Isolation of symbiosomes and the symbiosome membrane complex from the zoanthid Zoanthus robustus

The zoanthid Zoanthus robustus was used as a model organism to develop procedures for isolating pure symbiosomes and symbiosome membranes. The symbiosome is comprised of a zooxanthella (Symbiodinium sp.) cell that divides rarely and is separated from the host gastrodermal cytoplasm by a symbiosome multimembrane complex. Devising a method to isolate membranes at the interface between the symbiotic partners is a critical first step in characterising the molecular components involved in the metabolic trafficking necessary to sustain an effective symbiosis. After zoanthid gastrodermal cells were extracted, symbiosomes were released by mechanical disruption, recovered by centrifugation, and then purified using discontinuous sucrose gradient centrifugation. The material forming the membrane complex around symbiosomes proved highly resistant to disruption. Methods used to dissociate this interface from symbionts included (1) Triton X-100 detergent solubilisation, (2) osmotic shock with mechanical disruption, and (3) vigorous mechanical disruptions, where powerful shearing forces were used, combined with a series of sucrose density gradient centrifugation steps. The lipophilic styryl fluorochrome FM 1-43, at a concentration of 30 μM, selectively labelled the symbiosome membrane complex, both for isolated symbiosomes and those in hospite. Other cell membranes, including plasma membranes, endoplasmic reticulum, tonoplast, and organelle membranes, were not visibly labelled at this concentration. The selective labelling of the symbiosome membrane complex remained stable even after long exposure times (3 h). At 30 μM concentration, FM 1-43 also labelled symbiosome membrane fragments isolated using methods (1), (2) and (3). Method (3) proved to be the most effective in producing a fraction enriched in FM-143-labelled membrane material, which we call a symbiosome membrane complex. Transmission electron microscopy, together with confocal and conventional epifluorescence microscopy of the FM 1-43-stained preparations, was used to validate the purity of symbiosome preparations and to infer the complexity of the symbiosome membrane complex. This membrane complex has regions where the membranes contributed by the alga are appressed, and punctate regions whose function remains unclear.