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Tumor growth and metastasis strongly rely on cell–cell communication. One of the mechanisms by which tumor cells communicate involves the release and uptake of lipid membrane encapsulated particles full of bioactive molecules, called extracellular vesicles (EVs). EV exchange between cancer cells may induce phenotype changes in the recipient cells. Our work investigated the effect of EVs released by teratocarcinoma cells on glioblastoma (GBM) cells. EVs were isolated by differential centrifugation and analyzed through Western blot, nanoparticle tracking analysis, and electron microscopy. The effect of large EVs on GBM cells was tested through cell migration, proliferation, and drug-sensitivity assays, and resulted in a specific impairment in cell migration with no effects on proliferation and drug-sensitivity. Noticeably, we found the presence of the EGF-CFC founder member CRIPTO on both small and large EVs, in the latter case implicated in the EV-mediated negative regulation of GBM cell migration. Our data let us propose a novel route and function for CRIPTO during tumorigenesis, highlighting a complex scenario regulating its effect, and paving the way to novel strategies to control cell migration, to ultimately improve the prognosis and quality of life of GBM patients.

Protein S-palmitoylation is a reversible post-translational lipidation in which palmitic acid (16:0) is added to protein cysteine residue by a covalent thioester bond. This modification plays an active role in membrane targeting of soluble proteins, protein–protein interaction, protein trafficking, and subcellular localization. Moreover, palmitoylation is related to different diseases, such as neurodegenerative pathologies, cancer, and developmental defects. The aim of this research is to provide a straightforward and sensitive procedure to detect protein palmitoylation based on Acyl Biotin Exchange (ABE) chemistry. Our protocol setup consists of co-immunoprecipitation of native proteins (i.e., CD63), followed by the direct detection of palmitoylation on proteins immobilized on polyvinylidene difluoride (PVDF) membranes. With respect to the conventional ABE-based protocol, we optimized and validated a rapid semi-quantitative assay that is shown to be significantly more sensitive and highly reproducible.

The 2,2’4,4’-tetrabromodiphenyl ether (PBDE-47) is one of the most prominent PBDE congeners detected in the environment and in animal and human tissues. Animal model experiments suggested the occurrence of PBDE-induced immunotoxicity leading to different outcomes and recently we demonstrated that this substance can impair macrophage and basophil activities. In this manuscript, we decided to further examine the effects induced by PBDE-47 treatment on innate immune response by looking at the intracellular expression profile of miRNAs as well as the biogenesis, cargo content and activity of human M(LPS) macrophage cell-derived small extracellular vesicles (sEVs). Microarray and in silico analysis demonstrated that PBDE-47 can induce some epigenetic effects in M(LPS) THP-1 cells modulating the expression of a set of intracellular miRNAs involved in biological pathways regulating the expression of estrogen-mediated signaling and immune responses with particular reference to M1/M2 differentiation. In addition to the cell-intrinsic modulation of intracellular miRNAs, we demonstrated that PBDE-47 could also interfere with the biogenesis of sEVs increasing their number and selecting a de novo population of sEVs. Moreover, PBDE-47 induced the overload of specific immune related miRNAs in PBDE-47 derived sEVs. Finally, culture experiments with naïve M(LPS) macrophages demonstrated that purified PBDE-47 derived sEVs can modulate macrophage immune response exacerbating the LPS-induced pro-inflammatory response inducing the overexpression of the IL-6 and the MMP9 genes. Data from this study demonstrated that PBDE-47 can perturb the innate immune response at different levels modulating the intracellular expression of miRNAs but also interfering with the biogenesis, cargo content and functional activity of M(LPS) macrophage cell-derived sEVs.

Cellular, inter-organismal and cross kingdom communication via extracellular vesicles (EVs) is intensively studied in basic science with high expectation for a large variety of bio-technological applications. EVs intrinsically possess many attributes of a drug delivery vehicle. Beyond the implications for basic cell biology, academic and industrial interests in EVs have increased in the last few years. Microalgae constitute sustainable and renewable sources of bioactive compounds with a range of sectoral applications, including the formulation of health supplements, cosmetic products and food ingredients. Here we describe a newly discovered subtype of EVs derived from microalgae, which we named nanoalgosomes. We isolated these extracellular nano-objects from cultures of microalgal strains, including the marine photosynthetic chlorophyte Tetraselmis chuii, using differential ultracentrifugation or tangential flow fractionation and focusing on the nanosized small EVs (sEVs). We explore different biochemical and physical properties and we show that nanoalgosomes are efficiently taken up by mammalian cell lines, confirming the cross kingdom communication potential of EVs. This is the first detailed description of such membranous nanovesicles from microalgae. With respect to EVs isolated from other organisms, nanoalgosomes present several advantages in that microalgae are a renewable and sustainable natural source, which could easily be scalable in terms of nanoalgosome production., Journal of Extracellular Vesicles, 10 (6), ISSN:2001-3078

