Curvature sorting of peripheral proteins on solid-supported wavy membranes - PubMed (original) (raw)

. 2012 Sep 4;28(35):12838-43.

doi: 10.1021/la302205b. Epub 2012 Aug 23.

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Curvature sorting of peripheral proteins on solid-supported wavy membranes

Wan-Ting Hsieh et al. Langmuir. 2012.

Abstract

Cellular membrane deformation and the associated redistribution of membrane-bound proteins are important aspects of membrane function. Current model membrane approaches for studying curvature sensing are limited to positive curvatures and often require complex and delicate experimental setups. To overcome these challenges, we fabricated a wavy substrate by imposing a range of curvatures onto an adhering lipid bilayer membrane. We examined the curvature sorting of several peripheral proteins binding to the wavy membrane and observed them to partition into distinct regions of curvature. Furthermore, single-molecule imaging experiments suggested that the curvature sensing of proteins on low-curvature substrates requires cooperative interactions.

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Figures

Figure 1

Figure 1. Surface topography of wavy glass surface

(A) Schematic cross-section view of the surface with topography pattern of a wavelength of 1 µm and depth of 110 nm supporting a fluid lipid bilayer membrane. (B) Transmitted light and (C) AFM images of a wavy glass surface with average depth of 110 nm. (D) AFM height profile quantified from (C). Note that the aspect ratio of the surface profile is adjusted for display purposes. The average curvature range within a half wavelength highlighted in the cyan box is shown in the inset. The error bars represent standard deviations from five regions with identical curvatures on a single substrate.

Figure 2

Figure 2. Curvature sensing proteins exhibit preferential partitioning on wavy membranes

Proteins were incubated on fluid wavy membranes (containing a variety of negatively charged phospholipids or GM1 in a background of DOPC lipids) and visualized via confocal fluorescence microscopy. (A) Partitioning of ENTH-GFP into positive curvature regions identified by transmitted light imaging (see upper edge). Fluorescence distributions of (B) Endophilin N-BAR-Alexa Fluor 488 and (C) BIN1 N-BAR-Alexa Fluor 488 showing enrichment in positive curvature regions. (D) Preferential partitioning of CTB-Alexa Fluor 555 into negative curvature regions identified via transmitted light imaging (see upper edge). No significant curvature preference was observed for (E) streptavidin-FITC bound to membranes containing 1% cap-biotin PE, and (F) 0.1% of the lipid fluorophore Texas-Red DHPE in a wavy DOPC membrane.

Figure 3

Figure 3. Analysis of protein and lipid distributions with respect to membrane curvatures

Fluorescence images from confocal microscopy were normalized to the mean intensity of the image and analyzed as a function of membrane curvature. Left panel: ENTH (black), Endophilin N-BAR (red), BIN1 N-BAR (green), and CTB (cyan). Right panel: streptavidin (purple), and Texas-Red DHPE (blue). Error bars represent standard deviations from at least six different regions on the substrate for three different bilayer preparations.

Figure 4

Figure 4. ENTH domain exhibits lateral mobility in the wavy membrane and tether-GUV systems

(A) Time-lapse recovery of ENTH was examined from 2D photobleaching experiments. Error bars represent standard deviation from three bilayer preparations and five measurements in total. (B) Time-lapse recovery fraction of ENTH bound on the membrane tether generated from single GUV containing Texas-Red DHPE (red) and ENTH-GFP (green). Error bars represent standard deviations from three tethers. Gray lines in (A) and (B) represent the best fit curves. (C) Two representative trajectories of single ENTH domains on the wavy membrane. ENTH domain was observed to diffuse across both the positive-curvature (red dashed lines) and negative-curvature regions (blue dashed lines) of the wavy membrane. (D) The spatial distribution of single moving ENTH domains on the wavy membrane with positive and negative curvature regions. More than 400 steps and three different wavy membranes were analyzed.

Figure 5

Figure 5. Solid-supported wavy membrane shows higher capacity for curvature sorting compared to tether-GUV system

Normalized ENTH (squares) and CTB (triangles) intensities as a function of membrane curvature in solid-supported wavy membrane were compared with tether-GUV system (circles and diamonds; data reproduced from refs. 13 and 21, respectively). Experimental data points were normalized to the value found for zero curvature, and only the positive curvature regime is represented, in logarithmic form. The inset compares the slopes of linear sorting versus curvature plots.

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