HighlandReefer
Team RC
http://www.pnas.org/content/106/39/16574.full
approved July 28, 2009
From it in part (the entire article is available to read):
"Discussion
This is the first study to report intracellular pH (pHi) in anthozoans and symbiotic cnidarians and the first to use live cell confocal imaging in concert with a dual emission probe to monitor the physiology of coral and anemones. pHi has previously been determined in most model organisms that are routinely used for cell and developmental biology. However, data on pHi and many other physiological parameters are still lacking for cnidarians in general, despite growing interest in the cell biology of this group in both the fields of marine ecology and evolutionary-developmental biology (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 the limited number of studies that have undertaken imaging of physiological parameters in isolated living cells from corals and sea anemones (26, 27). Cell imaging is particularly challenging in symbiotic systems where the autofluorescence derived from the chlorophyll of intracellular algae can confound signals from many commercially available fluorescent dyes and the fact that algal cells occupy the majority of the cell volume leaving limited areas of animal cytoplasm to work with (28). The current study overcame these problems using confocal imaging to analyze each cell in 3D (Z-stack profile) and restrict pH determination to the regions of interest identified in host cytoplasm that did not overlap with algal symbionts. This approach allows the monitoring of intracellular parameters (in this case pHi) in both algal symbiont-containing and symbiont free cells. Overall, this work paves the way for future studies on symbiotic cnidaria utilizing the wide range of ratiometric fluorescent dyes designed to investigate numerous aspects of cell function including signal transduction, membrane potential, and the flux of metal and nonmetallic ions (29).
Values of pHi obtained for the reef coral S. pistillata (dark: 7.13 ± 0.24 and light: 7.41 ± 0.22) and the symbiotic sea anemone A. viridis (dark: 7.01 ± 0.27 and light: 7.29 ± 0.15) fall within the range typically observed for marine invertebrates, with low values having been observed in unfertilized sea urchin eggs (pH 6.7) (30) and higher values obtained in ascidian eggs (pH 7.6) (31). Two findings support the validity of our pHi measurements. First, perfusion experiments with NH4Cl cells displayed the classic pattern of alkalinization of pHi and return to previous pHi levels on removal of NH4Cl; this pattern has been observed before in more conventionally used cell types (21, 32). Secondly, the fact that light or dark treatment had no effect on pHi in A. viridis cells without algal symbionts excludes the possibility that differences observed in the SNARF-1 ratio between light and dark-symbiont-containing cells were caused by direct effects of light on the fluorescence behavior of the dye.
The pHi values obtained in the current study are markedly lower than the surrounding seawater pH of 8.1 revealing that coral and anemone cells maintain a strong gradient of pH between the outside medium and the cells. This finding and the observation that pHi was higher in light-incubated relative to dark-incubated symbiont-containing-cells have important implications when considering 2 processes that underpin coral reef ecosystems: photosynthesis and calcification. Cnidarian hosts supply DIC to their algal symbionts via a mechanism of DIC uptake and transport about which little is currently known (11, 33, 34). CO2 has been proposed as one species of DIC that enters hosts cells from the external environment (34, 35). Using the pHi measurements in the current study and a previous measurement of intracellular bicarbonate concentration [3.7 mM at minimum, (36)], it is possible to estimate the intracellular host CO2 concentration using the Henderson-Hasselbalch equation: pH = pK +log [HCO3−]/[CO2] with pK = 6.1 (37). This would indicate that if CO2 were to enter host cells by diffusion, as proposed by previous authors (34, 35), external CO2 concentrations would have to exceed a minimum of ca. 460 μM to achieve diffusion into host cells.
Although the route by which DIC enters host cells is not clear, the majority of host intracellular DIC would be predicted to be HCO3− at the values of pHi determined in the present study. Previous authors have proposed that within endodermal cells that contain algae, the carbon requirements of algal photosynthesis are supplied by dehydration of host cell HCO3− to CO2 by carbonic anhydrase close to the symbiotic algae resulting in the production of OH− (10). Previous observations that light-incubated endoderm cell layers excrete OH− leading to alkalinization of the cnidarian coelenteron cavity (10, 38) are supporting evidence for this mechanism. As the production of OH− within endoderm cells containing symbionts could be predicted to increase host cell pHi, the finding in the current study that pHi was significantly higher in light-incubated than dark-incubated symbiont-containing cells is important new evidence that also supports this proposed mechanism.