Safe, efficient and specific nano-delivery systems are essential for current and emerging therapeutics, precision medicine and other biotechnology sectors. Novel bio-based nanotechnologies have recently arisen, which are based on the exploitation of extracellular vesicles (EVs). In this context, it has become essential to identify suitable organisms or cellular types to act as reliable sources of EVs and to develop their pilot- to large-scale production. The discovery of new biosources and the optimisation of related bioprocesses for the isolation and functionalisation of nano-delivery vehicles are fundamental to further develop therapeutic and biotechnological applications. Microalgae constitute sustainable sources of bioactive compounds with a range of sectorial applications including for example the formulation of health supplements, cosmetic products or food ingredients. In this study, we demonstrate that microalgae are promising producers of EVs. By analysing the nanosized extracellular nano-objects produced by eighteen microalgal species, we identified seven promising EV-producing strains belonging to distinct lineages, suggesting that the production of EVs in microalgae is an evolutionary conserved trait. Here we report the selection process and focus on one of this seven species, the glaucophyte Cyanophora paradoxa, which returned a protein yield in the small EV fraction of 1 μg of EV proteins per mg of dry weight of microalgal biomass (corresponding to 109 particles per mg of dried biomass) and EVs with a diameter of 130 nm (mode), as determined by the micro bicinchoninic acid assay, nanoparticle tracking and dynamic light scattering analyses. Moreover, the extracellular nanostructures isolated from the conditioned media of microalgae species returned positive immunoblot signals for some commonly used EV-biomarkers such as Alix, Enolase, HSP70, and β-actin. Overall, this work establishes a platform for the efficient production of EVs from a sustainable bioresource and highlights the potential of microalgal EVs as novel biogenic nanovehicles.

Extracellular vesicles (EVs) are phospholipid bilayer enclosed structures containing specific proteins, nucleic acids, and metabolites, which are naturally released from cells as vehicles of intercellular communication. Post-translational modifications (PTMs) of EV proteins can regulate EVs biogenesis or play diverse roles in their biology, in both homeostatic and pathological conditions. Here, we focus on the mechanism and biological roles of post-translational lipidations of EV proteins, with a particular insight on their involvement in EV biogenesis and functions. We investigate various aspects of PTM by lipidation in depth, ranging from chemistry to technical advances for lipidation detection and recent biotech applications. Among lipidations on EV proteins, palmitoylation is the most widespread mechanism. EVs have been shown to be enriched of palmitoylated proteins, and the profile of EV palmitoyl proteome could contribute to early diagnosis in some kinds of cancer, suggesting that EV protein lipidation can be exploited as potential early tumor biomarker. Thus, the emerging evidence on EV protein lipidations could help us to better understand their physio/pathological function with implications for preclinical and clinical settings.

Epithelial-mesenchymal transition (EMT) is an evolutionarily conserved cellular program, which is a prerequisite for the metastatic cascade in carcinoma progression. Here, we evaluate the EMT process using the sea urchin Paracentrotus lividus embryo. In sea urchin embryos, the earliest EMT event is related to the acquisition of a mesenchymal phenotype by the spiculogenetic primary mesenchyme cells (PMCs) and their migration into the blastocoel. We investigated the effect of inhibiting the epidermal growth factor (EGF) signaling pathway on this process, and we observed that mesenchyme cell differentiation was blocked. In order to extend and validate our studies, we investigated the migratory capability and the level of potential epidermal growth factor receptor (EGFr) targets in a breast cancer cell line after EGF modulation. Altogether, our data highlight the sensitivity of the sea urchin embryo to anti-EMT drugs and pinpoint the sea urchin embryo as a valuable in vivo model system for studying EMT and the screening of anti-EMT candidates.

