Imaging Intracellular Ph in a Reef Coral and Symbiotic Anemone

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 compartmentalization of SNARF-1 began to occur in (Table 1). In both light and dark conditions pHi was not mitochondria and vacuoles after prolonged periods of greater significantly different in coral relative to anemone cells. than 90 min in perfusion chambers.

Manipulation of pHi by Perfusion with NH4Cl. To determine the Intracellular pH in Coral and Anemone Cells. pHi was measured by sensitivity of SNARF-1 fluorescence signal to induced pH changes, ratiometric analysis of SNARF-1 fluorescence at 585 and 640 nm the pHi of algal-containing A. viridis endoderm cells were experi- within a region of interest (ROI) in host cytoplasm that did not mentally manipulated by short term perfusion of 20 mM NH4Cl. overlap with algae in x, y, and z planes of each cell. Calibration Perfusion with NH4Cl significantly increased pHi by approximately

Venn et al. PNAS ͉ September 29, 2009 ͉ vol. 106 ͉ no. 39 ͉ 16575 Downloaded by guest on September 28, 2021 Fluorescence Intensity 7.5 AB CpHi * 0 500 1000 6.6 6.8 7.0 7.4 0 0 0.8 µm 7.3 0.5 0.5

7.2 ROI 1 1

2.0 µm 1.5 1.5 7.1 pHi 2 2 7.0

2.5 2.5 6.9 3.3 µm 3 3 6.8

3.5 3.5 6.7 4.5 µm 4 4 6.6 Cell depth (Z) µm (Z) depth Cell Cell depth (Z) µm 4.5 4.5 control 20mM washout NH4CL 5.7 µm 5 5 Fig. 3. The impact of NH4Cl on pHi (mean Ϯ SE) in endoderm cells in FSW 5.5 5.5 isolated from A. viridis.* indicates signiﬁcant difference to control as determined by Tukey’s post hoc analysis, following one way ANOVA. 6 6 6.9 µm 6.5 6.5 physiology of coral and anemones. pHi has previously been deter- 7 7 mined in most model organisms that are routinely used for cell and developmental biology. However, data on pHi and many other Fig. 2. Z (cell depth) proﬁle of an A. viridis endodermal cell. (A) Represen- physiological parameters are still lacking for cnidarians in general, tative Z stack slices with cell depth. White circle ϭ ROI in the host cytoplasm. despite growing interest in the cell biology of this group in both the (B) Fluorescence intensity of SNARF-1 at 585 (circles) and 640 nm (triangles). (C) fields of marine ecology and evolutionary-developmental biology pHi with cell depth. (2, 23). Part of the reason for this lack of data lies in difficulties in obtaining viable cnidarian cells in continuous culture (2, 24, 25) and 0.4 pH units (one way ANOVA, F , ϭ 10.90, P Ͻ 0.01) and the limited number of studies that have undertaken imaging of 2 16 physiological parameters in isolated living cells from corals and sea returned to previous levels 10 min following removal of NH4Cl by washing with filtered seawater (FSW) (Fig. 3). anemones (26, 27). Cell imaging is particularly challenging in symbiotic systems where the autofluorescence derived from the Spatial Patterns in pHi. Analysis of Z stack images revealed that chlorophyll of intracellular algae can confound signals from many the principle source of pH heterogeneity within the cell was commercially available fluorescent dyes and the fact that algal cells associated with the presence of symbiotic algal cells. In algae- occupy the majority of the cell volume leaving limited areas of containing endoderm cells isolated from both A. viridis and Stylophora pistillata the immediate area surrounding each alga was at lower pH than the rest of the host cytoplasm, at pH 6 or below the limits of our calibration using SNARF-1 (Fig. 4). The region of low pH was observed in both light and dark-incubated <6.0 6.5 7.0 >7.5 endoderm cells. pH Discussion This is the first study to report intracellular pH (pHi) in anthozoans A and symbiotic cnidarians and the first to use live cell confocal C imaging in concert with a dual emission probe to monitor the A Table 1. Intracellular pH in S. pistillata and A. viridis endoderm cells treated with 20 min dark or light incubation following loading with the pH sensitive dye SNARF-1 for 30 min in the dark. Superscripted letters indicate homogeneous subsets as determined by Tukey’s posthoc analysis following one way ANOVA Intracellular pH (Mean Ϯ SD n ϭ 20)

