Nature of curvature coupling of amphiphysin with membranes depends on its bound density - PubMed (original) (raw)

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

Benoît Sorre et al. Proc Natl Acad Sci U S A. 2012.

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.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

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 = Φmax/(1 + _K_d/_C_bulk) with Φmax = 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.

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 formula image, where A = 55 ± 2 pN1/2·nm1/2 and B = 0 without protein and A = 52 ± 2 × pN1/2·nm1/2 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 formula image. (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_B_T membrane (32 nm).

Fig. 3.

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 formula image with protein (□) or without (▪). The linear fit to the force, formula image, gives κ = 12 ± 2 k_B_T. (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 formula image.

Fig. 4.

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 formula image, where A = 56 ± 1 pN1/2 nm1/2, 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/4_πσ; in its presence, the radius was found using R t = f/2_πσ. (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_∗ = A_Φ_v + B, where A = (4 ± 1) × 10-8 Nm-1 μm-2 and B = (-2.7 ± 1.4) × 10-5 Nm-1.

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