Background Virtually all cell types have the capacity to secrete nanometer-sized extracellular vesicles, which have emerged in recent years as potent signal transducers and cell-cell communicators. The multifunctional protein Alix is a bona fide exosomal regulator and skeletal muscle cells can release Alix-positive nano-sized extracellular vesicles, offering a new paradigm for understanding how myofibers communicate within skeletal muscle and with other organs. S-palmitoylation is a reversible lipid post-translational modification, involved in different biological processes, such as the trafficking of membrane proteins, achievement of stable protein conformations, and stabilization of protein interactions. Methods Here, we have used an integrated biochemical-biophysical approach to determine whether S-palmitoylation contributes to the regulation of extracellular vesicle production in skeletal muscle cells. Results We ascertained that Alix is S-palmitoylated and that this post-translational modification influences its protein-protein interaction with CD9, a member of the tetraspanin protein family. Furthermore, we showed that the structural organization of the lipid bilayer of the small (nano-sized) extracellular vesicle membrane with altered palmitoylation is qualitatively different compared to mock control vesicles. Conclusions We propose that S-palmitoylation regulates the function of Alix in facilitating the interactions among extracellular vesicle-specific regulators and maintains the proper structural organization of exosome-like extracellular vesicle membranes. General Significance Beyond its biological relevance, our study also provides the means for a comprehensive structural characterization of EVs.

Cell surface expression of alpha-enolase, a glycolytic enzyme displaying moonlighting activities, has been shown to contribute to the motility and invasiveness of cancer cells through the protein non-enzymatic function of binding plasminogen and enhancing plasmin formation. Although a few recent records indicate the involvement of protein partners in the localization of alpha-enolase to the plasma membrane, the cellular mechanisms underlying surface exposure remain largely elusive. Searching for novel interactors and signalling pathways, we used low-metastatic breast cancer cells, a doxorubicin-resistant counterpart and a non-tumourigenic mammary epithelial cell line. Here, we demonstrate by a combination of experimental approaches that epidermal growth factor (EGF) exposure, like lipopolysaccharide (LPS) exposure, promotes the surface expression of alpha-enolase. We also establish Heat shock protein 70 (Hsp70), a multifunctional chaperone distributed in intracellular, plasma membrane and extracellular compartments, as a novel alpha-enolase interactor and demonstrate a functional involvement of Hsp70 in the surface localization of alpha-enolase. Our results contribute to shedding light on the control of surface expression of alpha-enolase in non-tumourigenic and cancer cells and suggest novel targets to counteract the metastatic potential of tumours.

The EGFR pathway is critical for cell fate specification throughout the development of several organisms. Here we identified in sea urchin an EGFR-related antigen maternally expressed and showing a dynamic pattern of localization during development. To investigate the role played by the EGFR in Paracentrotus lividus development we blocked its activity by using the EGFR kinase inhibitor AG1478. This treatment produces decrease of EGFR phosphorylation, and embryos with various defects especially in the endomesoderm territory until to obtain an animalized phenotype. These effects are rescued by the addition of TGF-alpha, an EGFR ligand. The role played by EGFR-like along the animal/vegetal axis was also detected, after AG1478 treatment, by the extended distribution of HE and decreased nuclearization of beta-catenin in vegetal cells. Moreover, inhibition of EGFR-like reduced ERK phosphorylation, necessary for cell fate specification in the micromeres and their derivates. Taken together these results indicate that EGFR-like activity is required both for A/V axis formation and endomesoderm differentiation.

Paracentrotus lividus eggs were divided by centrifugation into nucleated and anucleated halves. Fertilization and development of the two halves permitted us to establish that nucleated and anucleated fragments correspond, respectively, to the animal and vegetal parts. RNA was extracted from both egg halves and submitted to differential display. Northern blot analysis confirmed their maternal origin and showed that each transcript has a different expression pattern during development. By Northern blot and in situ hybridization experiments we ascertained that Bep2 and PlAn1 are localized in the animal part, whereas 16S rRNA, Plveg1, and L27 in the vegetal part, and that Plun1 is uniformly distributed. Moreover, by treating P. lividus eggs with detergent, in presence or not of drugs such as colchicine and cytochalasin B, we demonstrated the involvement of the cytoskeleton only in localization of Bep2, PlAn1, and Plun1, suggesting that different mechanisms are utilized for animal and vegetal distribution.