Species/ cell type Dark Light

S.pistillata containing algal symbionts 7.13 Ϯ 0.24AB 7.41 Ϯ 0.22C A. viridis containing algal symbionts 7.01 Ϯ 0.27A 7.29 Ϯ 0.15BC A. viridis with no algal symbionts 7.04 Ϯ 0.29A 7.00 Ϯ 0.33A Fig. 4. Ratiometric image of SNARF-1 emission (585 and 640 nm) in ϭ ϭ Filtered seawater 8.1 Ϯ 0.1 n/a endoderm cell isolated from A. viridis.C animal cytoplasm. A Algal symbiont. Note: Red ϭ algal autoﬂuorescence and is not pH related.

16576 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0902894106 Venn et al. Downloaded by guest on September 28, 2021 animal cytoplasm to work with (28). The current study overcame dark and light-incubated cells of both species. One possibility is these problems using confocal imaging to analyze each cell in 3D that this region is associated with the symbiosome membrane (Z-stack profile) and restrict pH determination to the regions of complex (39), as previous research indicates that the symbio- SEE COMMENTARY interest identified in host cytoplasm that did not overlap with algal some in A. viridis could be as low as pH 5.7 (40), and symbio- symbionts. This approach allows the monitoring of intracellular somes in other symbiotic associations have also been found to be parameters (in this case pHi) in both algal symbiont-containing and at low pH (41). Symbiosome pH is important to determine given symbiont free cells. Overall, this work paves the way for future the potential role of the symbiosome in the trafficking of studies on symbiotic cnidaria utilizing the wide range of ratiometric compounds between host and symbiont, but the current data do fluorescent dyes designed to investigate numerous aspects of cell not definitively show whether the low pH region is associated function including signal transduction, membrane potential, and the with the symbiosome membrane complex or the adjacent host flux of metal and nonmetallic ions (29). cytoplasm. Future work using pH sensitive dyes with a lower pKa Values of pHi obtained for the reef coral S. pistillata (dark: and recently developed symbiosome specific probes (42) have 7.13 Ϯ 0.24 and light: 7.41 Ϯ 0.22) and the symbiotic sea the potential to resolve this issue. Ϯ Ϯ anemone A. viridis (dark: 7.01 0.27 and light: 7.29 0.15) fall Turning to processes of calcification in corals, our findings within the range typically observed for marine invertebrates, must be taken into account when deciphering how ions are with low values having been observed in unfertilized sea urchin transported to and from the site of calcification. In particular, eggs (pH 6.7) (30) and higher values obtained in ascidian eggs our results have implications for the speciation of carbon used (pH 7.6) (31). Two findings support the validity of our pHi for calcification. At the cnidarian pHi determined in the current measurements. First, perfusion experiments with NH4Cl cells 2Ϫ Ϫ study, the intracellular availability of CO3 relative to HCO3 displayed the classic pattern of alkalinization of pHi and return would be even lower than predicted in previous studies (43), thus to previous pHi levels on removal of NH4Cl; this pattern has 2Ϫ precluding the direct secretion of CO3 from cells at the site of been observed before in more conventionally used cell types calcification. To confirm this, pHi must be determined in (21, 32). Secondly, the fact that light or dark treatment had calicoblastic cells. This research will be greatly facilitated by the no effect on pHi in A. viridis cells without algal symbionts recent development of protocols to maintain primary cultures of excludes the possibility that differences observed in the this cell type (25). SNARF-1 ratio between light and dark-symbiont-containing In conclusion, we report measurements of pHi in symbiotic cells were caused by direct effects of light on the fluorescence cnidarian cells using a cell imaging procedure that paves the way behavior of the dye. for future investigations into the regulation of pHi and other The pHi values obtained in the current study are markedly lower than the surrounding seawater pH of 8.1 revealing that physiological parameters in corals and other cnidarians. Given coral and anemone cells maintain a strong gradient of pH the importance of pHi in most elements of cell function, the between the outside medium and the cells. This finding and the observed differences in pHi between light and dark conditions observation that pHi was higher in light-incubated relative to may be a key regulator of cell physiology in symbiotic