Shedding of membrane vesicles containing HSP70 and FGF-2 from A6 stem cells.

Analysis of intracellular FGF-2 trafficking in Sk-Hep1 cells.

Mouse A6 stem cells release active FGF-2 into extracellular space through plasma membrane vesicles

In this study, mouse mesoangioblasts were seeded onto bidimensional matrices within three-dimensional porous scaffolds of poly (L-lactic acid) (PLLA), in the presence or absence of a type I collagen coating. The cells were observed under a scanning electron microscope and tested for their adhesion, survival and proliferation. Immunolocalization of heat shock protein (Hsp) 70, an abundant and ubiquitous intracellular protein in these cells, was also performed in sectioned cell-containing scaffolds under a confocal fluorescence microscope to determine if in situ analysis of intracellular constituents was feasible. The data show that PLLA films allow direct cell adhesion and represent an optim…

Analysis of FGF-2 traffiking in Sk-Hep1 cells

Proteomic analysis of extracellular vesicles shed in vitro by MDA MB 231 breast carcinoma cells

Cross talk between tumor cells and connective tissue plays a key role in tumor progression. The communication is due to the release of signalling molecules from both tumor cells and surrounding stromal cells. Several secreted proteins lack the N-terminal signal peptides and, therefore, they are secreted by alternative unconventional processes such as secretion mechanism mediated by vesicle shedding in the extracellular matrix. Actually, a certain number of proteins, playing roles in some aspects of tumor progression, have been found in shed vesicles. For example, EMMPRIN, carried out in vesicles shed by tumor cells, stimulates matrix metalloproteinase (MMP) production in stromal fibroblasts…

Type-II Transmembrane Prolyl Dipeptidases and Matrix Metalloproteinases in Membrane Vesicles of Active Endothelial Cells

Endothelia cells in sparse culture are migratory and increase the production of gelatinases of serine- and metallo-classes in membrane vesicles. Collectively, proteases associated with membrane vesicles degrade extracellular matrix components including type-I and type-IV collagens, laminin and fibronectin. Inhibitor studies suggest the existence of small gelatinases that were derived from these serine- and metallo-proteases. Thus, further studies are warranted to demonstrate the cooperative action of metallo- and serine proteases on cell surfaces and in extracellular vesicles during endothelial cell migration in 3D collagenous matrices, and potential proteolytic activation mechanism for the…

Shed membrane vesicles and selective localization of gelatinases and MMP-9/TIMP-1 complexes.

Vescicole extracellulari rilasciate da una linea di oligodendroglioma causano inibizione della crescita neuritica e morte neuronale per apoptosi

Cellule staminali A6 di topo producono vescicole che contengono HSP70 e FGF-2

Localization of neutral ceramidase and sphingosine kinase1 in hepatoma and breast carcinoma cells and their shed vesicles

Regulation of Macromolecular Synthesis during Sea Urchin Development

Immediately following fertilization the sea urchin egg enters a period of very rapid cell division that cleaves the egg cell into about one thousand proportionately smaller cells, which form the swimming blastula, i.e. a larval form that is less vulnerable to environmental injuries since it is capable of actively swimming away from them.

Utilization of C14-glucose for amino acids and protein synthesis by the sea urchin embryo

An acid extract from dissociation medium of sea urchin embryos, induces mesenchyme differentiation

Abstract When material extracted by 1 M acetic acid from the dissociation medium of sea urchin embryos is added at low concentrations to isolated primary mesenchyme cells, it induces skeletogenesis. The same material added to dissociated blastula cells, or to embryos at the blastula stage, stimulates skeleton formation and pigment cell differentiation. On dissociated cells, it also increases cell reaggregation, thymidine incorporation and survival. On embryos, it induces exogastrulation and appearence of extraembryonic pigment cells. The activity of the extract is resistant to raised temperatures and partially to tryptic digestion but is abolished by trypsin treatment followed by heating. T…

Clustering of type-II transmembrane serine proteases (TTSPs) and matrix metalloproteases (MMPs) in the cell surface and shed membrane vesicles of active endothelial cells

Comparative analyses of endothelial and tumoral cells cultured in 2D and 3D type-1 collagen fibril gels

An active form of sphingosine kinase-1 is released in the extracellular medium as component of membrane vesicles shed by two human tumor cell line.