The seagrass Posidonia oceanica plays a multifunctional role in the coastal area as an important and productive component of ecosystems in the Mediterranean Sea. We detected by RAPD analysis with two arbitrary primers genetic differences in P. oceanica collected from several sites in the Southern Mediterranean. By AMOVA analysis we observed a level of about 20% genetic difference among individuals within a population and 80% among populations. A common band of 200 bp was found in all the amplified samples. Cloning and sequencing analysis of this band revealed the presence of a simple tandem repeat sequence (minisatellite) that we called PoTR (Posidonia oceanica tandem repeat). Finally, the ability of PoTR to detect genetic variability in P. oceanica genome was demonstrated by the presence of amplification products of different lengths utilizing primers internal to this sequence.

In sea urchin embryos, the initial animal–vegetal (AV) axis is specified during oogenesis but the mechanism is largely unknown. By using chemical reagents such as lithium, it is possible to shift the principal embryonic territories toward a vegetal fate. We have investigated the possibility of obtaining the same morphological effect as with lithium by utilizing Fabs against the maternal Bep4 protein that is localized in the animal part of Paracentrotus lividus egg and embryos. Incubation of fertilized eggs with Fabs against Bep4 protein causes exogastrulation at 48 h of development of P. lividus embryos, similar to embryos treated with lithium. This vegetalizing effect was ascertained by utilizing territorial markers such as EctoV, EndoI, and Ig8. The effect of Fabs against Bep4 on gene expression was observed by monitoring spatial expression of the hatching enzyme gene. A decreased expression domain compared to its normal spatial distribution was detected and this effect was again comparable to those obtained with lithium treatment. Association of Bep4 with a cadherin was demonstrated by immunoprecipitation and immunostaining experiments, and an involvement in cell signaling is discussed. In addition, treatment of embryos with anti-Bep4 Fabs causes an enhancement in the level and an expansion in the pattern of nuclear β-catenin. Moreover, this treatment also provokes a decrease of β-catenin in adherens junctions. Together, these data indicate that anti-Bep4 Fabs provoke a shift of the animal–vegetal boundary toward the animal pole and suggest an active role of Bep4 protein in patterning along the AV axis.

The temporal expression of two cell surface proteins, called BEP1 and BEP4, during Paracentrosus lividus embryonic development was studied. These proteins are found in both monomeric and dimeric forms in egg and embryos and we have established that their specific form is related to their being in the cytoplasm or on the cell surface. The spatial distribution of BEP1 and BEP4 proteins in eggs and embryos was established by whole mount immunohistochemistry. These proteins are located in the animal part of unfertilized and fertilized eggs; thereafter they are much less represented in structures derived from the vegetal cells of the embryo such as the micromeres of the 16 cell stage, the primary mesenchyme of blastula and the gut of gastrula. At the prism stage BEP1 and BEP4 proteins are present to some ectodermal parts and thereafter, at the pluteus stage, to the oral region.

The maternal bep1 and bep4 mRNAs and their protein products are localized at the animal pole of Paracentrotus lividus eggs. We have examined the role of the cytoskeleton in localization both of bep RNAs and BEP proteins in unfertilized and fertilized eggs and in determining the polarity of P. lividus eggs. The use of drugs such as colchicine and cytochalasin B, which depolymerize microtubules and microfilaments respectively, revealed a perturbation of localization of bep1 and bep4 mRNAs. In contrast, the microfilament inhibitor cytochalasin B had no effect on localization of BEP1 and BEP4 antigens localization, which appears to be due only to microtubules. Moreover, the presence of bep mRNAs and BEP proteins in a microtubule preparation has been demonstrated. Maintenance of the asymmetric distribution of BEP proteins during cellular division of eggs and embryos, by association with mitotic spindle, is also shown.

