Midbody and primary cilium of neural progenitors release extracellular membrane particles enriched in the stem cell marker prominin-1

Expansion of the neocortex requires symmetric divisions of neuroepithelial cells, the primary progenitor cells of the developing mammalian central nervous system. Symmetrically dividing neuroepithelial cells are known to form a midbody at their apical (rather than lateral) surface. We show that apical midbodies of neuroepithelial cells concentrate prominin-1 (CD133), a somatic stem cell marker and defining constituent of a specific plasma membrane microdomain. Moreover, these apical midbodies are released, as a whole or in part, into the extracellular space, yielding the prominin-1–enriched membrane particles found in the neural tube fluid. The primary cilium of neuroepithelial cells also concentrates prominin-1 and appears to be a second source of the prominin-1–bearing extracellular membrane particles. Our data reveal novel origins of extracellular membrane traffic that enable neural stem and progenitor cells to avoid the asymmetric inheritance of the midbody observed for other cells and, by releasing a stem cell membrane microdomain, to potentially influence the balance of their proliferation versus differentiation

Human Prominin-1 (CD133) Is Detected in Both Neoplastic and Non-Neoplastic Salivary Gland Diseases and Released into Saliva in a Ubiquitinated Form.

Prominin-1 (CD133) is physiologically expressed at the apical membranes of secretory (serous and mucous) and duct cells of major salivary glands. We investigated its expression in various human salivary gland lesions using two distinct anti-prominin-1 monoclonal antibodies (80B258 and AC133) applied on paraffin-embedded sections and characterized its occurrence in saliva. The 80B258 epitope was extensively expressed in adenoid cystic carcinoma, in lesser extent in acinic cell carcinoma and pleomorphic adenoma, and rarely in mucoepidermoid carcinoma. The 80B258 immunoreactivity was predominately detected at the apical membrane of tumor cells showing acinar or intercalated duct cell differentiation, which lined duct- or cyst-like structures, and in luminal secretions. It was observed on the whole cell membrane in non-luminal structures present in the vicinity of thin-walled blood vessels and hemorrhagic areas in adenoid cystic carcinoma. Of note, AC133 labeled only a subset of 80B258-positive structures. In peritumoral salivary gland tissues as well as in obstructive sialadenitis, an up-regulation of prominin-1 (both 80B258 and AC133 immunoreactivities) was observed in intercalated duct cells. In most tissues, prominin-1 was partially co-expressed with two cancer markers: carcinoembryonic antigen (CEA) and mucin-1 (MUC1). Differential centrifugation of saliva followed by immunoblotting indicated that all three markers were released in association with small membrane vesicles. Immuno-isolated prominin-1-positive vesicles contained CEA and MUC1, but also exosome-related proteins CD63, flotillin-1, flotillin-2 and the adaptor protein syntenin-1. The latter protein was shown to interact with prominin-1 as demonstrated by its co-immunoisolation. A fraction of saliva-associated prominin-1 appeared to be ubiquitinated. Collectively, our findings bring new insights into the biochemistry and trafficking of prominin-1 as well as its immunohistochemical profile in certain types of salivary gland tumors and inflammatory diseases

Release of extracellular membrane vesicles from microvilli of epithelial cells is enhanced by depleting membrane cholesterol

AbstractWe previously reported on the occurrence of prominin-1-carrying membrane vesicles that are released into body fluids from microvilli of epithelial cells. This release has been implicated in cell differentiation. Here we have characterized these vesicles released from the differentiated Caco-2 cells. We find that in these vesicles, prominin-1 directly interacts with membrane cholesterol and is associated with a membrane microdomain. The cholesterol depletion using methyl-β-cyclodextrin resulted in a marked increase in their release, and a dramatic change in the microvillar ultrastructure from a tubular shape to a “pearling” state, with multiple membrane constrictions, suggesting a role of membrane cholesterol in vesicle release from microvilli

