Imaging Intracellular Ph in a Reef Coral and Symbiotic Anemone
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Imaging intracellular pH in a reef coral and symbiotic anemone A. A. Venn1, E. Tambutte´ , S. Lotto, D. Zoccola, D. Allemand, and S. Tambutte´ Centre Scientifique de Monaco, Avenue Saint Martin, MC98000 Monaco Edited by Paul G. Falkowski, Rutgers, The State University of New Jersey, New Brunswick, NJ, and approved July 28, 2009 (received for review March 16, 2009) The challenges corals and symbiotic cnidarians face from global global climate change (12). Oceanic absorption of atmospheric environmental change brings new urgency to understanding fun- anthropogenic CO2 is decreasing both the availability of car- damental elements of their physiology. Intracellular pH (pHi) bonate ions and seawater pH potentially compromising coral influences almost all aspects of cellular physiology but has never calcification (13–15), while rising sea surface temperatures affect been described in anthozoans or symbiotic cnidarians, despite its photosynthesis and cause dysfunction in the symbiotic relation- pivotal role in carbon concentration for photosynthesis and calci- ship between corals and their intracellular algae (bleaching) (16, fication. Using confocal microscopy and the pH sensitive probe 17). Quantification of pHi is a key step toward determining the carboxy SNARF-1, we mapped pHi in short-term light and dark- mechanistic basis behind the vulnerability of these physiological incubated cells of the reef coral Stylophora pistillata and the processes to environmental change. symbiotic anemone Anemonia viridis. In all cells isolated from both Progress in the understanding of the physiology of symbiotic species, pHi was markedly lower than the surrounding seawater cnidarians like corals depends on the development of tools to pH of 8.1. In cells that contained symbiotic algae, mean values of examine their cell biology (2). In many other biological systems pHi were significantly higher in light treated cells than dark treated understanding of pHi regulation and other physiological param- -cells (7.41 ؎ 0.22 versus 7.13 ؎ 0.24 for S. pistillata; and 7.29 ؎ 0.15 eters has improved significantly by the application of intracel versus 7.01 ؎ 0.27 for A. viridis). In contrast, there was no signif- lular probes that emit fluorescence at 2 wavelengths which can icant difference in pHi in light and dark treated cells without algal be calibrated to the concentration of specific ions in the cell (e.g., symbionts. Close inspection of the interface between host cyto- Hϩ) (18–20). When coupled with confocal microscopy, this plasm and algal symbionts revealed a distinct area of lower pH approach allows the mapping of dynamic physiological processes adjacent to the symbionts in both light and dark treated cells, across cell structures (21, 22) and has enormous potential utility possibly associated with the symbiosome membrane complex. in physiological studies of symbiotic cnidarians, which harbor These findings are significant developments for the elucidation of intracellular algae and thus require excellent spatial resolution to models of inorganic carbon transport for photosynthesis and distinguish between animal and algal compartments. calcification and also provide a cell imaging procedure for future In the current study, we adapted a live cell imaging procedure investigations into how pHi and other fundamental intracellular that allowed us to make the first pHi measurements in symbiotic parameters in corals respond to changes in the external environ- cnidarians and anthozoans. To do this, we observed endoderm ment such as reductions in seawater pH. cells from the reef coral Stylophora pistillata and the symbiotic anemone Anemonia viridis exposed to both light and dark ͉ ͉ ͉ ͉ cell biology cnidaria symbiosis photosynthesis climate change conditions. pHi was determined by loading cells with the pH sensitive fluorescent probe, carboxyseminaphthorhodafluor-1 ymbiotic cnidarians form the structural foundation of coral (SNARF-1), and analysis by confocal microscopy. Our in vivo Sreefs, which rank among the most biodiverse and ecologically cell imaging procedure was developed to allow spatial separation important ecosystems. Despite their environmental significance, of cnidarian host and symbiont cell compartments and has a wide key elements of coral physiology such as calcification and the range of potential applications in the study of coral and cnidarian symbiotic interactions between cnidarians and their intracellular physiology. algae (Symbiodinium) are poorly understood, largely due to knowledge gaps in fundamental aspects of cnidarian cell biology Results (1–3). Observation of Cell Preparations. Cell preparations isolated from Intracellular pH (pHi) modulates virtually all aspects of cell A. viridis