Exocytic Mechanisms of Storage and Release of Brevetoxin in the Dinoflagellate Karenia Brevis

Abstract

Photoreceptor light responses are encoded by changes in synaptic vesicle release. Release from rods is triggered by the opening of calcium channels beneath plate-like synaptic ribbons. Maintained depolarization can activate CICR and enhance release. Using total internal reflection fluorescence microscopy (TIRFM) to visualize release of single synaptic vesicles, we tested whether CICR enhances release from rods by stimulating fusion at nonribbon sites. Rods from salamander retina were loaded with activity-dependent dyes, FM1-43 or dextran-conjugated pHrodo, and visualized by TIRFM. Rods were depolarized with steps to À10 mV under voltage-clamp or by puff application of 50 mM KCl. CICR was activated with 10 mM ryanodine and inhibited with 100 mM ryanodine. Ribbon locations were identified with a fluorescent ribbonbinding peptide or from hot spots of depolarization-evoked calcium entry visualized with Fluo5F. In terminals loaded with FM1-43 or pHrodo, depolarization stimulated rapid disappearance of vesicles with kinetics similar to that measured electrophysiologically. Additionally, stimulation-evoked vesicle disappearance was blocked by Cd 2þ , indicating that it was due to calcium-dependent exocytosis. Vesicles docked for about 200 ms before fusion. Most release events occurred close to ribbons, but some also occurred further away. Activation of CICR with 10 mM ryanodine stimulated intracellular calcium increases and vesicle release. Ryanodine-evoked release events were less clustered than release evoked by depolarization, consistent with greater non-ribbon release. The spread of calcium evoked by 500 ms steps (but not 50 ms steps) was inhibited by blocking CICR with 100 mM ryanodine in the patch pipette. Release evoked by 500 ms steps also involved sites further from the ribbon than release evoked by 50 ms steps. These results indicate that the stimulation of CICR by maintained depolarization enhances release from rods by triggering fusion of vesicles at nonribbon sites.

Brevetoxin, the Dinoflagellate Neurotoxin, Localizes to Thylakoid Membranes and Interacts with the Light‐Harvesting Complex II (LHCII) of Photosystem II

“…Brevetoxins have also been demonstrated to aggregate into artificial lipid bilayers 29. Our findings contradict earlier localization studies: one which failed to identify brevetoxin in isolated chloroplasts by LC‐MS/MS,30 and a published abstract that reported that brevetoxins are located in secretory vesicles by using fluorescent anti‐brevetoxin antibodies 31. A follow‐up paper to the latter report has not yet appeared.…”

Differences in the toxicity of six Gambierdiscus (Dinophyceae) species measured using an in vitro human erythrocyte lysis assay

“…4). A recent report indicates that brevetoxins, which are structurally related to MTX, are retained in membrane lined vesicles (Vigna et al, 2012). If hemolytic compounds produced by Gambierdiscus are sequestered in similar structures or bound to proteins it could account for why they are not immediately released into the aqueous phase when cells are disrupted, but are instead released over a 24 incubation period.…”

Uptake and localization of fluorescently‐labeled Karenia brevis metabolites in non‐toxic marine microbial taxa

“…) and the secretory granules of K. brevis (Vigna et al. ). PbTx‐2 fluorophore conjugates exhibit classic brevetoxin pharmacology in vitro.…”