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This review explains the potential and the problems, and gives a brief …


Biology Articles » Methods & Techniques » FRAP analysis of photosynthetic membranes » Photosynthetic membranes as model systems for FRAP

Photosynthetic membranes as model systems for FRAP
- FRAP analysis of photosynthetic membranes

 

One great advantage of photosynthetic membranes is that some of the protein complexes are naturally fluorescent (Table 1), so their diffusion can be observed without any necessity for specific fluorophore binding or GFP gene fusions. In other respects, the majority of thylakoid membranes are far from ideal for FRAP measurements. The membrane systems are enclosed in bacteria or chloroplasts whose overall dimensions are usually rather small. Furthermore, most green plant thylakoid membranes have an intricate, convoluted structure, and extensive lateral heterogeneity on small scales (Mustardy and Garab, 2003). Thus they do not meet the basic requirements for quantitative FRAP, that the membrane geometry is predictable and the membrane environment is uniform over the area of the measurement. While it will not be possible to use FRAP to obtain accurate diffusion coefficients for components of typical green plant thylakoids, it will be possible to see if particular components are mobile or not, and to get a rough idea of time-scales.

By far the most regular thylakoid membranes are found in certain species of cyanobacteria, which have elongated cells, and thylakoid membrane whose conformation approximates to a set of concentric cylinders aligned along the long axis of the cell (Mullineaux and Sarcina, 2002). In contrast to typical green plants, there is no thylakoid membrane stacking and no extensive lateral heterogeneity, although there are some indications of differences in composition between the inner and outer membrane cylinders (Sherman et al., 1994). The preferred model organism is the cyanobacterium Synechococcus sp. PCC7942, which has such a membrane conformation and is also well-characterized and transformable. A complete genome sequence is now available (US Department of Energy Joint Genome Institute: http://genome.jgi-psf.org/draft_microbes/synel/synel.home.html). Synechococcus 7942 cells are generally about 3 µm long. In order to increase the area of thylakoid membrane relative to the bleach size, they were generally grown in the presence of cell division inhibitors: under these conditions, cells 10–20 µm long can be found in the culture (Sarcina and Mullineaux, 2000). Growth for about 24 h in the presence of 0.5% DMSO is an efficient way to produce elongated cells (Aspinwall et al., 2004). With cylindrical membranes like Synechococcus thylakoids, the best way to do a FRAP measurement is to bleach a narrow line across the cell, by scanning the confocal spot for about 1–3 s in the X-direction (Mullineaux et al., 1997; Fig. 1). Diffusion can then be followed in one dimension, along the long-axis of the cell (Mullineaux et al., 1997; Mullineaux and Sarcina, 2002). These measurements are fully quantitative. This system has been used to show that the phycobilisome light-harvesting antennae are highly mobile, and to investigate a number of factors that affect their diffusion coefficient (Sarcina et al., 2001; Aspinwall et al., 2004). It has also been shown that Photosystem II core complexes are astonishingly immobile: no diffusion can be detected even on very long time-scales (Sarcina et al., 2001). This raised the possibility that all the integral membrane complexes might be locked into a rigid array. However, it has recently been found that this is not the case: the IsiA protein, an additional chlorophyll-protein induced under iron-stress, is quite mobile (M Sarcina, CW Mullineaux, unpublished data). In addition, BODIPY FL C12, a lipid-soluble green-fluorescent dye, has been used to probe the mobility of thylakoid membrane lipids (Sarcina et al., 2003). It was shown that the diffusion coefficient is strongly increased in a transformant with more desaturated thylakoid membrane lipids, but that lipid lateral diffusion is rather slow in thylakoid membranes, compared with, for example, eukaryotic plasma membranes. This may reflect differences in membrane lipid composition, but also the exceptionally high protein content of thylakoid membranes (Sarcina et al., 2003). 

The author does not know of any green plant whose thylakoid membrane conformation is as amenable to FRAP as that of Synechococcus. However, some green algae have the advantages of large chloroplast size and low levels of membrane stacking and lateral heterogeneity. Mullineaux and Sarcina (2002) have started a study of Chlamydomonas reinhardtii. It was found that a proportion of the fluorescent chlorophyll in the membrane is mobile. Studies in a range of mutant backgrounds suggest that Photosystem II is immobile, as in cyanobacteria, but a part of the LHCII pool is mobile (M Sarcina, CW Mullineaux, unpublished results). Chloroplast division mutants (Osteryoung and Pyke, 1998) may, potentially, be useful systems for thylakoid FRAP in higher plants.


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