Nature of curvature coupling of amphiphysin with membranes depends on its bound density

Abstract

Cells are populated by a vast array of membrane-binding proteins that execute critical functions. Functions, like signaling and intracellular transport, require the abilities to bind to highly curved membranes and to trigger membrane deformation. Among these proteins is amphiphysin 1, implicated in clathrin-mediated endocytosis. It contains a Bin-Amphiphysin-Rvs membrane-binding domain with an N-terminal amphipathic helix that senses and generates membrane curvature. However, an understanding of the parameters distinguishing these two functions is missing. By pulling a highly curved nanotube of controlled radius from a giant vesicle in a solution containing amphiphysin, we observed that the action of the protein depends directly on its density on the membrane. At low densities of protein on the nearly flat vesicle, the distribution of proteins and the mechanical effects induced are described by a model based on spontaneous curvature induction. The tube radius and force are modified by protein binding but still depend on membrane tension. In the dilute limit, when practically no proteins were present on the vesicle, no mechanical effects were detected, but strong protein enrichment proportional to curvature was seen on the tube. At high densities, the radius is independent of tension and vesicle protein density, resulting from the formation of a scaffold around the tube. As a consequence, the scaling of the force with tension is modified. For the entire density range, protein was enriched on the tube as compared to the vesicle. Our approach shows that the strength of curvature sensing and mechanical effects on the tube depends on the protein density.

Fig. 1.

Two regimes for amphiphysin 1 binding to a GUV membrane DOPC∶DOPE∶DOPS (1∶1∶1). (A) Adsorption isotherm of amphiphysin 1 on GUV. The amphiphysin density, Φ_ν, deduced from the fluorescence signal, as a function of the protein bulk density C_bulk. Data are fitted with Φ_v = Φ_max/(1 + K_d/C_bulk) with Φ_max = 3,000 μm^-2 and K_d = 35 nm (error bars correspond to standard deviation. N = 6 vesicles for each point). (B) For C_bulk < K_d, the amphiphysin signal (green) is undetectable on the GUV, the fluorescent lipid signal is in red, and amphiphysin is visible on the tube. (C) At high C_bulk (here equal to 1 μM) amphiphysin binds to the GUV and forms tubes rich in amphiphysin (green fluorescence). (Scale bar: 5 μm.)

Fig. 2.

Low-density regime. (A) Force curve shift due to amphiphysin binding in the low-density regime. Here, Φ_v = 280 ± 100 μm^-2. The force is lower with protein (▪) than without (□). Data are fitted to , where A = 55 ± 2 pN^1/2·nm^1/2 and B = 0 without protein and A = 52 ± 2 × pN^1/2·nm^1/2 and B = -3.5 ± 2 pN with protein. (B) The radius, R_t, versus tension, σ, with protein (○) or without (●). Radius is deduced from fluorescence. (C) Linear variation of the sorting ratio as a function of 1/R_t. Data correspond to five independent experiments. A fit using Eq. 3 gives . (D) Variation of R_t, at fixed tension σ = 2 × 10^-5 N/m, versus Φ. The data are fitted to R_t = 32 - (38 ± 5) × 10^-3Φ_v nm (line), as deduced from Eq. 2. Round symbol corresponds to expected radius for a 10 k_BT membrane (32 nm).

Fig. 3.

The dilute limit. The results presented in this figure correspond to a single GUV with Φ_v < 50 μm^-2. (A) No difference between the tube force as a function of with protein (□) or without (▪). The linear fit to the force, , gives κ = 12 ± 2 k_BT. (B) The radius, R_t, versus tension, σ, with protein (empty symbols) or without (full symbols). Radius is deduced either from fluorescence (round symbols) or from force (square symbols) measurements. (C) Amphiphysin density on the tube, Φ_t, versus tube curvature, 1/R_t. R_t was found from force measurements. A linear fit yields Φ_t = A/R_t μm^-2, where A = 29 ± 2 μm^-1. (D) Linear variation of the sorting ratio as a function of 1/R_t. Data correspond to five independent experiments. A fit using Eq. 4 gives .

Fig. 4.

High-density regime. The experiments presented in A and B correspond to a single GUV with Φ_v = 1,100 ± 100 μm^-2. (A) The force is lower with protein (▪) than without (□). Force data without protein are fitted to , where A = 56 ± 1 pN^1/2 nm^1/2, and without to f = B(σ - σ^∗), where B = 67 ± 4 nm and σ^∗ = (1.0 ± 0.9) × 10^-5 N/m (the asterisk denotes σ^∗). (B) R_t versus σ with no protein (empty symbols) and with protein (full symbols). The radius is found from fluorescence (round symbols) or from force (square symbols) measurements. With no protein, the radius was determined from the force according to R_t = f/4πσ; in its presence, the radius was found using R_t = f/2πσ. (C) R_t as a function of Φ_v, as measured by fluorescence. (D) Tension at zero force, σ^∗, versus Φ_v. Data were fitted to Eq. 6, neglecting the logarithmic term, σ^∗ = AΦ_v + B, where A = (4 ± 1) × 10^-8 Nm^-1 μm^-2 and B = (-2.7 ± 1.4) × 10^-5 Nm^-1.