Close inspection of the interface by ratiometric imaging between the algae and host cell cytoplasm revealed that pH was low (<6) in the immediate area surrounding each alga in both dark and light-incubated cells of both species. One possibility is that this region is associated with the symbiosome membrane complex (39), as previous research indicates that the symbiosome in A. viridis could be as low as pH 5.7 (40), and symbiosomes in other symbiotic associations have also been found to be at low pH (41). Symbiosome pH is important to determine given the potential role of the symbiosome in the trafficking of compounds between host and symbiont, but the current data do not definitively show whether the low pH region is associated with the symbiosome membrane complex or the adjacent host cytoplasm. Future work using pH sensitive dyes with a lower pKa and recently developed symbiosome specific probes (42) have the potential to resolve this issue.
Turning to processes of calcification in corals, our findings must be taken into account when deciphering how ions are transported to and from the site of calcification. In particular, our results have implications for the speciation of carbon used for calcification. At the cnidarian pHi determined in the current study, the intracellular availability of CO32− relative to HCO3− would be even lower than predicted in previous studies (43), thus precluding the direct secretion of CO32− from cells at the site of calcification. To confirm this, pHi must be determined in calicoblastic cells. This research will be greatly facilitated by the recent development of protocols to maintain primary cultures of this cell type (25).
In conclusion, we report measurements of pHi in symbiotic cnidarian cells using a cell imaging procedure that paves the way for future investigations into the regulation of pHi and other physiological parameters in corals and other cnidarians. Given the importance of pHi in most elements of cell function, the observed differences in pHi between light and dark conditions may be a key regulator of cell physiology in symbiotic cnidarians. This possibility requires further research into pHi dynamics, building on the single time point measurements made in the current study, for a more compete understanding of the interactions between host pHi and photosynthesis. An additional research priority must also be to determine how changes in external parameters such as seawater acidification impact pHi in calicoblasts and other cell types. The information arising from investigations such as these will be essential for determining the mechanistic basis behind vulnerability of coral physiology to aspects of global environmental change and improving predictions about the future status of coral reefs."
approved July 28, 2009
From it in part (the entire article is available to read):
"Discussion
This is the first study to report intracellular pH (pHi) in anthozoans and symbiotic cnidarians and the first to use live cell confocal imaging in concert with a dual emission probe to monitor the physiology of coral and anemones. pHi has previously been determined in most model organisms that are routinely used for cell and developmental biology. However, data on pHi and many other physiological parameters are still lacking for cnidarians in general, despite growing interest in the cell biology of this group in both the fields of marine ecology and evolutionary-developmental biology (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 the limited number of studies that have undertaken imaging of physiological parameters in isolated living cells from corals and sea anemones (26, 27). Cell imaging is particularly challenging in symbiotic systems where the autofluorescence derived from the chlorophyll of intracellular algae can confound signals from many commercially available fluorescent dyes and the fact that algal cells occupy the majority of the cell volume leaving limited areas of animal cytoplasm to work with (28). The current study overcame these problems using confocal imaging to analyze each cell in 3D (Z-stack profile) and restrict pH determination to the regions of interest identified in host cytoplasm that did not overlap with algal symbionts. This approach allows the monitoring of intracellular parameters (in this case pHi) in both algal symbiont-containing and symbiont free cells. Overall, this work paves the way for future studies on symbiotic cnidaria utilizing the wide range of ratiometric fluorescent dyes designed to investigate numerous aspects of cell function including signal transduction, membrane potential, and the flux of metal and nonmetallic ions (29).
Values of pHi obtained for the reef coral S. pistillata (dark: 7.13 ± 0.24 and light: 7.41 ± 0.22) and the symbiotic sea anemone A. viridis (dark: 7.01 ± 0.27 and light: 7.29 ± 0.15) fall within the range typically observed for marine invertebrates, with low values having been observed in unfertilized sea urchin eggs (pH 6.7) (30) and higher values obtained in ascidian eggs (pH 7.6) (31). Two findings support the validity of our pHi measurements. First, perfusion experiments with NH4Cl cells displayed the classic pattern of alkalinization of pHi and return to previous pHi levels on removal of NH4Cl; this pattern has been observed before in more conventionally used cell types (21, 32). Secondly, the fact that light or dark treatment had no effect on pHi in A. viridis cells without algal symbionts excludes the possibility that differences observed in the SNARF-1 ratio between light and dark-symbiont-containing cells were caused by direct effects of light on the fluorescence behavior of the dye.
