4.1. Biotinylation.

Approximately 1×10⁷ SGCs were first suspended in 1 mL ASW. After the addition of 10 µL biotin-XX sulfosuccinimidyl ester (Invitrogen, F-20650) stock solution (1 µg/µL, prepared in anhydrous DMSO), the cell suspension was incubated on ice for 30 min to inhibit membrane endocytosis [14]. The biotinylation reaction was terminated with 50 mM glycine at 4°C for 15 min. Cells were then pelleted (100×g for 5 min at 4°C) and washed with ASW. SGCs without biotinylation were used as controls.

4.2. Confocal fluorescent microscopic examinations.

To check whether biotinylation was successful on the SGC surfaces, 1×10⁶ biotinylated SGCs (1×10⁶ non-biotinylated SGCs were used as controls.) were suspended in 100 µL FSW. Then, 1 µL of 1 ng/µL Alexa Fluor® 488 conjugated streptavidin (Invitrogen) was added, and the mixture was incubated at room temperature (RT) for 30 min in the dark. Afterwards, the stained cells were washed with FSW and examined on a confocal microscope (Carl Zeiss, LSM510, Oberkochen, Germany).

4.3. Transmission Electron Microscopy (TEM).

The biotinylated SGCs were fixed in an ice-cold fix solution of 2.5% glutaraldehyde, 2% paraformaldehyde, 0.2 M phosphate saline buffer (PBS), and 6% sucrose for 3 hr. They were then rinsed thrice with “washing buffer” (1% bovine serum albumin (BSA) and 0.1% gelatin in PBS, (pH 7.4) for 5 min. The cells were then incubated with the same washing buffer containing 30 µg/mL streptavidin conjugated with 10 nm colloidal gold (Invitrogen) for 1 hr at RT. After rinsing with washing buffer to remove unbound streptavidin, cells were post-fixed with 1% osmium tetroxide in 0.05 M phosphate buffer at 4°C for 2 hr. Cells were then washed with distilled water and pre-stained with 0.2% uranyl acetate in 70% ethanol overnight in the dark. The cells were then washed thrice with distilled water and dehydrated in a graded aqueous ethanol series (50, 70, 80, 90, 95, and 100%; 20 min at each step) at 4°C. The solvent was changed to acetone in a graded acetone/ethanol series (33%, 50%, 66%, 100% acetone; 20 min each step). Cells were then infiltrated with Spurr’s resin in acetone (33, 66, and 100% Spurr’s resin for 1 hr at each step) and embedded in gelatin capsules, which were polymerized at 70°C for 8 hrs. Afterwards, ultra-thin sections (70–80 nm) were made from the polymerized sample block and mounted on formvar-coated copper grids (300 mesh, Electron Microscopy Sciences, Hatfield, PA, USA). The specimens were developed for 4 min in silver enhancer reagent (Li silver enhancement kit, cat. number L-24919, Invitrogen) and then washed twice with deionized water for 5 minutes. After drying on filter paper for 10 min, the sections were stained with 2.5% uranyl acetate in methanol, washed with methanol, and stained with 0.4% lead citrate. After complete drying, grids were observed with a JEM-1400 transmission electron microscope (JEOL, Japan).

4.4. 2D SDS-PAGE analysis of biotinylated proteins.

Biotinylated SGCs were prepared as described above and suspended in 550 µL modified isotonic RadioImmunoPrecipitation Assay (RIPA) buffer (50 mM Tris, pH 7.4, 0.25% Na-deoxycholate, 150 mM NaCl, 1% NP-40, 1 mM EDTA, 1 mM Na₃VO₄, 1 mM NaF, 1000 mOsm.) containing a protease inhibitor cocktail (Roche, Basel, Switzerland). To this cell suspension, 1.5 g glass beads (Sigma-Aldrich, G 9268, 425–600 µm, U.S. sieve) were added, and the mixture was homogenized thrice in a TissueLyser LT (Invitrogen) containing liquid nitrogen for 5 min. Subsequently, the proteins were collected from the supernatant after centrifugation at 10,000×g at 4°C for 15 min. The dissolved salts were removed by trichloroacetic acid precipitation according to a published procedure [15], and the protein pellet was re-dissolved in rehydration solution (8 M urea, 2% CHAPS, and 20 mM DTT) for 1 hr and spun at 10,000×g at 4°C for 15 min. The concentration of soluble protein was quantified using a 2-D Quant kit (GE Healthcare, Piscataway, NJ, USA) according to the manufacturer’s recommendations.