cnidarians. dark-incubated symbiont-containing-cells have important impli- This possibility requires further research into pHi dynamics, cations when considering 2 processes that underpin coral reef building on the single time point measurements made in the ecosystems: photosynthesis and calcification. Cnidarian hosts current study, for a more compete understanding of the inter- supply DIC to their algal symbionts via a mechanism of DIC actions between host pHi and photosynthesis. An additional uptake and transport about which little is currently known (11, research priority must also be to determine how changes in external parameters such as seawater acidification impact pHi in 33, 34). CO2 has been proposed as one species of DIC that enters hosts cells from the external environment (34, 35). Using the pHi calicoblasts and other cell types. The information arising from measurements in the current study and a previous measurement investigations such as these will be essential for determining the of intracellular bicarbonate concentration [3.7 mM at minimum, mechanistic basis behind vulnerability of coral physiology to SCIENCES (36)], it is possible to estimate the intracellular host CO2 aspects of global environmental change and improving predic- concentration using the Henderson-Hasselbalch equation: pH ϭ tions about the future status of coral reefs. pK ϩlog [HCO Ϫ]/[CO ] with pK ϭ 6.1 (37). This would indicate APPLIED BIOLOGICAL 3 2 Materials and Methods that if CO2 were to enter host cells by diffusion, as proposed by Coral and Anemone Culture. Colonies of S. pistillata and individuals of A. viridis previous authors (34, 35), external CO2 concentrations would have to exceed a minimum of ca. 460 ␮M to achieve diffusion were maintained at the Centre Scientifique de Monaco in aquaria supplied with flowing seawater from the Mediterranean Sea (exchange rate 2% hϪ1)at into host cells. Ϯ ␮ Although the route by which DIC enters host cells is not clear, a salinity of 38.2 ppt, pH 8.1 0.1 under an irradiance of 300 mol photons mϪ2 sϪ1 (S. pistillata)or100␮mol photons mϪ2 sϪ1 (A. viridis). Temperature the majority of host intracellular DIC would be predicted to be Ϯ Ϯ Ϫ was maintained at 25 0.5 °C for S. pistillata and 21 1 °C for A. viridis. Corals HCO3 at the values of pHi determined in the present study. and anemones were fed 3 times a week with both frozen krill and live Artemia Previous authors have proposed that within endodermal cells salina nauplii. that contain algae, the carbon requirements of algal photosyn- Ϫ thesis are supplied by dehydration of host cell HCO3 to CO2 by Preparation of Cells. Branch tips of S. pistillata were removed using surgical carbonic anhydrase close to the symbiotic algae resulting in the bone cutters and the tissue removed by gentle brushing with a soft bristle Ϫ production of OH (10). Previous observations that light- toothbrush in 0.45 ␮m FSW. For A. viridis, cells were prepared by scraping incubated endoderm cell layers excrete OHϪ leading to alkalin- endoderm cells from longitudinally sectioned tentacles and suspending in ization of the cnidarian coelenteron cavity (10, 38) are support- FSW (44). Suspensions of anemone or coral cells were then filtered through a ing evidence for this mechanism. As the production of OHϪ 40 ␮m mesh made up to a volume of 45 ml and centrifuged at 350 g, for 4 min, at 20 °C. For confocal microscopy, the resulting pellets were resuspended in 2 within endoderm cells containing symbionts could be predicted 6 Ϫ1 to increase host cell pHi, the finding in the current study that pHi ml of FSW to obtain a density of approximately 10 cells ml and loaded into perfusion chambers that were constructed from silane slides with glass cov- was significantly higher in light-incubated than dark-incubated erslips mounted on thin runners of scotch tape sealed to the slide on 2 sides by symbiont-containing cells is important new evidence that also silicone grease (45). In the following procedures exposure of cells to fluores- supports this proposed mechanism. cence dyes and buffer solutions was achieved by perfusion, allowing the Close inspection of the interface by ratiometric imaging elimination of further centrifugation steps which reduce the yield of viable between the algae and host cell cytoplasm revealed that pH was endoderm cells. Viability staining using a Live/Dead Viability Kits (Invitrogen) low (Ͻ6) in the immediate area surrounding each alga in both confirmed cells remained viable in perfusion chambers for at least4hinFSW.