The sea urchin provides a relatively simple and tractable system for analyzing the early stages of embryo development. Here, we use the sea urchin species, Paracentrotus lividus, to investigate the role of Alix in key stages of embryogenesis, namely the egg fertilization and the first cleavage division. Alix is a multifunctional protein involved in different cellular processes including endocytic membrane trafficking, filamentous (F)-actin remodeling, and cytokinesis. Alix homologues have been identified in different metazoans; in these organisms, Alix is involved in oogenesis and in determination/differentiation events during embryo development. Herein, we describe the identification of the sea urchin homologue of Alix, PlAlix. The deduced amino acid sequence shows that Alix is highly conserved in sea urchins. Accordingly, we detect the PlAlix protein cross-reacting with monoclonal Alix antibodies in extracts from P. lividus, at different developmental stages. Focusing on the role of PlAlix during early embryogenesis we found that PlAlix is a maternal protein that is expressed at increasingly higher levels from fertilization to the 2-cell stage embryo. In sea urchin eggs, PlAlix localizes throughout the cytoplasm with a punctuated pattern and, soon after fertilization, accumulates in larger puncta in the cytosol, and in microvilli-like protrusions. Together our data show that PlAlix is structurally conserved from sea urchin to mammals and may open new lines of inquiry into the role of Alix during the early stages of embryo development.

Microsurgery experiments demonstrate that the animal side of the unfertilized sea urchin Paracentrotus lividus egg coincides with the side of the egg pronucleus location. It is demonstrated by means of in situ hybridization and immunostaining of whole mounts of animal or vegetal halves that the previously identified bep 1 and bep4 RNAs and their proteins are located in the animal part of the unfertilized egg and much less in the vegetal part. The addition of Fabs against BEP1 and BEP4 causes exogastrulation.

We have isolated and characterized a cDNA clone corresponding to a new member of bep (butanol, extracted, proteins) Paracentrotus lividus multigene family coding for cell surface proteins. The cDNA, called bep3, encodes a 370 amino acid protein and shares the same structural organization in the coding region with other members of the same gene family already characterized. Expression of this clone studied by Northern blot and by whole mount hybridization shows that the bep3 messenger is transcribed during oogenesis and utilized till the gastrula stage, whereas at the prism stage, unlike other members of the same gene family, new synthesis of messenger occurs. By whole mount hybridization spatial distribution of bep3 messenger in egg and embryos is established. This messenger appears located in the animal half of the unfertilized egg and moves to the cortical zone after fertilization ; it is not present in the structures derived by the vegetal part of the embryo, such as the micromeres of the 16-cell stage, the primary mesenchyme cells of the blastula, and the primary intestine of the gastrula. At the prism stage instead, hybridization of bep3 messenger is restricted to the part of the embryo that will give origin to the oral region as successively confirmed by hybridization at the pluteus stage. The result of whole mount hybridization was confirmed by Northern blot hybridization of separated meso-macromere and micromere RNAs. A Southern blot experiment demonstrates that bep3 is codified by a single copy gene. Conservation of the bep multigene family in several Mediterranean and Japanese sea urchin species has also been analyzed.

It is demonstrated that the animal side of the unfertilizedParacentrotus egg can be identified for the location of the female pronucleus on that side. It was therefore possible to demonstrate that the previously described bep RNAs and proteins are exclusively located in the animal part of the unfertilized egg. These RNAs are anchored to this part of the egg because they are binding to the cytoskeleton, probably through their 3’UTR.

Alix/AIP1 is a multifunctional adaptor protein that participates in basic cellular processes, including membrane trafficking and actin cytoskeleton assembly, by binding selectively to a variety of partner proteins. However, the mechanisms regulating Alix turnover, subcellular distribution, and function in muscle cells are unknown. We now report that Alix is expressed in skeletal muscle throughout myogenic differentiation. In myotubes, a specific pool of Alix colocalizes with Ozz, the substrate-binding component of the muscle-specific ubiquitin ligase complex Ozz-E3. We found that interaction of the two endogenous proteins in the differentiated muscle fibers changes Alix conformation and promotes its ubiquitination. This in turn regulates the levels of the protein in specific subcompartments, in particular the one containing the actin polymerization factor cortactin. In Ozz(-/-) myotubes, the levels of filamentous (F)-actin is perturbed, and Alix accumulates in large puncta positive for cortactin. In line with this observation, we show that the knockdown of Alix expression in C2C12 muscle cells affects the amount and distribution of F-actin, which consequently leads to changes in cell morphology, impaired formation of sarcolemmal protrusions, and defective cell motility. These findings suggest that the Ozz-E3 ligase regulates Alix at sites where the actin cytoskeleton undergoes remodeling.