A 13 cm DryStrip (pH 4–7) (GE Healthcare) was rehydrated in an IPGphor isoelectric focusing (IEF) system (GE Healthcare) (13 h at 50 V) with 450 µg soluble proteins mixed with 0.5% IPG buffer (pH 4–7) (GE Healthcare). IEF was performed with the following protocol: 1 h at 300 V (step), 1 h at 1000 V (gradient), 2 h at 4000 V (gradient), 1 h at 8000 V (gradient), and 4 h at 8000 V (step). Afterwards, the IPG strips were equilibrated in 1% DTT equilibration buffer (6 M urea, 2% SDS, 30% glycerol, 50 mM Tris-HCl [pH 8.8], and 0.008% bromophenol blue) for 15 min, followed by 2.5% iodoacetamide (IAA) equilibration buffer for 15 min. The equilibrated IPG strips were then placed onto a 14% polyacrylamide gel for the second-dimensional separation.

Biotinylated proteins on the 2-D SDS-PAGE gels were stained with streptavidin–Alexa Fluor® 488 (Invitrogen) and modified according to the methods described in a previous report [9], [16]. First, the gel was washed with phosphate buffered saline (PBS) for 5 min and immersed in 20 µg/ml streptavidin–Alexa Fluor® 488 for 30 min in the dark. The gel was then washed sequentially for 30 min with PBS containing 0.1% Tween-20 (thrice) and then PBS only (twice). The green fluorescent biotinylated protein spots were detected by a fluorescence image scanner (Typhoon TRIO, GE Healthcare) with an excitation wavelength of 488 nm and an emission wavelength of 526 nm. The total protein quantity of the same gel was then examined by SYPRO® Ruby gel staining according to the manufacturer’s instructions (Invitrogen). The distribution of red fluorescence protein spots was detected by the Typhoon TRIO scanner with an excitation wavelength of 532 nm and an emission wavelength of 610 nm.

4.5. Identification of biotinylated proteins by LC-MS/MS analysis.

The biotinylated protein spots were identified by LC-MS/MS according to the methods described in a previous report [9]. Only biotinylated protein spots repeatedly detected using the streptavidin–Alexa Fluor® 488 conjugate were selected for identification. Briefly, the spots were excised from the gels, washed with 50% ACN buffer, dehydrated with 100% ACN, vacuum-dried, and then digested by trypsin. Peptides were extracted with ACN/TFA/ddH₂O (50∶5:45 v/v/v), and evaporated to complete dryness under a vacuum. The samples were subsequently dissolved in formic acid/ACN/ddH₂O (0.1∶50:49.9 v/v/v) and analyzed by LC-nanoESI-MS/MS.

MS/MS ion searches were performed on the processed spectra against the 23,677 predicted proteins of Acropora digitifera (http://marinegenomics.oist.jp/genomes/downloads?project_id=3; adi_v1.0.1.prot.fa.gz (genome assembly version 1.0)) [17] using the MASCOT search program. First, the 23,677 predicted proteins were annotated by sequence homolog match in NCBI non-redundant protein sequences (nr) database (database releasing date: 2011/06) using BlastP (E value cutoff: 1E⁻⁵) [18]. For identifying possible functional domains, we conducted RPS-BLAST on Conserved Domain Database (CDD) with predicted proteins [19]. Orthologous assignment and mapping of the predicted proteins to the biological pathways were performed using KEGG Automatic Annotation Server [20]. Secondly, the acquired MS/MS sequences were blasted the annotated proteome of Acropora digitifera, as acquired above. The peptide tolerance parameter was 20 ppm, the MS/MS tolerance was 1 Da, and up to one missed cleavage was allowed. Variable modifications were oxidation (M) and carbamidomethyl (C), and fixed modifications were biotin (K) or biotin (N-terminal), or none. The criteria for the positive identification of proteins were set as follows: (i) the MOWSE score against a matched protein was higher than 23 or (ii) the matched protein had the same molecular weight (MW) or pI as the SGC biotinylated protein, or (iii) the SGC biotinylated protein aligned significantly to a published cnidarian protein sequence. Possible transmembrane domains of the identified proteins were predicted by TMpred (http://www.ch.embnet.org/software/TMPRED_form.html). Finally, the identified coral proteins blasted NCBInr database with default setting to further identify protein names/species/GI numbers with the highest identity (%) among marine species. Identified proteins were further analyzed by Protein Knowledgebase (UniProtKB) (http://www.uniprot.org/uniprot/) in order to determine their possible functions.