Venn et al. PNAS ͉ September 29, 2009 ͉ vol. 106 ͉ no. 39 ͉ 16577 Downloaded by guest on September 28, 2021 Dye Loading. Loading of SNARF-1 into cnidarian cells was achieved using the range pH 6–8.5 by washing endoderm cells in calibration buffer adjusted to cell permeant acetoxymethyl ester acetate of SNARF-1 (SNARF-1 AM). Solu- the desired pH by the addition of HCl or NaOH. The ionophore nigericin was tions were prepared from 20 mM stock in DMSO with working concentrations added at 30 ␮M to equilibriate pHi with the surrounding media and eliminate at 10 ␮M SNARF-1 a.m., 0.01% Pluronic F-127, and 0.1% DMSO in FSW for 30 pH gradients within the cell (18). Calibration buffers contained approximate ϩ min dark incubation at 21–24 °C. Concentrations fell within the range used in cytosolic concentrations of major ions in cnidarian cells (49, 50) (60 mM Na , ϩ Ϫ previous investigations (18). Inspection of cells treated with 5 ␮M digitonin in 200 mM K , 190 mM Cl , 25 mM Pipes (pH 6- 7.5) or Tricine (pH 8–8.5). Ϫ1 FSW to permeablize the cell membrane confirmed that any unhydrolyzed Mannitol was added to adjust osmolarity to 1100 mosm l . SNARF-1 a.m. did not accumulate with cell compartments. Calibration curves of pHi in S. pistillata and A. viridis endoderm cells are Host Cell Nuclei and Mitochondria Were Labeled by Incubation in 10 ␮g/ml provided as Fig. S1. Hoechst 33342 and 5 ␮M Rhodamine 123 in FSW for 10 min. After dye loading, cells were washed with 2 ml FSW. All dyes used for microscopy were purchased Image Analysis. After inspection of each cell in x, y and z planes, SNARF-1 Ϯ from Invitrogen. Other chemicals were purchased from Sigma-Aldrich. fluorescence intensity at 585 and 640 10 nm was recorded within an ROI selected in an area of host cytoplasm that did not overlap with algal cells. Examination of cells not loaded with SNARF-1 confirmed that algal autofluo- Confocal Microscopy. Confocal microscopy was conducted using a Leica SP5 rescence was not detectable in ROIs in this location. R was calculated after confocal microscope (Leica) equipped with UV and visible laser lines. Cells subtracting background fluorescence recorded in a second ROI in the sur- loaded with SNARF-1 a.m. were excited at 543 nm at a laser power at 25%, scan rounding cell media. speed 400htz, and pinhole set at 1.51 units using a X100-fold oil immersion The 585/640 nm fluorescence intensity ratio (R) was related to pH by the lens. Each cell was analyzed by capturing 15 frames in the x/y plane, through- following equation: out the depth (z plane) of the cell (z-stack profile). Prior optimization of settings ensured no loss of the SNARF-1 signal by photobleaching during the ϭ Ϫ Ϫ Ϫ ϫ pH pKA log [R RB/RA R FB(␭2)/FA(␭2)] 12 sec required for acquisition of each z-stack. SNARF-1 fluorescence emission was captured in 2 channels at 585 and 640 Ϯ 10 nm whilst simultaneously where (F) is fluorescence intensity measured at 640 nm (␭2) and the subscripts monitoring in transmission. As SNARF-1 a.m. has been shown previously to be A and B represent the limiting values at the acidic and basic end points of the responsive to temperature (46); temperature was maintained between 22 and titration respectively (i.e., pH 6 and 8.5). 24 °C during observation of cells. Procedures used for calibration and image analysis were validated by In cells containing algal symbionts, the use of 543 nm as the excitation checking the methods on a mammalian cell line (Murine C2C12 mesenchymal wavelength minimized chlorophyll autofluorescence, as 543 nm lies outside of cells) in PBS buffer. pHi was determined to be in the range of pH 7–7.2 which absorption spectra of chlorophyll a and in a low region of absorption of the fits the values determined previously for murine cells (e.g., 46). Peridinin-Chlorophyll-Protein complex (47).

