Visualizing structural dynamics of thylakoid membranes

Abstract

To optimize photosynthesis, light-harvesting antenna proteins regulate light energy dissipation and redistribution in chloroplast thylakoid membranes, which involve dynamic protein reorganization of photosystems I and II. However, direct evidence for such protein reorganization has not been visualized in live cells. Here we demonstrate structural dynamics of thylakoid membranes by live cell imaging in combination with deconvolution. We observed chlorophyll fluorescence in the antibiotics-induced macrochloroplast in the moss Physcomitrella patens. The three-dimensional reconstruction uncovered the fine thylakoid membrane structure in live cells. The time-lapse imaging shows that the entire thylakoid membrane network is structurally stable, but the individual thylakoid membrane structure is flexible in vivo. Our observation indicates that grana serve as a framework to maintain structural integrity of the entire thylakoid membrane network. Both the structural stability and flexibility of thylakoid membranes would be essential for dynamic protein reorganization under fluctuating light environments.

Figure 1. Thylakoid membrane network revealed by confocal microscopy in combination with 3D deconvolution.

(a–d) The normal chloroplasts in P. patens protonema grown on the agar media in a glass-bottom dish were directly observed. (e–i) The formation of macrochloroplasts was induced by growing on the agar media containing 1 mM ampicillin in a glass-bottom dish. (a), (e) The differential interference contrast images. (b), (f) The chlorophyll fluorescence images. (c), (g) The reconstructed 3D images of chlorophyll fluorescence. (d), (h) The enlarged images of the squares in (c) and (g), respectively. (i) Surface plot after linear contrast adjustments of the enlarged image of the square in (h). The arrows and arrowheads indicate the dot-like and thread-like structures, respectively. Scale bars, 5 μm.

Figure 2. The spatiotemporal dynamics of thylakoid membrane structure in the macrochloroplast under the control conditions.

(a) The 3D time-lapse images of the P. patens macrochloroplast showed the random oscillation of thylakoid membrane structure (see also Supplementary Movie 1). Numbers indicate the elapsed time in seconds during the observation. (b) The kymograph of the square in (a) suggested that the location of the dot-like structures were mostly unchanged during the observation. (c) Surface plot of kymograph in (b). Arrows indicate the directions for fluorescence intensity (i), the distance (d), and the time (t). (d) The entire thylakoid membrane network of the initial image was almost the same as the sum image of the 28 recorded images during the observation for 196 s. Scale bars, 2 μm.

Figure 3. Structural dynamics of the thread-like structures.

(a) Chl fluorescence signal indicating the dot-like (black) and thread-like structures (transparent green) was extracted from Supplementary Movie 1. The structures observed in the previous panel are faintly outlined in the next panel to compare the structural changes between the sequential images. Numbers indicate the elapsed time in seconds during the observation. Scale bar, 2 μm. (b) The enlarged images of the square in (a) with the transparent green indicating the thread-like structures. M, the merged image, focusing on the structural variations of the dot-like structures (transparent grey) during the observation. Numbers indicate the elapsed time in seconds during the observation. Scale bar, 1 μm.

Figure 4. The enhanced structural dynamics of thylakoid membranes in the lincomycin-treated macrochloroplast.

(a) The 3D time-lapse imaging of the P. patens macrochloroplast treated with 1 mM lincomycin showed more dynamic movement of thylakoid membrane structure as compared to the control conditions (see also Supplementary Movie 2). Numbers indicate the elapsed time in seconds during the observation. (b) The kymograph of the square in (a) shows that the location of the dot-like structures was randomly changed during the observation. (c) Surface plot of kymograph in (b). Arrows indicate the directions for fluorescence intensity (i), the distance (d), and the time (t). (d) The entire thylakoid membrane network of the initial image became mostly vague in the sum image of the 28 recorded images during the observation for 196 s. (e) Chl fluorescence signal indicating the dot-like (black) and thread-like structures (transparent green) was extracted from Supplementary Movie 2. The structures observed in the previous panel were faintly outlined in the next panel to compare the structural changes between the sequential images as in Fig. 3a. Numbers indicate the elapsed time in seconds during the observation. Scale bars, 2 μm.