Sample CLSM images of CHO-K1 cells transiently transfected with NBCe1-A.

<p>(A) NBCe1-A-EGFP. (B) NBCe1-A-α-bungarotoxin mutant expressed in cells, and labeled with Alexa 488-α-bungarotoxin conjugate. In both cases, basal membranes of highly adherent cells was imaged. The cells were chemically fixed prior to imaging. The pixel size is 0.046 µm. The images were taken under the identical acquisition condition so that valid comparisons between different images could be made. The image contrast was enhanced in both panel A and B for visualization purposes.</p

SpIDA measurements of NBCe1-A monomer-dimer density in expression systems (CHO-K1 cells) and native tissues.

<p>SpIDA was applied to CLSM images assuming a monomer-dimer mixture model (Equation 4). The values of monomeric, dimeric and total subunit number density were obtained for NBCe1-A in heterologous expression systems and tissues. The recovered number density of monomeric NBCe1-A in tissues is expected to be overestimated based on the presence of non-specific antibody binding. The error bars represent the standard error of the mean obtained from multiple cells. All of the measurements were carried out under identical collection conditions.</p

Sample CLSM images of native NBCe1-A on basolateral cell membranes of rat kidney.

<p>Post fixation, tissues were immunolabeled with the primary anti-wt-NBCe1-A antibody (I) and the secondary (II) Alexa Fluor 488 conjugated antibody. Panel A shows non-specific secondary antibody staining in the absence of the primary. Panel B shows kidney cells immunostained with both antibodies. The pixel size is 0.0921 µm. The images were taken at identical acquisition conditions so valid comparisons between different images could be made. The image contrast was enhanced for visualization and comparison purposes.</p

Dual-color CLSM images of HEK293 cells transiently expressing NBCe1-A-EGFP immunostained with Alexa Fluor 647 conjugated antibody.

<p>Post fixation, cells were immunolabeled with the primary anti-wt-NBCe1-A antibody and the secondary Alexa Fluor 647 conjugated antibody. Panel A shows the fluorescence image taken in channel 0 (green), Panel B shows the image recorded in channel 1 (red), and Panel C is the overlap of the two. The image contrast was enhanced for better visualization of non-specific binding of the secondary Alexa Fluor 647 conjugated antibody to HEK293 cells. The pixel size is 0.0921 µm.</p

Characterization of the shot noise profiles for PMT detector.

<p>Plot of the variance as a function of the mean intensity for uniform illumination acquired with CLSM. Only the initial part of the data was taken into account for linear fitting.</p

Quantal brightness of Alexa Fluor 488 measured for 2D samples prepared with different values of concentration in 3D solution.

<p>(A) Mean intensity measured from ROIs. (B) The values of the quantal brightness measured with fluorescent image moment analysis and SpIDA as a function of the dye concentration in solution. The error bars were calculated as the standard error of multiple images taken at different locations in the sample.</p

The measurements of quantal brightness of monomeric EGFP for two distinct sets of imaging conditions (referred as “Set I” and “Set II” throughout the text).

<p>Set I corresponds to pixel size of 0.046 µm, with the scan speed set to “fast” (18.1 µm/µs), and Set II - pixel size of 0.0921 µm, with the scan speed set to “slow” (10.1 µm/µs). The values of ε were recovered with both fluorescence moment image analysis and SpIDA for comparison purposes. The error bars were calculated as the standard error of images of multiple cells. A) measured quantal brightness for various values of the mean intensity; B) average values (with error bars) for sets I & II.</p

Spatial fluorescence intensity moment analysis of the NBCe1-A oligomerization states.

<p>The percent occurrence graphs (A–C) show the recovered values of quantal brightness normalized to MEU. The experiments were carried out on fixed cells. All of the measurements summarized in each histogram were carried under identical collection conditions.</p

Single Particle Electron Microscopy Analysis of the Bovine Anion Exchanger 1 Reveals a Flexible Linker Connecting the Cytoplasmic and Membrane Domains

<div><p>Anion exchanger 1 (AE1) is the major erythrocyte membrane protein that mediates chloride/bicarbonate exchange across the erythrocyte membrane facilitating CO₂ transport by the blood, and anchors the plasma membrane to the spectrin-based cytoskeleton. This multi-protein cytoskeletal complex plays an important role in erythrocyte elasticity and membrane stability. An in-frame AE1 deletion of nine amino acids in the cytoplasmic domain in a proximity to the membrane domain results in a marked increase in membrane rigidity and ovalocytic red cells in the disease Southeast Asian Ovalocytosis (SAO). We hypothesized that AE1 has a flexible region connecting the cytoplasmic and membrane domains, which is partially deleted in SAO, thus causing the loss of erythrocyte elasticity. To explore this hypothesis, we developed a new non-denaturing method of AE1 purification from bovine erythrocyte membranes. A three-dimensional (3D) structure of bovine AE1 at 2.4 nm resolution was obtained by negative staining electron microscopy, orthogonal tilt reconstruction and single particle analysis. The cytoplasmic and membrane domains are connected by two parallel linkers. Image classification demonstrated substantial flexibility in the linker region. We propose a mechanism whereby flexibility of the linker region plays a critical role in regulating red cell elasticity.</p> </div

Single-particle reconstruction of bovine AE1.

<p>(<b>a</b>) Schematic illustration of the OTR data collection method. For each target sample area, two micrographs were recorded with the grid tilted at −45° and +45°, respectively. (<b>b</b>) 3D map generated by averaging 25 OTR maps. Two orthogonal views, defined as front view (left panel) and side view (right panel), are shown. (<b>c</b>) Final map obtained by merging 174,197 particle images with single particle reconstruction method. The map in (<b>c</b>) is shown in the same orientations as in (<b>b</b>). (<b>d</b>) Fourier shell correlation (FSC) coefficient between two reconstructions obtained from even- and odd-numbered particle images. The effective resolution is estimated to be 2.4 nm using the 0.5 FSC cut-off. (<b>e</b>) Comparisons of the computed projection, class average, and raw particle. Four representative views (top, tilt, front, and side) are show from left to right, respectively. (<b>f</b>) Euler angle distribution of classified particles. The brightness of each point indicates the number of particles used in the class average in that orientation.</p