We report the nucleotide sequence of a fragment of DNA derived from a sea urchin genomic clone containing the cell surface Bep4 (butanol-extracted protein 4)-encoding gene. The structural gene is interrupted by four introns and the promoter region contains TATA and CAAT consensus motifs. The transcription start point (tsp) was also determined. Remarkable homologies, between Bep4 and other proteins known to be involved in cell interactions, were observed regarding two poteintial Ca2+-binding sites and the corresponding DNA consensus sequences. We also report the conservation of the bep4 gene and its corresponding Bep4 protein between various sea urchin species by way of Southern and Western blotting.

We have localized, byin situ hybridization, two messengers, called bepl and bep4 (butanol extracted proteins), coding for cell surface proteins, involved in cell interaction, in sections of eggs of the sea urchinP. lividus. These maternal RNAs are spatially distributed in a gradient with a maximum at one pole of the egg. These results represent the first demonstration of the existence of a gradient of informational molecules in the sea urchin egg,i.e., the biological material for which the gradient theory was first proposed.

We have identified and partially characterised two antigens, extracted with 3% butanol, from Paracentrotus lividus embryos dissociated at the blastula stage, and encoded by the cDNA clones previously described as bep1 and bep4 (bep-butanol extracted proteins). The cDNA fragments containing the specific central portions of bep1 and bep4 were expressed as MS2 polymerase fusion proteins in Escherichia coli. These two fusion proteins, called 1C1 (bep1) and 4A1 (bep4), were injected subcutaneously into rabbits and the corresponding polyclonal antibodies generated. Western blot analysis of proteins, extracted with 3% butanol, from sea urchin embryos at the blastula stage (b.e.p.), established that both antibodies recognize two 33 KDa proteins. Reducing and non-reducing electrophoretic conditions show that both antibodies against bep1 and bep4 related proteins react also with a protein band of a molecular weight 66 KDa, indicating that these two antigens probably exist as dimers. Immunolocalization with anti 1C1 and 4A1 antibodies shows the presence of the related antigens also on the cell surface. Fab fragments of the polyclonal antibodies against 1C1 and 4A1 inhibited reaggregation of sea urchin embryonic cells, dissociated from blastula stage embryos. This prevention of reaggregation indicates that these proteins probably play a role in cell interaction during sea urchin embryonic development.

In Paracentrotus lividus eggs, there are RNAs localized at the animal and vegetal poles. During the first cell division, some of these RNAs are associated with the mitotic spindle, whereas others are free in the cytoplasm. Among the RNAs bound to mitotic apparatus (MA), we have found the mitochondrial 16S rRNA. By immunohistochemistry we have also detected hsp60, a mitochondrial membrane protein, localized around the MA, suggesting that the entire mitochondria are associated with it. Western blotting of proteins prepared by cellular fractionation after detergent treatment of P. lividus eggs revealed that both hsp60 and cytochrome c are not associated with cytoskeletal elements. All the above data have been confirmed by immunoblot analyses of preparations of microtubules and MA in which the presence of hsp60 and cytochrome c were detected only in the MA fraction. Moreover, mitochondrial succinate dehydrogenase activity was determined in MA and cytoplasm fractions during the first cell division, and the localization and vitality of the organelles were also confirmed by in vivo staining with Mito red. A possible role for mitochondria in the asymmetric distribution of RNAs and in cell division is discussed.

Localization of Paracentrotus lividus bep maternal mRNAs at the animal pole occurs by association with the cytoskeleton and involves a 54-kDa protein, called LP54, that is able to bind to the 3' untranslated regions (UTRs) of bep mRNAs. We describe here the isolation and purification of this protein. Antibodies raised against purified LP54 allowed us to establish its localization in P. lividus eggs and embryos. This localization coincides with the mRNAs with which it is associated, that is, the animal pole in the egg, and, after fertilization, the regions derived from this part of the egg, and finally the oral ectoderm of the pluteus. Association with the cytoskeleton was shown by the copurification of LP54 in a microtubule preparation. Involvement in bep mRNA localization was demonstrated by microinjection of anti-LP54 antibodies in P. lividus eggs, which caused alteration of spatial distribution of bep3 mRNA.