Cell nuclei stained with Hoechst 33342 were visualized using UV excitation Manipulation of Intracellular pH by NH4Cl. Endoderm cells were suspended in with image capture at 450 nm Ϯ 10 nm. Mitochondria stained with Rhodamine FSW and dark loaded in perfusion chambers as described above and pHi 123 were visualized by excitation at 496 nm and captured at 530 nm Ϯ 10 nm determined (n ϭ 8 cells). Cells were then perfused with FSW containing 20 mM and in transmission. When imaging host nuclei and mitochondria, autoﬂu- NH4Cl and left for 10 min before pHi was determined again. NH4CL was then oresence of intracellular algae was captured in a separate channel at 670 Ϯ 10 washed out by perfusion with 5 ml FSW, left for 10 min and pHi determined nm and used to identify the position of algal symbionts within the cell. for a third time.

Light and Dark Incubation of Cells. After dark loading with SNARF-1 and Transmission Electron Microscopy (TEM). Fixation and observation of samples for perfusion with 2 ml FSW (described above), cell preparations were incubated TEM was carried out by methods described in ref. 51. Briefly, cell pellets were fixed for a further 20 min in the dark or under illumination from a fiber optic light at 4 °C with 4% glutaraldehyde in 0.085 M Sorensen phosphate buffer at pH 7.8 source (Bioblock Scientific, SFBN Mechanik GmbH). The duration of light with 0.5 M sucrose. Samples were rinsed in Sorensen buffer, then postfixed for 1 h incubation was chosen based on previous research showing that light- at ambient temperature with 1% osmium tetroxide in Sorensen phosphate dependant increases in coelenteron pH reached a maximum after 20 min light buffer. Samples were dehydrated by transfer through a graded series of ethanol exposure in tentacles of A. viridis (38). During light incubation, A. viridis or S. ending with a concentration of 100%. Following dehydration, samples were pistillata cells were exposed to levels of photosynthetically active radiation embedded in Epon resin. Ultrathin sections were cut with a diamond knife, (PAR) that matched the conditions under which the anemones and corals were transferred to copper grids coated with Formvar, stained with uranyl acetate and Ϫ Ϫ lead citrate, rinsed in distilled water, and observed with a CM12 Phillips TEM at 80 cultured (100 and 300 ␮mol photons m 2 s 1, respectively). During confocal kV at the Centre Commun de Microscopie Appliqueé at the University of Nice- analysis, light-incubated cells continued to receive light while locating and Sophia Antipolis, Nice, France. moving between cells in the preparation, but the light source was turned off TEM examination of cells immediately after centrifugation, following load- during the 12 sec required for each z-stack profile acquisition. All pH mea- ing with SNARF-1 a.m. and 90 min post SNARF-1 a.m. loading, confirmed there surements were performed within 20 min following light or dark illumination, were no impacts of these preparation steps on cell ultrastructure. with no loss of SNARF-1 signal in either 585 and 640 nm channels. ACKNOWLEDGMENTS. We thank Evelyn Houliston and Jonathan Erez for Calibration of pH in Cnidarian Cells. Measurements of intracellular pH were advice on microscopy, Sophie Pagnotta for assistance with TEM, and Mia performed by ratiometric analysis of SNARF-1 fluorescence (48). The ratio of Hoogenboom and 2 anonymous reviewers for comments on the manuscript. fluorescence intensity of SNARF-1 at 585/640 nm was calibrated in vivo in the This research was funded by the government of the Principality of Monaco.

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