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 FGF-2 trafficking toward the cell periphery was supported by coimmunoprecipitation and immune localization experiments. Colocalization of FGF-2 granules moving to the cell periphery and FM4-64-labelled intracellular lipids were not observed. Ouabain and methylamine, two inhibitors of FGF-2 release, were analyzed for their effects on FGF-2 intracellular localization and on vesicle shedding. Ouabain inhibited FGF-2 movements toward the cell periphery. The FGF-2 content of shed vesicles was therefore reduced. Methylamine inhibited vesicle shedding; in its presence, FGF-2 clustered at the cell periphery, but the rate of its release decreased. FGF-2 targeting to the nucleus and nucleolus was not affected by cytocalasin B, whereas it was inhibited by drugs that modify microtubule dynamics. Neither ouabain, nor methylamine interfered with FGF-2 translocation to the nucleus and nucleolus. FGF-2 targeting to the budding vesicles and to the nucleus and nucleolus is therefore mediated by fundamentally different mechanisms.
Taverna
Simona
S
Dipartimento di Biologia Cellulare e dello Sviluppo, Università di Palermo, Italy.
Rigogliuso
Salvatrice
S
Dipartimento di Biologia Cellulare e dello Sviluppo, Università di Palermo, Italy.
Salamone
Monica
M
Dipartimento di Biologia Cellulare e dello Sviluppo, Università di Palermo, Italy.
Vittorelli
Maria Letizia
ML
Dipartimento di Biologia Cellulare e dello Sviluppo, Università di Palermo, Italy.
D000818:Animals; D002417:Cattle; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism; D003599:Cytoskeleton / Q000378:metabolism; D016222:Fibroblast Growth Factor 2 / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007424:Intracellular Fluid / Q000737:chemistry / Q000378:metabolism*; D021381:Protein Transport / Q000502:physiology; D051381:Rats
D016222:Fibroblast Growth Factor 2