Live Imaging at the Onset of Cortical Neurogenesis Reveals Differential Appearance of the Neuronal Phenotype in Apical versus Basal Progenitor Progeny

The neurons of the mammalian brain are generated by progenitors dividing either at the apical surface of the ventricular zone (neuroepithelial and radial glial cells, collectively referred to as apical progenitors) or at its basal side (basal progenitors, also called intermediate progenitors). For apical progenitors, the orientation of the cleavage plane relative to their apical-basal axis is thought to be of critical importance for the fate of the daughter cells. For basal progenitors, the relationship between cell polarity, cleavage plane orientation and the fate of daughter cells is unknown. Here, we have investigated these issues at the very onset of cortical neurogenesis. To directly observe the generation of neurons from apical and basal progenitors, we established a novel transgenic mouse line in which membrane GFP is expressed from the beta-III-tubulin promoter, an early pan-neuronal marker, and crossed this line with a previously described knock-in line in which nuclear GFP is expressed from the Tis21 promoter, a pan-neurogenic progenitor marker. Mitotic Tis21-positive basal progenitors nearly always divided symmetrically, generating two neurons, but, in contrast to symmetrically dividing apical progenitors, lacked apical-basal polarity and showed a nearly randomized cleavage plane orientation. Moreover, the appearance of beta-III-tubulin–driven GFP fluorescence in basal progenitor-derived neurons, in contrast to that in apical progenitor-derived neurons, was so rapid that it suggested the initiation of the neuronal phenotype already in the progenitor. Our observations imply that (i) the loss of apical-basal polarity restricts neuronal progenitors to the symmetric mode of cell division, and that (ii) basal progenitors initiate the expression of neuronal phenotype already before mitosis, in contrast to apical progenitors

Extracellular vesicles and intercellular communication within the nervous system

Extracellular vesicles (EVs, including exosomes) are implicated in many aspects of nervous system development and function, including regulation of synaptic communication, synaptic strength, and nerve regeneration. They mediate the transfer of packets of information in the form of nonsecreted proteins and DNA/RNA protected within a membrane compartment. EVs are essential for the packaging and transport of many cell-fate proteins during development as well as many neurotoxic misfolded proteins during pathogenesis. This form of communication provides another dimension of cellular crosstalk, with the ability to assemble a “kit” of directional instructions made up of different molecular entities and address it to specific recipient cells. This multidimensional form of communication has special significance in the nervous system. How EVs help to orchestrate the wiring of the brain while allowing for plasticity associated with learning and memory and contribute to regeneration and degeneration are all under investigation. Because they carry specific disease-related RNAs and proteins, practical applications of EVs include potential uses as biomarkers and therapeutics. This Review describes our current understanding of EVs and serves as a springboard for future advances, which may reveal new important mechanisms by which EVs in coordinate brain and body function and dysfunction

Peripheral blood biomarkers in multiple sclerosis.

Multiple sclerosis is the most common autoimmune disorder affecting the central nervous system. The heteroge-neity of pathophysiological processes in MS contributes to the highly variable course of the disease and unpre-dictable response to therapies. The major focus of the research on MS is the identification of biomarkers inbiologicalfluids, such as cerebrospinalfluid or blood, to guide patient management reliably. Because of the diffi-culties in obtaining spinalfluid samples and the necessity for lumbar puncture to make a diagnosis has reduced,the research of blood-based biomarkers may provide increasingly important tools for clinical practice. However,currently there are no clearly established MS blood-based biomarkers. The availability of reliable biomarkerscould radically alter the management of MS at critical phases of the disease spectrum, allowing for interventionstrategies that may prevent evolution to long-term neurological disability. This article provides an overview ofthis researchfield and focuses on recent advances in blood-based biomarker researc

Extracellular Vesicles in Liver Diseases: Meeting Report from the International Liver Congress 2018

