini et al., 2006; Huang et al., 2007). Once released, they can be purified from axonemes by differential centrifugation.Each of these methods may enrich for different populations of axonemal and membrane proteins and lipids. The different solubility of membranes may reveal local differences in lipid or protein composition (Bloodgood, 2009). The ectosomes contain most but not all surface proteins found on purified Chlamydomonas flagella (Dentler, 2013). The ectosomes vesicles may be enriched in different soluble flagellar proteins than those trapped as vesicles are released from purified flagella. The detergent-solubilized “membrane+matrix” will contain all soluble membrane proteins as well as all of the soluble proteins in the flagellar compartment. In this paper, a method to purify ectosomes vesicles released from the tips of living Chlamydomonas cells is presented as are two methods to release flagellar membrane vesicles and proteins from purified flagella., 真核纤毛/鞭毛是分析膜运输，膜组装和各种信号转导分子的功能的理想细胞器。纤毛是半岛细胞器，并且膜脂质，膜蛋白和微管相关组分通过由基底体/过渡区和紧密相关的睫状膜形成的区域选择性地转运到纤毛中。纤毛可以从许多生物体中分离而不破坏细胞，并且许多将快速再生纤毛（具有睫状膜脂质和蛋白质）以替代释放的纤毛。尽管它们容易分离，但我们对调节脂质和蛋白质转运到睫状膜的机制的理解相对较少（Pazour和Bloodgood，2008; Bloodgood，2009; Bloodgood，2012）。 鞭毛脱落的膜囊泡，也称为来自鞭毛尖端的卵母细胞（Wood等人，2013），并且可以从培养基中纯化这些囊泡而没有损伤或去鞭毛细胞（McLean等人， ，1974; Bergman et al。，1975; Snell，1976; Kalshoven et al。，1990）。基于脱落囊泡上的生物素化蛋白质与从纯化鞭毛和细胞体分离的生物素化蛋白质的比较，外周体含有大多数但不是全部的鞭毛表面蛋白质，并且没有主要的细胞体蛋白质（Dentler，2013）。虽然ectosomes只从衣原体细胞中纯化，但初步证据表明类似的囊泡从四膜虫纤毛释放（Dentler，未发表）。 鞭毛（和睫状体）膜或膜蛋白也可以从纯化的鞭毛/纤毛释放。大多数膜蛋白可以通过用非离子去污剂[Triton X-100或X-114或Nonidet P-40（NP-40）]提取纯化的纤毛并将微管（轴突）造粒来溶解。然而，不是所有的膜都被去污剂释放（Dentler，1980），并且上清液还包含所有未附着到微管的鞭毛蛋白。通过搅动鞭毛，通常使用低浓度的非离子型洗涤剂或冷冻融化，可以从鞭毛中释放完整的膜囊泡（Witman等人，1972; Snell，1976; Dentler，1980 ; Dentler，1995; Bloodgood和May，1982; Pasquale和Goodenough，1987; Iomini等人，2006; Huang等人，2007）。一旦释放，可以通过差速离心从轴突中纯化它们。这些方法中的每一种可以富集轴突和膜蛋白和脂质的不同群体。膜的不同溶解度可以揭示脂质或蛋白质组成的局部差异（Bloodgood，2009）。外来体含有在纯化的衣藻鞭毛上发现的大多数但不是全部表面蛋白（Dentler，2013）。外来体囊泡可以富集在不同的可溶性鞭毛蛋白中，而不是当从纯化的鞭毛中释放囊泡时捕获的那些。洗涤剂增溶的"膜+基质"将包含鞭毛区室中的所有可溶性膜蛋白以及所有可溶性蛋白。 在本文中，提出了从活的衣藻细胞的末端释放的外来体囊泡的方法，以提供从纯化的鞭毛中释放鞭毛膜囊泡和蛋白质的两种方法。

Oligodendroglioma cells synthesize the differentiation-specific linker histone H1° and release it into the extracellular environment through shed vesicles

Chromatin remodelling can be involved in some of the epigenetic modifications found in tumor cells. One of the mechanisms at the basis of chromatin dynamics is likely to be synthesis and incorporation of replacement histone variants, such as the H1° linker histone. Regulation of the expression of this protein can thus be critical in tumorigenesis. In developing brain, H1° expression is mainly regulated at the post-transcriptional level and RNA-binding proteins (RBPs) are involved. In the past, attention mainly focused on the whole brain or isolated neurons and little information is available on H1° expression in other brain cells. Even less is known relating to tumor glial cells. In this study we report that, like in maturing brain and isolated neurons, H1° synthesis sharply increases in differentiating astrocytes growing in a serum-free medium, while the corresponding mRNA decreases. Unexpectedly, in tumor glial cells both H1° RNA and protein are highly expressed, in spite of the fact that H1° is considered a differentiation-specific histone variant. Persistence of H1° mRNA in oligodendroglioma cells is accompanied by high levels of H1° RNA-binding activities which seem to be present, at least in part, also in actively proliferating, but not in differentiating, astrocytes. Finally, we report that oligodendroglioma cells, but not astrocytes, release H1° protein into the culture medium by shedding extracellular vesicles. These findings suggest that deregulation of H1° histone expression can be linked to tumorigenesis.