CLUSTERING OF SPECIFIC MOLECULES IN SHED VESICLES.

In several tumor cell lines serum addition causes release of vesicles that bud from the cellsurface and can be purified from cell conditionated media. These vesicles are known to beinvolved in cell migration and tumor progression.We recently demonstrated that FGF-2, a growth factor devoid of the classical signalingsequence, is secreted as a component of these vesicles. In order to analyze how molecules areclustered in shed vesicles we followed their intracellular movements by immunofluorescencetechniques. The role of cytoskeletal components was analyzed using molecules such aspaclitaxol, nocodazole, colchicin and cytochalasin which destabilize their organization. In theabsence of serum, no clear localization of FGF-2 was observed. After serum addition, FGF-2was localized partially in the nucleus and nucleolus, and partially in granules near the plasmamembrane. Nocodazole and paclitaxol, which interfere with microtubular organization, inhibitFGF-2 nuclear localization but do not appear to modify FGF-2 movements toward the plasmamembrane. Cytocalasine, which interferes with actin polymerization, decreases FGF-2clustering in granules localized near the cell membrane. In summary, microtubularorganization seems to be required for FGF-2 nuclear localization while actin filaments appearto be needed for FGF-2 translocation toward the plasma membrane.In a different set of experiments, we analyzed localization of neutral ceramidase (ncDase) andof Sphingosine Kinase (SphK). Ceramidase catalyzes ceramide hydrolysis giving rise tosphingosine, which in turn can be phosphorilated to S1P by SphK. Sp1P is an importantsignaling molecule involved in induction of cell migration and apoptosis. SphK-1 was knownto be shed into the extracellular medium by an unconventional mechanism, we hypothesizedthat shed vesicles could vehicle its release.We therefore analyzed the localization of membrane-bound isoforms of ceramidase (ncDase)and of SphK (SphK-1 and SphK-2) by western blotting and Immunofluorescence techniques.Immunolocalization showed that ncDase is located into the plasma membrane and in cellularextensions. The concentration of ncDase was found to be higher in extracts of shed vesiclesthan in cell extracts. SphK-1 was found to be localized in plasma membrane and in vesicles,which appear to be enriched in this enzyme. SphK-2 was preferentially located in the nucleusand it was not detected in vesicles. In conclusion, ncDase and SphK were found to beclustered in shed vesicles. In order to analyze the role of SphK-1, either in the sheddingphenomenon or in vesicle functions, we used transiently transfected SK-Hep1 cells, whichoverexpress SphK or express a non-functional mutant of this enzyme.