Expression of sphingosine kinase-1 (SphK-1) correlates with a poor survival rate of tumor patients. This effect is probably due to the ability of SphK-1 to be released into the extracellular medium where it catalyzes the biosynthesis of sphingosine-1-phosphate (S1P), a signaling molecule endowed with profound proangiogenic effects. SphK-1 is a leaderless protein which is secreted by an unconventional mechanism. In this paper, we will show that in human hepatocarcinoma Sk-Hep1 cells, extracellular signaling is followed by targeting the enzyme to the cell surface and parallels targeting of FGF-2 to the budding vesicles. We will also show that SphK-1 is present in a catalitycally active form i…

Ectosomes containing HSP70 and FGF-2 are released from mouse A6 stem cells

Shedding of Membrane Vesicles Mediates Fibroblast Growth Factor-2 Release from Cells

Fibroblast growth factor-2 (FGF-2), a polypeptide with regulatory activity on cell growth and differentiation, lacks a conventional secretory signal sequence, and its mechanism of release from cells remains unclear. We characterized the role of extracellular vesicle shedding in FGF-2 release. Viable cells released membrane vesicles in the presence of serum. However, in serum-free medium vesicle shedding was dramatically down-regulated, and the cells did not release FGF-2 activity into their conditioned medium. Addition of serum to serum-starved cells rapidly induced intracellular FGF-2 clustering under the plasma membrane and into granules that colocalized with patches of the cell membrane …

CLUSTERING OF TYPE-II TRANSMEMBRANE SERINE PROTEASES (TTSPS) AND MATRIX METALLOPROTEINASES (MMPS) IN THE CELL SURFACE AND SHED MEMBRANE VESICLES OF ACTIVE ENDOTHELIAL CELLS.

Urokinase Plasminogen Activator and Gelatinases Are Associated with Membrane Vesicles Shed by Human HT1080 Fibrosarcoma Cells

Membrane vesicles are shed by tumor cells both in vivo and in vitro. Although their functions are not well understood, it has been proposed that they may play multiple roles in tumor progression. We characterized membrane vesicles from human HT1080 fibrosarcoma cell cultures for the presence of proteinases involved in tumor invasion. By gelatin zymography and Western blotting, these vesicles showed major bands corresponding to the zymogen and active forms of gelatinase B (MMP-9) and gelatinase A (MMP-2) and to the MMP-9. tissue inhibitor of metalloproteinase 1 complex. Both gelatinases appeared to be associated with the vesicle membrane. HT1080 cell vesicles also showed a strong, plasminoge…

Vesicles shed by G26/24 oligodendroglioma cells, added to fetal murin cortical neurons, inhibit neurite sprouting and induce neuronal death

Proteolytic enzymes in membrane domains of endothelial cells cultured in type-1 collagen 3D gels

Proteolytic enzymes in membrane domains of endothelial cells cultured in type-I collagen 3D gels

Intracellular trafficking of endogenous fibroblast growth factor‐2

We have previously reported how the release of fibroblast growth factor-2 (FGF-2) is mediated by shed vesicles. In the present study, we address the question of how newly synthesized FGF-2 is targeted to the budding vesicles. Considering that in vitro cultured Sk-Hep1 hepatocarcinoma cells release FGF-2 and shed membrane vesicles only when cultured in the presence of serum, we added serum to starved cells and monitored intracellular movements of the growth factor. FGF-2 was targeted both to the cell periphery and to the nucleus and nucleolus. Movements toward the cell periphery were not influenced by drugs affecting microtubules, but were inhibited by cytocalasin B. Involvement of actin in …

Localisation of neutral ceramidase and sphingosine kinase 1 in hepatoma and breastr carcinoma cells and their shed vesicles

Meccanismi non convenzionali di secrezione dell’FGF-2

Proteomic profiling of vesicles released by 8701-bc cells

8701-BC cells were shown to release “membrane vesicles” playing a role in tumor progression mechanisms. On the other hand, production of “exosomes”, smaller vesicles known to be involved in immune response activation, had not been revealed. The first goal of this study was to separate different vesicle populations from 8701-BC cell conditioned medium. To this aim, the medium was differentially centrifuged. Western analysis revealed that the 15,000xg pelletted fraction contains β1-integrin, which had been shown to be clustered in membrane vesicles shed by 8701-BC cells, but not Hsc70, a protein found in exosomes. On the contrary, Hsc70 is detectable while β1-integrin is not present in the fr…

Clustering of type-II transmembrane serine proteases (TTSPs) and matrix metalloproteinases (MMPs) in the cell surface and shed membrane vesicles of active endothelial cells.

Comparative analyses of endothelial and tumoral cells cultured in 2D and 3D type-I collagen fibril gels.

CLUSTERING OF PROTEOLYTIC ENZYMES ON SPECIALIZED PLASMA MEMBRANE DOMAINS OF ENDOTHELIAL CELLS. ROLE IN ANGIOGENESIS

CLUSTERING OF SPECIFIC MOLECULES IN SHED VESICLES.

In several tumor cell lines serum addition causes release of vesicles that bud from the cell surface and can be purified from cell conditionated media. These vesicles are known to be involved in cell migration and tumor progression. We recently demonstrated that FGF-2, a growth factor devoid of the classical signaling sequence, is secreted as a component of these vesicles. In order to analyze how molecules are clustered in shed vesicles we followed their intracellular movements by immunofluorescence techniques. The role of cytoskeletal components was analyzed using molecules such as paclitaxol, nocodazole, colchicin and cytochalasin which destabilize their organization. In the absence of se…