Extracellular vesicles (EVs) are small and heterogeneous membrane-bound structures released by cells and found in all biological fluids. They are effective intercellular communicators, acting on a number of close and/or distant target cells. EV cargo may reflect the cell of origin as well as the specific stress that induces their formation and release. They transport a variety of bioactive molecules, including messenger RNA, noncoding RNAs, proteins, lipids, and metabolites, that can be transferred among cells, regulating various cell responses. Alteration in the concentration and composition of EVs in biological fluids is a typical hallmark of pathologies in different liver diseases. Circulating EVs can serve as biomarkers or as messengers following uptake by other cells. This review is a meeting report from the International Liver Congress 2018 (European Association for the Study of the Liver) celebrated in Paris (Symposium: Extracellular vesicles and signal transmission) that discusses the role of EVs in several liver diseases, highlighting their potential value as disease biomarkers and therapeutic opportunities

Proliferating versus differentiating stem and cancer cells exhibit distinct midbody-release behaviour

The central portion of the midbody, a cytoplasmic bridge between nascent daughter cells at the end of cell division, has generally been thought to be retained by one of the daughter cells, but has, recently, also been shown to be released into the extracellular space. The significance of midbody-retention versus -release is unknown. Here we show, by quantitatively analysing midbody-fate in various cell lines under different growth conditions, that the extent of midbody-release is significantly greater in stem cells than cancer-derived cells. Induction of cell differentiation is accompanied by an increase in midbody-release. Knockdown of the endosomal sorting complex required for transport family members, Alix and tumour-suppressor gene 101, or of their interaction partner, centrosomal protein 55, impairs midbody-release, suggesting mechanistic similarities to abscission. Cells with such impaired midbody-release exhibit enhanced responsiveness to a differentiation stimulus. Taken together, midbody-release emerges as a characteristic feature of cells capable of differentiation

The morphology and biochemistry of nanostructures provide evidence for synthesis and signaling functions in human cerebrospinal fluid

<p>Abstract</p> <p>Background</p> <p>Cerebrospinal fluid (CSF) contacts many brain regions and may mediate humoral signaling distinct from synaptic neurotransmission. However, synthesis and transport mechanisms for such signaling are not defined. The purpose of this study was to investigate whether human CSF contains discrete structures that may enable the regulation of humoral transmission.</p> <p>Methods</p> <p>Lumbar CSF was collected prospectively from 17 participants: with no neurological or psychiatric disease, with Alzheimer's disease, multiple sclerosis, or migraine; and ventricular CSF from two cognitively healthy participants with long-standing shunts for congenital hydrocephalus. Cell-free CSF was subjected to ultracentrifugation to yield supernatants and pellets that were examined by transmission electron microscopy, shotgun protein sequencing, electrophoresis, western blotting, lipid analysis, enzymatic activity assay, and immuno-electron microscopy.</p> <p>Results</p> <p>Over 3,600 CSF proteins were identified from repeated shotgun sequencing of cell-free CSF from two individuals with Alzheimer's disease: 25% of these proteins are normally present in membranes. Abundant nanometer-scaled structures were observed in ultracentrifuged pellets of CSF from all 16 participants examined. The most common structures included synaptic vesicle and exosome components in 30-200 nm spheres and irregular blobs. Much less abundant nanostructures were present that derived from cellular debris. Nanostructure fractions had a unique composition compared to CSF supernatant, richer in omega-3 and phosphoinositide lipids, active prostanoid enzymes, and fibronectin.</p> <p>Conclusion</p> <p>Unique morphology and biochemistry features of abundant and discrete membrane-bound CSF nanostructures are described. Prostaglandin H synthase activity, essential for prostanoid production and previously unknown in CSF, is localized to nanospheres. Considering CSF bulk flow and its circulatory dynamics, we propose that these nanostructures provide signaling mechanisms <it>via </it>volume transmission within the nervous system that are for slower, more diffuse, and of longer duration than synaptic transmission.</p