Thylakoid membrane perforations and connectivity enable intracellular traffic in cyanobacteria

Abstract

Cyanobacteria, the progenitors of plant and algal chloroplasts, enabled aerobic life on earth by introducing oxygenic photosynthesis. In most cyanobacteria, the photosynthetic membranes are arranged in multiple, seemingly disconnected, concentric shells. In such an arrangement, it is unclear how intracellular trafficking proceeds and how different layers of the photosynthetic membranes communicate with each other to maintain photosynthetic homeostasis. Using electron microscope tomography, we show that the photosynthetic membranes of two distantly related cyanobacterial species contain multiple perforations. These perforations, which are filled with particles of different sizes including ribosomes, glycogen granules and lipid bodies, allow for traffic throughout the cell. In addition, different layers of the photosynthetic membranes are joined together by internal bridges formed by branching and fusion of the membranes. The result is a highly connected network, similar to that of higher-plant chloroplasts, allowing water-soluble and lipid-soluble molecules to diffuse through the entire membrane network. Notably, we observed intracellular membrane-bounded vesicles, which were frequently fused to the photosynthetic membranes and may play a role in transport to these membranes.

Figure 1

Perforations in the thylakoid membranes of cyanobacteria. Tomographic slices (∼12 nm) from dual-axis tomograms of cryo-immobilized, freeze-substituted cells of (A) Synechococcus sp PCC 7942 and (B) Microcoleus sp. The complete sets of tomographic slices used to generate the volumes are provided (Supplementary Movies S1 and S2). A total of six tomograms and about 500 thin-section slices were examined and used in the analysis. In both species, the thylakoid membranes (T) are perforated at multiple sites (red arrowheads). Labels: CW, cell wall, both the inner and outer cell membranes are apparent; R, ribosomes; G, polysaccharide (glycogen) granules; C, carboxysomes; P, extracellular polysaccharides, which envelope filaments of Microcoleus sp (Ohad et al, 2005); asterisk, thylakoid membrane extension crossing the cell. The small black dots seen around the cells are gold particles used for image alignment. Scale bars, 200 nm (A); 500 nm (B). (C) Model of the thylakoid membranes of Synechococcus sp PCC 7942 generated from the tomographic data shown in (A). The inset shows a rotated view of the upper left hole. Ribosomes (white) and polysaccharide granules (light green) are seen near and inside the perforations.

Figure 2

Types of perforations in the thylakoid membranes. The major types of perforations observed in the thylakoid membranes of Synechococcus sp PCC 7942 and Microcoleus sp are shown. Models (A) generated from a tomogram of a Microcoleus sp cell (not shown), (B) generated from Supplementary Movie S2; (C) an enlargement of the lower perforation seen in Figure 1C; thin-section micrographs of Microcoleus sp cells (D, E), and a tomographic slice (∼12 nm; F, an enlargement of Figure 1A). Scale bars, 200 nm (D); 80 nm (E); 100 nm (F).

Figure 3

Thylakoid membranes are interconnected. Models (A) generated from a tomogram of a Synechococcus cell (not shown), (B) generated from a tomogram of a Microcoleus cell (see Supplementary Movie S2); and thin-section micrographs of Microcoleus sp cells (C, D), showing how different layers of the thylakoid membranes are connected to each other. Connections involve splitting or branching of the membranes (red arrowheads) and subsequent folding or fusion. A similar mode of connectivity, involving bifurcations and fusion of the membranes, has also been observed in the thylakoid membranes of higher-plant chloroplasts (Shimoni et al, 2005). Scale bars, 100 nm.

Figure 4

Vesicles in Microcoleus sp cells. Thin-section micrographs (A, B) showing vesicles in which the delimiting bilayer is clearly visualized (A), and vesicles spread out throughout the center and periphery of the cell (B). Tomographic slices (∼12 nm; C, D; see complete tomograms provided, Supplementary Movies S3 and S4) of a vesicle found in the center of the cell (C) and vesicles connected to each other and to the thylakoid membrane network by protruding lamella (D). Inset in (D) shows a model of the vesicle marked by a red asterisk, depicting its connections to the flanking lamellae. The volume of the vesicle could not be reconstructed in whole, as the vesicle was not fully contained in the semi-thick section used for tomography. Scale bars, 200 nm (A); 500 nm (B); 300 nm (C, D). See also Supplementary Figure S3.

Figure 5

Perforations and connectivity in the cyanobacterial thylakoid membranes. An artistic view of cyanobacterial thylakoid membranes, combining features observed in the two species examined in this work, including perforations, gaps, bifurcations and fusion. The result is a highly connected but perforated network of lamellae sharing a single, continuous lumen and allowing for unperturbed transport of molecules and macromolecules throughout the entire cell volume. A configuration capturing many of these features has been proposed by Mullineaux (1999). Note that layers represent paired membranes enclosing the thylakoid lumen (blue). Other views of the model are provided in Supplementary Movie S5.