Immunogold labeling of phycobilisomes in a cyanobacterium

Abstract

Phycobilisomes are pigment-protein complexes that act as the major light- harvesting antenna, in addition to chlorophyll, for photosynthesis in red algae and Cyanobacteria.In vivo, phycobilisomes transfer their energy mainly to the photosystem II reaction center to which they are assumed to be attached. This assumption will be tested by the use of gold labeling of antibodies raised against photosystem II reaction center components to localize the reaction centers and to compare the location of the antibodies with that of the phycobili- somes. In Cyanobacteria phycobi1isomes are difficult to fix particularly under conditions required for immunocytochemistry. Here, two methods for efficient fixation of phycobi1isomes are described, together with immunocytochemical results to confirm the identity of the phycobilisomes.For phycobilisome detection,Synechocvstissp. PCC 6803 cells were either freeze substituted or fixed in a high molarity buffer followed by cryomicrotomy. For freeze substitution the cells were collected, subjected to ultra-rapid freezing with a slammer device and a copper block at liquid helium temperature, placed in 5% acrolein in ethanol at -85°C for 4 days, and embedded in LR White. For cryo- sectioning the cells were fixed with 1% glutaraldehyde in 0.75M sodium potassium phosphate buffer, pH 7.4, for 2 h, enrobed in 10% gelatin, infused with 1.6M sucrose in 0.1M sodium phosphate buffer,pH 7.2, for 3 h, and frozen in liquid freon. Cryosections were prepared with an RMC CR2000 cryoultramicrotomy unit on an MT-6000 ultramicrotome. In both procedures 0.05% Carnation nonfat dried milk in 0.1M sodium phosphate buffer was used to block nonspecific binding of the anti serum. Sections were treated with rod-phycocyanin (phycobilin protein) anti - serum followed by protein A conjugated to lOnm gold particles. Subsequently the plastic sections were stained with uranyl acetate and lead citrate. The cryosections were subsequently treated with 1% OSO4, 1% tannic acid, uranyl acetate, and alkaline bismuth, dehydrated in ethanol, and embedded in LR White. Sections were observed in a Philips EM201.