For example, a variant of FGF2 lacking the two key residues responsible for its interaction with the Na,K ATPase exhibits a 30-40% reduction in plasma membrane recruitment and translocation efficiency ). However, these components are not essential for FGF2 membrane translocation in reconstituted systems. Also, we hypothesize that the fast kinetics of this process observed in intact cells is driven by cellular redox systems facilitating the efficient oxidative dimerization of FGF2 disulfide bridges in combination with the asymmetric nature of the plasma membrane. Current views describe the plasma membrane as a composite material, where the exoplasmic leaflet primarily ensures functional barrier integrity, while the cytoplasmic leaflet contributes to membrane fluidity . Due to technical limitations, this duality is often lost in in vitro systems, where synthetic liposomes are typically produced with symmetric membranes. For example, a symmetric distribution of SM-which in living cells is predominantly located in the outer leaflet-can lead to excessive membrane rigidity when interacting with cholesterol. This rigidity disrupts the dual nature of the plasma membrane, and it might create an additional barrier that hinders the FGF2 translocation process. Focusing on PI(4,5)P 2 , the minimal requirement for FGF2 translocation, its asymmetric distribution within the plasma membrane can play a fundamental role in the membrane translocation mechanism. Our current model proposes that FGF2 undergoes oligomerization after interacting with the inner leaflet of the plasma membrane. Notably, a single FGF2 molecule can recruit up to 5 PI(4,5)P 2 molecules through high-and low-affinity binding sites . Given that FGF2 is known to oligomerize in a membrane-dependent manner into at least tetramers , this would result in the accumulation of approximately 20 PI(4,5)P 2 molecules in a spatially confined area. The localized accumulation of PI(4,5)P 2 molecules near the protein is expected to result in a local concentration of ~20 mol%. The inner leaflet is enriched with ~30 mol% of PE lipids . Both PI(4,5)P 2 and PE are conical, non-bilayer lipids with intrinsic curvature properties. This combination will likely generate significant membrane stress and induce curvature (Figure , left panel), thereby creating conditions that locally destabilize the bilayer and facilitate the membrane pore formation. Additionally, PI(4,5)P 2 carries a highly negative charge under physiological conditions, which would create a local charge gradient across the membrane. This gradient will likely drive the directionality of the translocation process and increase its likelihood, as illustrated in Figure , left panel. Contrarily, a symmetric distribution of PI(4,5)P 2 would neutralize this gradient, suppressing curvature formation (Figure , right panel). The long acyl chains of PI(4,5)P 2 (18:0 and 20:4) could interdigitate from both sides of the membrane, leading to a symmetrical accumulation that cancels out the curvature-inducing effects, as hypothesized in Figure , right panel. Furthermore, heparan sulfate chains, which are also negatively charged, are positioned on the outer leaflet of the plasma membrane. A highly negative lipid such as PI(4,5)P 2 in the outer leaflet would reduce the proximity of the heparan sulfate chains to the membrane surface due to electrostatic repulsion, as schematized in the right panels of Figure . Notably, FGF2 translocation depends on membrane-proximal heparan sulfate proteoglycans (HSPGs) on the cell surface, which capture and disassemble FGF2 oligomers. Among the diverse sub-classes of HSPGs, including syndecans, perlecans, and glypicans, we unexpectedly discovered that Glypican-1 (GPC1) is the principal and rate-limiting factor driving the unconventional secretion of FGF2 . This is due to its stronger binding affinity for FGF2 and proximity to the membrane surface. Exploring the role of PI(4,5)P 2 asymmetry in synthetic and cellular systems is essential to validate the proposed mechanism. Specifically, developing a method to generate PI(4,5)P 2 -asymmetric vesicles would allow for investigations of FGF2 pore formation and translocation kinetics. By mimicking the natural asymmetry of PI(4,5)P 2 in the inner plasma membrane leaflet, we hypothesize that this approach will accelerate the kinetics of FGF2 oligomerization and translocation, as the spatially constrained accumulation of PI(4,5)P 2 and the resulting membrane stress and charge gradient would facilitate membrane curvature and pore opening. Conversely, disrupting PI(4,5)P 2 asymmetry in living cell systems could offer deeper insights into its critical role in unconventional FGF2 secretion. We predict that the loss of PI(4,5)P 2 asymmetry would neutralize the charge gradient, reduce membrane curvature, and increase the distance of heparan sulfate chains from the membrane surface. This, in turn, would slow down the kinetics of FGF2 secretion. ,5)P 2 in the inner leaflet causes an extreme charge gradient and curvature-dependent stress in the asymmetric membrane. In the symmetric membrane PI(4,5)P 2 accumulates in both inner and outer leaflet due to interdigitation, due to which the charge gradient and stress are canceled out in the symmetric PI(4,5)P 2 membrane; (C) Presence of symmetric PI(4,5)P 2 in the membrane will cause the repulsion between PI(4,5)P 2 on the outer leaflet and heparan sulfate chains, resulting in the orientation of heparan sulfate chains farther from the membrane, translocating FGF2 presumably slower comparative to the asymmetric system.