A shift away from mutualism under food-deprived conditions in an anemone-dinoflagellate association.

The mutualistic symbiosis between anthozoans and intra-gastrodermal dinoflagellates of the family Symbiodiniaceae is the functional basis of all coral reef ecosystems, with the latter providing up to 95% of their fixed photosynthate to their hosts in exchange for nutrients. However, recent studies of sponges, jellyfish, and anemones have revealed the potential for this mutualistic relationship to shift to parasitism under stressful conditions. Over a period of eight weeks, we compared the physiological conditions of both inoculated and aposymbiotic anemones (Exaiptasia pallida) that were either fed or starved. By the sixth week, both fed groups of anemones were significantly larger than their starved counterparts. Moreover, inoculated and starved anemones tended to disintegrate into "tissue balls" within eight weeks, and 25% of the samples died; in contrast, starved aposymbiotic anemones required six months to form tissue balls, and no anemones from this group died. Our results show that the dinoflagellates within inoculated anemones may have posed a fatal metabolic burden on their hosts during starvation; this may be because of the need to prioritize their own metabolism and nourishment at the expense of their hosts. Collectively, our study reveals the potential of this dynamic symbiotic association to shift away from mutualism during food-deprived conditions.

Coral lipid bodies as the relay center interconnecting diel-dependent lipidomic changes in different cellular compartments.

Lipid bodies (LBs) in the coral gastrodermal tissues are key organelles in the regulation of endosymbiosis and exhibit a diel rhythmicity. Using the scleractinian Euphyllia glabrescens collected across the diel cycle, we observed temporally dynamic lipid profiles in three cellular compartments: host coral gastrodermal cells, LBs, and in hospite Symbiodinium. Particularly, the lipidome varied over time, demonstrating the temporally variable nature of the coral-Symbiodinium endosymbiosis. The lipidome-scale data highlight the dynamic, light-driven metabolism of such associations and reveal that LBs are not only lipid storage organelles but also act as a relay center in metabolic trafficking. Furthermore, lipogenesis in LBs is significantly regulated by coral hosts and the lipid metabolites within holobionts featured predominantly triacylglycerols, sterol esters, and free fatty acids. Given these findings through a time-varied lipidome status, the present study provided valuable insights likely to be crucial to understand the cellular biology of the coral-Symbiodinium endosymbiosis.

A detailed observation of the ejection and retraction of defense tissue acontia in sea anemone (Exaiptasia pallida).

Acontia, located in the gastrovascular cavity of anemone, are thread-like tissue containing numerous stinging cells which serve as a unique defense tissue against predators of the immobile acontiarian sea anemone. Although its morphology and biological functions, such as defense and digestion, have been studied, the defense behavior and the specific events of acontia ejection and retraction are unclear. The aim of this study is to observe and record the detailed process of acontia control in anemones. Observations reveal that the anemone, Exaiptasia pallida, possibly controls a network of body muscles and manipulates water pressure in the gastrovascular cavity to eject and retract acontia. Instead of resynthesizing acontia after each ejection, the retraction and reuse of acontia enables the anemone to respond quickly at any given time, thus increasing its overall survivability. Since the Exaiptasia anemone is an emerging model for coral biology, this study provides a foundation to further investigate the biophysics, neuroscience, and defense biology of this marine model organism.

Transmission of a heterologous clade C Symbiodinium in a model anemone infection system via asexual reproduction.

Anemones of genus Exaiptasia are used as model organisms for the study of cnidarian-dinoflagellate (genus Symbiodinium) endosymbiosis. However, while most reef-building corals harbor Symbiodinium of clade C, Exaiptasia spp. anemones mainly harbor clade B Symbiodinium (ITS2 type B1) populations. In this study, we reveal for the first time that bleached Exaiptasia pallida anemones can establish a symbiotic relationship with a clade C Symbiodinium (ITS2 type C1). We further found that anemones can transmit the exogenously supplied clade C Symbiodinium cells to their offspring by asexual reproduction (pedal laceration). In order to corroborate the establishment of stable symbiosis, we used microscopic techniques and genetic analyses to examine several generations of anemones, and the results of these endeavors confirmed the sustainability of the system. These findings provide a framework for understanding the differences in infection dynamics between homologous and heterologous dinoflagellate types using a model anemone infection system.

A Compartmental Comparison of Major Lipid Species in a Coral-Symbiodinium Endosymbiosis: Evidence that the Coral Host Regulates Lipogenesis of Its Cytosolic Lipid Bodies.