The selected spots on the 2D SDS-PAGE gels were circled, and the spot density was analyzed with ImageMaster (GE Healthcare).

2.1. Multifunctional chaperones: cell-cell recognition and regulation of membrane dynamics.

Four proteins involved in protein folding were identified, including heat shock protein (HSP) 60, HSP70, calreticulin and protein disulfide isomerase (PDI). HSPs function as molecular chaperones and respond to a variety of stressors, including temperature changes, cellular energy depletion, osmolarity changes, and toxic substance exposure [22], [23]. During the daytime, hyperoxic stress can characterize certain SGCs due to build-up of high oxygen concentrations stemming from Symbiodinium photosynthesis. These stress/chaperone-related proteins are involved with refolding of proteins that are denatured by reactive oxygen species (ROS) and prevention of their aggregation and are thus important for the stability of cnidarian–dinoflagellate endosymbioses [22], [24].

Besides these chaperone functions, the HSP60 proteins on the SGC surface could be involved in Symbiodinium recognition and consequent phagocytosis. HSP60 has been reported to specifically bind with lipopolysaccharides [25]. The Symbiodinium-host recognition process involves lectin/polysaccharide interactions [25], and HSP60 may therefore aid in the regulation of this interaction. Furthermore, as HSP60 was found to enhance phagocytic activity in U937 cells [23], its presence on the surface of SGC plasma membranes may implicate its role in phagocytosis.

Calreticulin, which was also found on the membrane surface of SGCs, binds oligosaccharides with terminal glucose residues [26] and is involved in the biosynthesis of a variety of molecules such as ion channels, surface receptors, integrins, and transporters [27]. Consequently, calreticulin on the surface of SGCs may also function in the recognition of Symbiodinium during the initial stages of the endosymbiosis. In addition, a calreticulin homolog that is involved in Ca²⁺ homeostasis and biomineralization has been found in corals [27], [28]. Therefore, calreticulin on the SGC surface may act to regulate Ca²⁺ concentration, a process that could even be linked to calcification.

2.2. The role of actins in membrane remodeling and regulation of phagocytic activity^. Symbiodinium (size ∼8–10 µm) typically occupy the majority of the volume of the host gastrodermal cell in which they reside (Fig. 1). In order for the coral host gastrodermal cell to maintain a normal physiology with such a bulky structure inside its cytoplasm, a unique intracellular architecture is required. Actin filament remodeling at cell surfaces is fundamental to regulating membrane elasticity and cell morphology [29], [30]. The present study identified three actin protein spots, with inferred molecular weights ranging from 44 to 47 kDa and pIs from 5.2 to 6.0 (Table 1). Besides their roles in signal transduction and protein biosynthesis, Rho family GTPases have also been shown to regulate the actin cytoskeleton and cell adhesion through specific targets in mammalian cells [31]. As both actin and GTPase were highly biotinylated (see the “Relative ratio (folds) of biotinylated vs total proteins” column in Table 1.), they may be involved in the cytoskeleton remodeling that would be necessitated by both phagocytosis and cell division of Symbiodinium with the SGC. Indeed, the cytoskeletal architecture must be fundamentally altered during the transition from a SGC housing one Symbiodinium cell to one housing multiple endosymbionts (Fig. 1) [32].