Different Conformational Subensembles of the Intrinsically Disordered Protein α-Synuclein in Cells

Abstract

The intrinsically disordered protein α-synuclein (αS) is thought to play an important role in cellular membrane processes. Although in vitro experiments indicate that this initially disordered protein obtains structure upon membrane binding, NMR and EPR studies in cells could not single out any conformational subensemble. Here we microinjected small amounts of αS, labeled with a Förster resonance energy transfer (FRET) pair, into SH-SY5Y cells to investigate conformational changes upon membrane binding. Our FRET studies show a clear conformational difference between αS in the cytosol and when bound to small vesicles. The identification of these different conformational subensembles inside cells resolves the apparent contradiction between in vitro and in vivo experiments and shows that at least two different conformational subensembles of αS exist in cells. The existence of conformational subensembles supports the idea that αS can obtain different functions which can possibly be dynamically addressed with changing intracellular physicochemical conditions.

Figure 1

Localization of αS in cells. (A) Confocal microscopy image of rat primary neurons immuno-stained for αS (red), actin filaments (green), and nuclei (blue) (scale bar is 10 μm). (B) In the αS fluorescence from image A, here shown in a separate image, the presence of both a diffuse background fluorescence and distinct puncta is clearly visible. (C) Confocal microscopy image of a single SH-SY5Y cell that was microinjected with αS-AF488. The αS localization pattern is the same as that of primary neurons. The αS is visible as a diffuse background and distinct high-intensity puncta (scale bar is 5 μm).

Figure 2

Membrane-bound αS. To confirm that the distinct puncta in the cells represent the vesicle-bound αS population, colocalization with the membrane marker WGA-AF647 was studied. (A) Confocal fluorescence image of SH-SY5Y cells microinjected with αS-AF488 (green) and stained with WGA-AF647 (red). Independent excitation and detection of the injected αS-AF488 (excitation 485 nm, detection 550/88 nm) and WGA-AF647 (excitation 640 nm, detection >665 nm) resulted in moderate colocalization. However, the two dyes form an efficient FRET pair, and this may render a fraction of the αS-AF488 invisible. (B) Scatter plots of the WGA-AF647 fluorescence intensity versus αS-AF488 fluorescence intensity. Photobleaching of WGA-AF647 dequenched the emission of αS-AF488, confirming the formation of a FRET pair and thus the nanometer proximity of the two dyes. The dequenching of the αS-AF488 fluorescence is visible as a shift of the intensity distribution. The original distribution presented in green changes upon bleaching to the distribution presented in orange. Data points were obtained per pixel. (C) Colocalization image obtained by combining the image of the initial WGA-AF647 fluorescence with the image of the αS-AF488 fluorescence after dequenching. Colocalization of αS-AF488 with the membrane is visible in yellow. In both images the position of the nucleus is indicated with a blue transparent oval; the scale bar is 5 μm.

Figure 3

(A) Schematic of the antiparallel helices (left) and a representation of a disordered conformation of the FRET labeled αS. Differences in distance between the red and green emitting fluorophores give different FRET, here in the cartoon depicted by different relative sizes of the donor and acceptor emission halos. (B) Composite donor and acceptor fluorescence image of a single cell microinjected with the FRET-labeled αS. Regions of low FRET (green) and high FRET (yellow-red) can be discriminated. The position of the nucleus is indicated with a transparent blue oval (scale bar is 5 μm). (C) Histogram of the FRET index for αS in the cytoplasm (green) and on vesicles (red). (D) Cumulative probability histogram of the FRET index for αS in the cytoplasm (dotted green), on fibrils (dot-dashed blue), and on vesicles (dashed red). The autofluorescence index of unlabeled cells is indicated in solid black.