The lipid body (LB) formation in the host coral gastrodermal cell cytoplasm is a hallmark of the coral-Symbiodinium endosymbiosis, and such lipid-based entities are not found in endosymbiont-free cnidarian cells. Therefore, the elucidation of lipogenesis regulation in LBs and how it is related to the lipid metabolism of the host and endosymbiont could provide direct insight to understand the symbiosis mechanism. Herein, the lipid composition of host cells of the stony coral Euphyllia glabrescens, as well as that of their cytoplasmic LBs and in hospite Symbiodinium populations, was examined by high performance liquid chromatography (HPLC) and gas chromatography/mass spectrometry (GC/MS), and six major lipid species were identified: wax esters, sterol esters, triacylglycerols, cholesterols, free fatty acids, and phospholipids. Their concentrations differed significantly between host coral cells, LBs, and Symbiodinium, suggesting compartmental regulation. WE were only present in the host coral and were particularly highly concentrated in LBs. Amongst the four species of WE, the monoene R = C18:1/R = C16 was found to be LB-specific and was not present in the host gastrodermal cell cytoplasm. Furthermore, the acyl pool profiles of the individual LB lipid species were more similar, but not equal to, those of the host gastrodermal cells in which they were located, indicating partially autonomous lipid metabolism in these LBs. Nevertheless, given the overall similarity in the host gastrodermal cell and LB lipid profiles, these data suggest that a significant portion of the LB lipids may be of host coral origin. Finally, lipid profiles of the in hospite Symbiodinium populations were significantly distinct from those of the cultured Symbiodinium, potentially suggesting a host regulation effect that may be fundamental to lipid metabolism in endosymbiotic associations involving clade C Symbiodinium.

Enhancement of neurite outgrowth in neuron cancer stem cells by growth on 3-D collagen scaffolds.

Collagen is one component of the extracellular matrix that has been widely used for constructive remodeling to facilitate cell growth and differentiation. The 3-D distribution and growth of cells within the porous scaffold suggest a clinical significance for nerve tissue engineering. In the current study, we investigated proliferation and differentiation of neuron cancer stem cells (NCSCs) on a 3-D porous collagen scaffold that mimics the natural extracellular matrix. We first generated green fluorescence protein (GFP) expressing NCSCs using a lentiviral system to instantly monitor the transitions of morphological changes during growth on the 3-D scaffold. We found that proliferation of GFP-NCSCs increased, and a single cell mass rapidly grew with unrestricted expansion between days 3 and 9 in culture. Moreover, immunostaining with neuronal nuclei (NeuN) revealed that NCSCs grown on the 3-D collagen scaffold significantly enhanced neurite outgrowth. Our findings confirmed that the 80 µm porous collagen scaffold could enhance attachment, viability and differentiation of the cancer neural stem cells. This result could provide a new application for nerve tissue engineering and nerve regeneration.

Assessment of metabolic modulation in free-living versus endosymbiotic Symbiodinium using synchrotron radiation-based infrared microspectroscopy.

The endosymbiotic relationship between coral hosts and dinoflagellates of the genus Symbiodinium is critical for the growth and productivity of coral reef ecosystems. Here, synchrotron radiation-based infrared microspectroscopy was applied to examine metabolite concentration differences between endosymbiotic (within the anemone Aiptasia pulchella) and free-living Symbiodinium over the light-dark cycle. Significant differences in levels of lipids, nitrogenous compounds, polysaccharides and putative cell wall components were documented. Compared with free-living Symbiodinium, total lipids, unsaturated lipids and polysaccharides were relatively enriched in endosymbiotic Symbiodinium during both light and dark photoperiods. Concentrations of cell wall-related metabolites did not vary temporally in endosymbiotic samples; in contrast, the concentrations of these metabolites increased dramatically during the dark photoperiod in free-living samples, possibly reflecting rhythmic cell-wall synthesis related to light-driven cell proliferation. The level of nitrogenous compounds in endosymbiotic cells did not vary greatly across the light-dark cycle and in general was significantly lower than that observed in free-living samples collected during the light. Collectively, these data suggest that nitrogen limitation is a factor that the host cell exploits to induce the biosynthesis of lipids and polysaccharides in endosymbiotic Symbiodinium.

Lipid bodies in coral-dinoflagellate endosymbiosis: proteomic and ultrastructural studies.

Gastrodermal lipid bodies (LBs) are organelles involved in the regulation of the mutualistic endosymbiosis between reef-building corals and their dinoflagellate endosymbionts (genus Symbiodinium). As their molecular composition remains poorly defined, we herein describe the first gastrodermal LB proteome and examine in situ morphology of LBs in order to provide insight into their structure and function. After tissue separation of the tentacles of the stony coral Euphyllia glabrescens, buoyant LBs of the gastroderm encompassing a variety of sizes (0.5-4 µm in diameter) were isolated after two cycles of subcellular fractionation via stepwise sucrose gradient ultracentrifugation and detergent washing. The purity of the isolated LBs was demonstrated by their high degree of lipid enrichment and as well as the absence of contaminating proteins of the host cell and Symbiodinium. LB-associated proteins were then purified, subjected to SDS-PAGE, and identified by MS using an LC-nano-ESI-MS/MS. A total of 42 proteins were identified within eight functional groups, including metabolism, intracellular trafficking, the stress response/molecular modification and development. Ultrastructural analyses of LBs in situ showed that they exhibit defined morphological characteristics, including a high-electron density resulting from a distinct lipid composition from that of the lipid droplets of mammalian cells. Coral LBs were also characterized by the presence of numerous electron-transparent inclusions of unknown origin and composition. Both proteomic and ultrastructural observations seem to suggest that both Symbiodinium and host organelles, such as the ER, are involved in LB biogenesis.

