The actin cytoskeleton organization in symbiotic marine dinoflagellates is largely undescribed; most likely, due to their intense pigment autofluorescence and cell walls that block fluorescent probe access. Using a freeze-fracture and fixation procedure, we observed the actin cytoskeleton of Symbiodinium kawagutii cultured in vitro with fluorescently labeled phalloidin and by indirect immunofluorescence with monoclonal antibodies specific for actin. The cytoskeleton appeared as an organized network with interconnected cortical and cytoplasmic thick filaments, along with some intertwined fine filaments. It showed a grid-type, reticular pattern organized in a lattice-like structure within the cell and throughout the cytoplasm. This organization was similar when the observations were done with either fluorescein isothiocyanate (FITC)-phalloidin or anti-actin, although the latter showed a more evenly distributed fluorescence characteristic of nonpolymerized actin. The network organization collapsed upon treatment with latrunculin, resulting in bright foci and diffuse fluorescence. A similar effect was obtained upon butanedione monoxime treatment, except that no bright foci were observed. We have been able to successfully visualize the actin cytoskeleton of S. kawagutii cells using fluorescence-based procedures. This is the first report on the visualization of the organization of the actin cytoskeleton under various conditions in these walled, highly autofluorescent cells.

Background Some genera of the family Symbiodiniaceae establish mutualistic endosymbioses with various marine invertebrates, with coral being the most important ecologically. Little is known about the biochemical communication of this association and the perception and translation of signals from the environment in the symbiont. However, specific phosphorylation/dephosphorylation processes are fundamental for the transmission of external signals to activate physiological responses. In this work, we searched phosphorylatable proteins in amino acids of Ser, Thr and Tyr from three species of the family Symbiodiniaceae, Symbiodinium kawagutii, Symbiodinium sp. Mf11 and Symbiodinium microadriaticum. Methods We used specific antibodies to the phosphorylated aminoacids pSer, pThr and pTyr to identify proteins harboring them in total extracts from three species of Symbiodinium in culture. Extractions were carried out on logarithmic phase growing cultures under a 12 h light/dark photoperiod. Various light/dark, nutritional and other stimuli were applied to the cultures prior to the extractions, and proteins were subjected to SDS-PAGE and western immunoblotting. Partial peptide sequencing was carried out by MALDI-TOF on specific protein spots separated by 2D electrophoresis. Results At 4 h of the light cycle, several Thr-phosphorylated proteins were consistently detected in the three species suggesting a genus-dependent expression; however, most Ser- and Tyr-phosphorylated proteins were species-specific. Analysis of protein extracts of S. microadriaticum cultures demonstrated that the level of phosphorylation of two Thr-phosphorylated proteins with molecular weights of 43 and 75 kDa, responded inversely to a light stimulus. The 43 kDa protein, originally weakly Thr-phosphorylated when the cells were previously adapted to their 12 h dark cycle, underwent an increase in Thr phosphorylation when stimulated for 30 min with light. On the other hand, the 75 kDa protein, which was significantly Thr-phosphorylated in the dark, underwent dephosphorylation in Thr after 30 min of the light stimulus. The phosphorylation response of the 43 kDa protein only occurred in S. microadriaticum, whereas the dephosphorylation of the 75 kDa protein occurred in the three species studied suggesting a general response. The 75 kDa protein was separated on 2D gels as two isoforms and the sequenced spots corresponded to a BiP-like protein of the HSP70 protein family. The presence of differential phosphorylations on these proteins after a light stimulus imply important light-regulated physiological processes in these organisms.

A photosystem II component, the PsbO protein is essential for maximum rates of oxygen production during photosynthesis, and has been extensively characterized in plants and cyanobacteria but not in symbiotic dinoflagellates. Its close interaction with D1 protein has important environmental implications since D1 has been identified as the primary site of damage in endosymbiotic dinoflagellates after thermal stress. We identified and biochemically characterized the PsbO homolog from Symbiodinium kawagutii as a 28-kDa protein, and immunolocalized it to chloroplast membranes. Chloroplast association was further confirmed by western blot on photosynthetic membrane preparations. TX-114 phase partitioning, chromatography, and SDS-PAGE for single band separation and partial peptide sequencing yielded peptides identical or with high identity to PsbO from dinoflagellates. Analysis of a cDNA library revealed three genes differing by only one aminoacid residue in the in silico-translated ORFs despite greater differences at nucleotide level in the untranslated, putative regulatory sequences. The consensus full amino acid sequence displayed all the characteristic domains and features of PsbO from other sources, but changes in functionally critical, highly conserved motifs were detected. Our biochemical, molecular, and immunolocalization data led to the conclusion that the 28-kDa protein from S. kawagutii is the PsbO homolog, thereby named SkPsbO. We discuss the implications of critical amino acid substitutions for a putative regulatory role of this protein.