Asymmetric divisions that produce two distinct cells play a fundamental role in generating different cell types during development. Here we investigate the role of the cortex region and mitotic apparatus in asymmetrical localization and segregation of Bep4 protein in Paracentrotus lividus egg. By centrifugation of eggs with or without drugs we established an involvement of the cortex region in localization of Bep4 protein, confirmed by immunohistochemistry of isolated cortex. Association with the mitotic apparatus during cell division permits selective partitioning of Bep4 protein into the daughter cells. Direct association with spindle was also demonstrated both by Western blot and immunohistochemestry after isolation of the mitotic apparatus.

We have identified the presence of type I collagen (COLL1alpha) mRNA in Paracentrotus lividus unfertilized egg, indicating a maternal origin of this mRNA. By in situ whole mount hybridization the spatial distribution of COLL1alpha mRNA in egg and embryo at different developmental stages was established. Moreover, the presence of COLL1alpha gene in Paracentrotus lividus genome was analyzed by Southern blot experiments. The localization pattern indicates that the maternal mRNA is placed in the fertilized egg in a fixed position, relative to the embryonic axes. Furthermore, the embryonic expression is spatially restricted during development, suggesting involvement in sea urchin embryo cell specification events. The presence of two bands in Southern blot hybridization may indicate that two genes specific for COLL1alpha are present in the sea urchin genome.

We demonstrated that two mRNAs that are synthesized during the vitellogenic period of oogenesis and that code for cell surface proteins are asymmetrically distributed in the unfertilized egg of Paracentrotus lividus. At fertilization, these RNAs rapidly localize in the cortical zone at the animal pole of the egg. They are then detected in the mesomeres and the macromeres, but not in the micromeres, and thereafter are found in the ectoderm but not in the vegetal plate, mesenchyme cells, or early intestine. They disappear in late gastrula. The proteins synthesized by these mRNAs show the same territorial location during the period examined here, which included the unfertilized egg and the 16-blastomere stage. These conclusions were reached on the basis of in situ hybridization and immunostaining experiments, as well as Northern and Western blot analyses of isolated blastomeres. The possible significance of this asymmetric distribution of these two mRNAs and proteins in the establishment of the animal/vegetal axis is discussed.

Bep mRNAs are localized at the animal pole of P. lividus eggs. In the present communication the secondary structures of the 3′UTRs of the bep1, bep3 and bep4 mRNAs are reported. The minimal lenghts of these regions required to bind the 54-kDa protein, previously shown to be involved in localization and anchoring of these RNAs, is estimated. Microinjection of the bep3 3′UTR into egg shows that this RNA fragment is also able to become localized to one of the egg poles, as happens for the entire bep3 RNA.

Several cell types secrete small membranous vesicles that contain cell-specific collections of proteins, lipids, and genetic material. The function of these vesicles is to allow cell-to-cell signaling and the horizontal transfer of their cargo molecules. Here, we demonstrate that muscle cells secrete nano-sized vesicles and that their release increases during muscle differentiation. Analysis of these nanovesicles allowed us to characterize them as exosome-like particles and to define the potential role of the multifunctional protein Alix in their biogenesis.

Paracentrotus lividus unfertilized eggs were centrifuged in a sucrose gradient, so to split each into two parts: a nucleated light fragment and an anucleated heavy fragment. Northern blot analyses utilizing a bep4 probe as animal marker and H2A histone gene and 12S-mit RNA as controls indicate that the eggs are elongated along the animal-vegetal axis during centrifugation and thereafter split into an animal and a vegetal half. Treatment of the eggs with colchicine before centrifugation abolishes the animal localization of bep4 mRNA.

Bep mRNAs, i.e., maternal messengers coding for cell surface proteins, are localized in the animal part of Paracertrotus lividus egg and embryos. Here we have examined the involvement of the cytoskeleton in asymmetric distribution of bep3 mRNA. Moreover, in order to understand whether and how cis- and trans-acting factors are necessary for bep3 mRNA localization, we have looked for in vitro-specific interactions between egg proteins and bep3 mRNA. By northwestern assay we have identified a 54-kDa protein that binds to the 3'UTR of bep3 mRNA. This 54-kDa protein also permits association of 3'UTR of bep3 with cytoskeleton elements, indicating its involvement in the localization process. Binding of 54-kDa protein to 3'UTR of bep1 and bep4 has also been demonstrated, suggesting that a binding motif is shared with these other two mRNAs of the same gene family. Northwestern analyses carried out utilizing proteins extracted from different developmental stages indicate that the 54-kDa protein is the only protein able to bind to the 3'UTR of bep3.