and S. pistillata included a variety of cell types including metabolism, including membrane channel functioning, the struc- endoderm cells that contained 1 to 3 algae and cells without algal tural and functional properties of enzymes, intracellular signal- symbionts (Fig. 1). ing, and ion availability (4, 5). As a consequence, pHi must be Cells that contained 2 algae were the most suitable for tightly controlled and changes in pHi are minimized by intra- measurements of pHi in algae-containing cells for 2 reasons. cellular buffers and regulation by ion carriers on the cell First, this cell type contained a larger area of host cytoplasm membrane (6, 7). In symbiotic cnidarians such as corals, pHi is available for pHi measurements than single alga cells [in which thought to be linked to processes of photosynthesis and calcifi- the cytoplasm was closely stretched around the alga (Fig. 1, cation by influencing the flux of ions between host, symbiont, and the surrounding environment (6, 8). This particularly con- cerns the transport of dissolved organic carbon (DIC) between Author contributions: A.A.V., D.A., and S.T. designed research; A.A.V., E.T., S.L., D.Z., and Ϫ cell layers and the speciation of DIC between CO2, HCO3 , and S.T. performed research; A.A.V., E.T., and S.T. analyzed data; and A.A.V., D.A., and S.T. 2Ϫ wrote the paper. CO3 (9–11). Measurements of pHi in cnidarian cells are therefore an essential step for elucidating how DIC is transferred The authors declare no conflict of interest. from the surrounding seawater to the respective sites of photo- This article is a PNAS Direct Submission. synthesis and calcification. See Commentary on page 16541. Understanding the links between pHi and processes of calci- 1To whom correspondence should be addressed. E-mail: alex@centrescientifique.mc. fication and photosynthesis is becoming increasingly important This article contains supporting information online at www.pnas.org/cgi/content/full/ given the challenges corals and symbiotic cnidarians face under 0902894106/DCSupplemental. 16574–16579 ͉ PNAS ͉ September 29, 2009 ͉ vol. 106 ͉ no. 39 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0902894106 Downloaded by guest on September 28, 2021 SEE COMMENTARY Fig. 1. Cnidarian endoderm cells isolated from A. viridis with and without symbiotic algae. (Row A–D) Different cell types: (A) endoderm cells without symbiotic algae. (B) endoderm cells containing one alga. C ϭ animal cytoplasm A ϭ intracellular symbiotic alga. (C) endoderm cells with 2 algae. (D) endoderm cells with 3 algae. (Column 1) TEM images. (Column 2) confocal images of cells stained with Hoechst 33342 and Rhodamine 123. (Column 3) Confocal images of cells loaded with SNARF-1 a.m. Colors in confocal images represent the following: Red ϭ autofluorescence of chlorophyll in intracellular algae, blue ϭ nuclei stained with Hoechst 33342, green ϭ mitochondria stained with Rhodamine 123, yellow ϭ cnidarian cytoplasm stained with SNARF-1. SCIENCES RowB)] and secondly, double cells were more numerous in cell curves of pHi with the SNARF-1 signal are given for S. pistillata APPLIED BIOLOGICAL preparations than cells that contained 3 algae. and A. viridis as supporting information (SI) Fig. S1. In the case of A. viridis preparations, cells with no intracellular Analysis of vertically-stacked horizontal slices through the cell algae could be identified to be of endodermal origin because the (z stack profiles) showed that pHi was stable (Ϯ pH 0.1) toward isolation procedure allowed separation of this layer from other the center of most cells where SNARF-1 fluorescence was host tissues. The presence of these cells allowed comparisons of greatest (Fig. 2). Exceptions to this rule occurred with the pHi to be made between endoderm cells with and without presence of acidic vacuoles which had a pH of 6 or below the intracellular algae. limits of our calibration. Analysis by TEM and staining with Hoecsht 33342 and Using this approach, pHi was determined in endoderm cells of rhodamine 123 identified the presence of nuclei and mitochon- S. pistillata and A. viridis that had been incubated for 20 min under light or dark conditions (Table 1). In both species, pHi was dria in all cell types, which were generally located between the significantly higher in light-incubated cells that contained algal algal cells in host cells with 2 or more algae (Fig. 1, Column 2). symbionts than dark-incubated symbiont-containing cells (one The cytoplasm of host cells stained evenly with SNARF-1 with way ANOVA, F5,114 ϭ 8.135, P Ͻ 0.001). However, when light little or no exclusion or concentration of the dye within cell and dark-incubated symbiont free cells isolated from A. viridis compartments such as nuclei or mitochondria (Fig. 1, Column 3), were compared, no significant difference in pHi was found although