Improved proteome coverage by using high efficiency cysteinyl peptide enrichment: the human mammary epithelial cell proteome.

Automated multidimensional capillary liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been increasingly applied in various large scale proteome profiling efforts. However, comprehensive global proteome analysis remains technically challenging due to issues associated with sample complexity and dynamic range of protein abundances, which is particularly apparent in mammalian biological systems. We report here the application of a high efficiency cysteinyl peptide enrichment (CPE) approach to the global proteome analysis of human mammary epithelial cells (HMECs) which significantly improved both sequence coverage of protein identifications and the overall proteome coverage. The cysteinyl peptides were specifically enriched by using a thiol-specific covalent resin, fractionated by strong cation exchange chromatography, and subsequently analyzed by reversed-phase capillary LC-MS/MS. An HMEC tryptic digest without CPE was also fractionated and analyzed under the same conditions for comparison. The combined analyses of HMEC tryptic digests with and without CPE resulted in a total of 14 416 confidently identified peptides covering 4294 different proteins with an estimated 10% gene coverage of the human genome. By using the high efficiency CPE, an additional 1096 relatively low abundance proteins were identified, resulting in 34.3% increase in proteome coverage; 1390 proteins were observed with increased sequence coverage. Comparative protein distribution analyses revealed that the CPE method is not biased with regard to protein M(r) , pI, cellular location, or biological functions. These results demonstrate that the use of the CPE approach provides improved efficiency in comprehensive proteome-wide analyses of highly complex mammalian biological systems.

Multidimensional proteome analysis of human mammary epithelial cells.

Recent multidimensional liquid chromatography MS/MS studies have contributed to the identification of large numbers of expressed proteins for numerous species. The present study couples size exclusion chromatography of intact proteins with the separation of tryptically digested peptides using a combination of strong cation exchange and high resolution, reversed phase capillary chromatography to identify proteins extracted from human mammary epithelial cells (HMECs). In addition to conventional conservative criteria for protein identifications, the confidence levels were additionally increased through the use of peptide normalized elution times (NET) for the liquid chromatographic separation step. The combined approach resulted in a total of 5838 unique peptides identified covering 1574 different proteins with an estimated 4% gene coverage of the human genome, as annotated by the National Center for Biotechnology Information (NCBI). This database provides a baseline for comparison against variations in other genetically and environmentally perturbed systems. Proteins identified were categorized based upon intracellular location and biological process with the identification of numerous receptors, regulatory proteins, and extracellular proteins, demonstrating the usefulness of this application in the global analysis of human cells for future comparative studies.

Induced autocrine signaling through the epidermal growth factor receptor contributes to the response of mammary epithelial cells to tumor necrosis factor alpha.

In contrast to the well known cytotoxic effects of tumor necrosis factor (TNF) alpha in many mammary cancer cells, we have found that TNF stimulates the proliferation and motility of human mammary epithelial cells (HMECs). Since the response of HMECs to TNF is similar to effects mediated by epidermal growth factor receptor (EGFR) activation, we explored the potential role of cross-talk through the EGFR signaling pathways in mediating cellular responses to TNF. Using a microarray enzyme-linked immunoassay, we found that exposure to TNF stimulated the dose-dependent shedding of the EGFR ligand transforming growth factor alpha (TGFalpha). Both proliferation and motility of HMECs induced by TNF was prevented either by inhibiting membrane protein shedding with a metalloprotease inhibitor, by blocking epidermal growth factor receptor (EGFR) kinase activity, or by limiting ligand-receptor interactions with an antagonistic anti-EGFR antibody. EGFR activity was also necessary for TNF-induced release of matrix metalloprotease-9, thought to be an essential regulator of mammary cell migration. The cellular response to TNF was associated with a biphasic temporal pattern of extracellular signal-regulated kinase (ERK) phosphorylation, which was EGFR-dependent and modulated by inhibition of metalloprotease-mediated shedding. Significantly, the late phase of ERK phosphorylation, detectable within 4 h after exposure, was blocked by the metalloprotease inhibitor batimastat, indicating that autocrine signaling through ligand shedding was responsible for this secondary wave of ERK activity. Our results indicate a novel and important role for metalloprotease activation and EGFR transmodulation in mediating the cellular response to TNF.

Detection of in situ labeled cell surface proteins by mass spectrometry: application to the membrane subproteome of human mammary epithelial cells.

Characterization of the surface exposed membrane subproteome of human mammary epithelial cells (strain 184 A1L5) implemented lysine specific in situ labeling of the proteins using sulfosuccinimidyl-6-(biotinamido)hexanoate, followed by enrichment of the biotinylated, tryptically digested peptides, and then liquid chromatography-tandem mass spectrometry analysis of the labeled peptides. Probing the membrane subproteome in this manner yielded unambiguous identification of proteins situated on the cell surface. The method reported can be adapted to include stable isotope labeling of proteins for quantitation of changes occurring on the cell surface in response to specific perturbations.