A key observation in elucidating the FGF2 secretion pathway was the ability of FGF2 to physically traverse the membranes of affinity-purified plasma membrane vesicles with an inside-out topology ). This suggested unconventional secretion of FGF2 from cells to be mediated by direct translocation across plasma membranes. This conclusion could be confirmed by TIRF imaging of individual FGF2 membrane translocation events at the plasma membrane in real time with single-molecule resolution . Further key insights that led to the unraveling of this secretory mechanism were its dependence on the phosphoinositide PI(4,5)P 2 at the inner leaflet ) and heparan sulfate proteoglycans at the outer leaflet of the plasma membrane . The interaction of FGF2 with PI(4,5)P 2 was then shown to trigger FGF2 oligomerization, which led to the formation of lipidic membrane pores implicated in FGF2 membrane translocation . Using giant unilamellar vesicles with an inside-out topology containing PI(4,5)P 2 on their surfaces and luminal encapsulation of heparin molecules with high affinity towards FGF2, the minimal machinery required for directional translocation of FGF2 across membranes could be defined . These studies revealed PI(4,5)P 2 and heparan sulfates from proteoglycans on opposing sides of the plasma membrane along with the ability of FGF2 to oligomerize and to form lipidic membrane pores to be the minimal machinery driving FGF2 membrane translocation. NMR binding studies of FGF2 towards PI(4,5)P 2 versus heparin established these interactions to be mutually exclusive, providing a compelling data set that explains directionality of FGF2 membrane translocation from the cytoplasm into the extracellular space . The functional oligomeric state of FGF2 was found to be highly dynamic, with about 4 to 8 FGF2 subunits required to trigger the formation of a membrane pore . The building blocks of pore-forming FGF2 oligomers were found to be disulfide-bridged dimers that form at the inner plasma membrane leaflet, followed by assembly into higher oligomers with a membrane pore-forming activity . It has been hypothesized that FGF2 oligomers cause the local and asymmetric accumulation of the non-bilayer lipid PI(4,5)P 2 at the inner plasma membrane leaflet, which triggers the opening of a lipidic membrane pore with a toroidal architecture. The discovery of PI(4,5)P 2 -induced oligomerization triggering the formation of a membrane pore through which FGF2 reaches the extracellular space also explained earlier findings demonstrating that FGF2 membrane translocation does not involve unfolded intermediates of FGF2 . With all of these aspects in mind, the unusual secretory pathway of FGF2 can be considered a protein self-translocation mechanism that, depending on PI(4,5)P 2 and heparan sulfates on opposing sides of the plasma membrane, is based upon the ability of FGF2 to trigger a membrane pore through which it is capable of physically traversing the plasma membrane .