Ezrin constructs. Schematic representations of ezrin fused to GFP. Ezrin contains an N-terminal membrane-binding domain, followed by an α-helical domain and a C-terminal actin-binding domain. GFP is represented by a star.

Classification of relaxation processes. Elementary situations used for modeling recovery curves. (A) Diffusion in the cytosol with a diffusion coefficient D_cyt. (B) Membrane diffusion with a coefficient D_mb. (C) Slow membrane diffusion or immobility. If D ≪ d⋅T (≈0.25 μm²⋅s⁻¹), molecules appear immobile during the observation time of T_mes = 10 s. (D–F) Two-compartment situations, where recovery quantitatively reflects the kinetic parameters k⁺, k⁻ of reaction-limited relaxations, either (D) between a membrane-diffusive and a cytoplasmic population, (E) a slowly or nondiffusing membrane population and a cytoplasmic population, or (F) two membrane populations, one diffusing and one virtually immobile (see Supporting Methods on the PNAS web site).

Dynamic behavior of ezrin-GFP mutants in microvilli. (a) Ezrin-Δ53-GFP (black), ezrin-GFP and ezrin-GFPi (gray) in microvilli (n = 172 and SEM = 1.2% for ezrin-Δ53). (b) Ezrin-Δ53-GFP (black) fitted with diffusive relaxation (D = 0.5, 0.8 and 1 μm²⋅s⁻¹) (gray). (c) Ezrin-Τ567Α-GFP (black, n = 115 and SEM = 1.6%), ezrin-Δ53-GFP (gray, Bottom) and ezrin-GFP (gray, Upper) in microvilli. The recovery of ezrin-T567A was analyzed by successive subtractions, first of 45% of D = 0.8 μm²⋅s⁻¹, second of 15% of D = 10 μm²⋅s⁻¹. Possible fits after these two subtractions with a slow diffusive process (D = 0.1, μm²⋅s⁻¹) or a reaction-limited process (τ = 7.7 s) are shown (both fits overlap: black). (e) Ezrin-T567D-GFP (black, n = 34 and SEM = 1.5%), ezrin-GFPi and GFP- actin (gray) in microvilli. The recovery curve of immobile proteins is shown (gray, Bottom). (f) Fit of ezrin-T567D-GFP (gray) with a slow diffusive process (D = 0.3 μm²⋅s⁻¹) or a reaction-limited process of τ = 3 s (black lines).

Subcellular localization of actin and ezrin constructs fused to GFP in LLC-PK1 cells. Images were acquired by two-photon microscopy, using a 900-nm pulsed laser delivering 70-fs pulses, with a ×63 oil-immersion objective (Zeiss). Individual cells were imaged successively in two planes: through the apical surface (simple letters) and through the middle plane of the cells (primed letters). GFP-actin (a and a′), ezrin-GFP (b and b′), ezrin-GFPi (c and c′), ezrin-Δ53-GFP (d and d′), ezrin-Τ567Α-GFP (e and e′), and ezrin-T567D-GFP (f and f′). (Bar = 10 μm.)

Dynamic behavior of GFP-actin, ezrin-GFP, and ezrin-GFPi in the cytoplasm and in the microvilli. (a–f) Local measurements. For all figures, mean recovery curves are drawn. (a) Actin-GFP in microvilli, before (Upper) and after (Lower) jasplakinolide treatment (n = 73 and SEM = 2%). (b) Numerical fits of GFP-actin recovery (black), with D = 5, 10 and 20 μm²⋅s⁻¹ (gray). (c) Cytoplasmic ezrin-GFP (gray) (n = 53, SEM = 2%) fitted with a diffusive curve of D = 30 μm²⋅s⁻¹ (black). (d) Ezrin-GFP (Upper, gray, n = 117 and SEM = 1.4%) and ezrin-GFPi (Bottom, gray, n = 118 and SEM = 1.5%) in microvilli. Black: fits of the short time regions with diffusive curves of D = 10 μm²⋅s⁻¹. (e) The difference between the ezrin-GFP recovery curve and the fast-diffusing curve of D = 10 μm²⋅s⁻¹ (gray) was fitted with a slow diffusive process of D = 0.1 μm²⋅s⁻¹ or with reaction-limited exchange process modeled by an exponential of τ = 5.7 s (both fits overlap: black). (f) The difference between the ezrin-GFPi recovery curve and the fast-diffusing curve of D = 10 μm²⋅s⁻¹ (gray) was fitted with a slow diffusive process of D = 0.1 μm²⋅s⁻¹ or an exchange process of τ = 4.8 s (both fits overlap: black). (g) Synopsis of the different time domains of relaxation, accessible either by local analysis or time-lapse imaging FRAP experiments, with arbitrary vertical scale. (h) Relaxation in a typical time-lapse imaging FRAP experiment. The profiles are similar for ezrin-GFP and -GFPi, shown here. The recovery was analyzed in terms of the average fluorescence intensity profile (y, arbitrary units) along the direction perpendicular to the bleached band. Successive profiles before photobleaching and at 7 s, 14 s, 30 s, 1 min, and 2 min after photobleaching are shown.

Proposed model for ezrin association with the membrane. We propose that cytosolic ezrin first binds to membrane, entering state E1 with high lateral mobility. From that state on, ezrin enters state E2, whereby its mobility is much reduced, with a lifetime of 3–5 s that might be interpreted as the typical time of a reverse transition leading ezrin backwards to E1. The fraction of ezrin molecules that do not dissociate over that time scale proceeds irreversibly to the E3 state with virtually zero mobility. E3 might dissociate slowly, on the order of minutes, most likely to the cytosolic pool E0. (Plain arrows represent the main elements of the model and dashed arrows open alternative possibilities.)