The pHi values obtained in the current study are markedly lower than the surrounding seawater pH of 8.1 revealing that coral and anemone cells maintain a strong gradient of pH between the outside medium and the cells. This finding and the observation that pHi was higher in light-incubated relative to dark-incubated symbiont-containing-cells have important implications when considering 2 processes that underpin coral reef ecosystems: photosynthesis and calcification. Cnidarian hosts supply DIC to their algal symbionts via a mechanism of DIC uptake and transport about which little is currently known (11, 33, 34). CO2 has been proposed as one species of DIC that enters hosts cells from the external environment (34, 35). Using the pHi measurements in the current study and a previous measurement of intracellular bicarbonate concentration [3.7 mM at minimum, (36)], it is possible to estimate the intracellular host CO2 concentration using the Henderson-Hasselbalch equation: pH = pK +log [HCO3−]/[CO2] with pK = 6.1 (37). This would indicate that if CO2 were to enter host cells by diffusion, as proposed by previous authors (34, 35), external CO2 concentrations would have to exceed a minimum of ca. 460 μM to achieve diffusion into host cells.
Although the route by which DIC enters host cells is not clear, the majority of host intracellular DIC would be predicted to be HCO3− at the values of pHi determined in the present study. Previous authors have proposed that within endodermal cells that contain algae, the carbon requirements of algal photosynthesis are supplied by dehydration of host cell HCO3− to CO2 by carbonic anhydrase close to the symbiotic algae resulting in the production of OH− (10). Previous observations that light-incubated endoderm cell layers excrete OH− leading to alkalinization of the cnidarian coelenteron cavity (10, 38) are supporting evidence for this mechanism. As the production of OH− within endoderm cells containing symbionts could be predicted to increase host cell pHi, the finding in the current study that pHi was significantly higher in light-incubated than dark-incubated symbiont-containing cells is important new evidence that also supports this proposed mechanism.
Close inspection of the interface by ratiometric imaging between the algae and host cell cytoplasm revealed that pH was low (<6) in the immediate area surrounding each alga in both dark and light-incubated cells of both species. One possibility is that this region is associated with the symbiosome membrane complex (39), as previous research indicates that the symbiosome in A. viridis could be as low as pH 5.7 (40), and symbiosomes in other symbiotic associations have also been found to be at low pH (41). Symbiosome pH is important to determine given the potential role of the symbiosome in the trafficking of compounds between host and symbiont, but the current data do not definitively show whether the low pH region is associated with the symbiosome membrane complex or the adjacent host cytoplasm. Future work using pH sensitive dyes with a lower pKa and recently developed symbiosome specific probes (42) have the potential to resolve this issue.
Turning to processes of calcification in corals, our findings must be taken into account when deciphering how ions are transported to and from the site of calcification. In particular, our results have implications for the speciation of carbon used for calcification. At the cnidarian pHi determined in the current study, the intracellular availability of CO32− relative to HCO3− would be even lower than predicted in previous studies (43), thus precluding the direct secretion of CO32− from cells at the site of calcification. To confirm this, pHi must be determined in calicoblastic cells. This research will be greatly facilitated by the recent development of protocols to maintain primary cultures of this cell type (25).
In conclusion, we report measurements of pHi in symbiotic cnidarian cells using a cell imaging procedure that paves the way for future investigations into the regulation of pHi and other physiological parameters in corals and other cnidarians. Given the importance of pHi in most elements of cell function, the observed differences in pHi between light and dark conditions may be a key regulator of cell physiology in symbiotic cnidarians. This possibility requires further research into pHi dynamics, building on the single time point measurements made in the current study, for a more compete understanding of the interactions between host pHi and photosynthesis. An additional research priority must also be to determine how changes in external parameters such as seawater acidification impact pHi in calicoblasts and other cell types. The information arising from investigations such as these will be essential for determining the mechanistic basis behind vulnerability of coral physiology to aspects of global environmental change and improving predictions about the future status of coral reefs."