Our current understanding of carbon exchange between partners in the Symbiodinium-cnidarian symbioses is still limited, even though studies employing carbon isotopes have made us aware of the metabolic complexity of this exchange. We examined glycerol and glucose metabolism to better understand how photosynthates are exchanged between host and symbiont. The levels of these metabolites were compared between symbiotic and bleached Exaiptasia pallida anemones, assaying enzymes directly involved in their metabolism. We measured a significant decrease of glucose levels in bleached animals but a significant increase in glycerol and G3P pools, suggesting that bleached animals degrade lipids to compensate for the loss of symbionts and seem to rely on symbiotic glucose. The lower glycerol 3-phosphate dehydrogenase but higher glucose 6-phosphate dehydrogenase specific activities measured in bleached animals agree with a metabolic deficit mainly due to the loss of glucose from the ruptured symbiosis. These results corroborate previous observations on carbon translocation from symbiont to host in the sea anemone Exaiptasia, where glucose was proposed as a main translocated metabolite. To better understand photosynthate translocation and its regulation, additional research with other symbiotic cnidarians is needed, in particular, those with calcium carbonate skeletons.

Immunofluorescence or fluorescent probe procedures with dinoflagellate algae have been hampered by their intrinsic pigment autofluorescence and rigid cell walls that cannot be digested by cellulolytic or pectinolytic enzymes. Highly autofluorescent, chlorophyll-containing cells from Symbiodinium were fixed and treated by a freeze-fracture procedure on microscope slides, intended to preserve their structure, break the cell wall, eliminate endogenous fluorescent pigments, and provide accessibility to antibodies. Formaldehyde fixation was carried out on intact cells in suspension, followed by freeze-fracture in liquid nitrogen. Finally, they were subjected through a series of tests for pigment extraction to eliminate the cell autofluorescence. Incubation in 70% isopropanol, followed by NaBH 4 after freeze-fracture on glass slides, proved to be the most effective treatment to remove the fluorescence from endogenous pigments and free aldehyde groups remaining after the fixation. The procedure was effective in preserving the cell structures during the lengthy incubations, and the cells were suitable for both, immunolocalization of internal antigens, and visualization of targets of fluorescent probes. The procedure was equally successful when applied to other Symbiodinium species. These results provide a powerful tool for performing immunofluorescence and fluorescent probe detection on these highly autofluorescent, chlorophyll-containing cells.

Specific phosphorylation/dephosphorylation processes are fundamental for the transduction of external stimuli into physiological responses. A few of these processes appear to be modulated by light in cultured Symbiodinium microadriaticum since the BiP-like protein SmicHSP75 undergoes threonine dephosphorylation upon light stimuli. Several isoforms of the protein are encoded in the S. microadriaticum genome and thus, we identified and heterologously expressed a specific sequence corresponding to the previously identified SmicHSP75 isoform to obtain a highly specific antibody. We then determined by western blot analysis, that the detected light-stimulated changes in SmicHSP75 threonine phosphorylation were not due to changes in the protein expression and explored further the effect of lower than normal and higher stressful temperature, on the phosphorylation levels of the protein. Upon long-term (12 h) exposure of the cells to the low temperature of 21ºC under darkness, the protein was found significantly phosphorylated; however, light exposure for 30 min caused a dephosphorylation effect like the 26ºC control treatment. On the other hand, in cells exposed to 32ºC for 12 h under darkness, the highly Thr-phosphorylated SmicHSP75 was converted to a low-level phosphorylated protein. Likewise, short term (30 min) exposure to 32ºC under dark conditions caused dephosphorylation of the protein, similar to what was observed upon long-term exposure to 32ºC and upon light stimulation of cells under the normal temperature of 26ºC. These data suggested activation/inactivation of the chaperone function of SmicHSP75 by regulation of its Thr phosphorylation levels under heat stress conditions in Symbiodinium microadriaticum, independent of changes in protein expression.