Safely ensconced in their protective cyst shells, brine shrimp embryos are some of the toughest creatures on the planet, happily surviving for years where other animals would suffocate. Researchers have painstakingly worked out that a key factor for the hardy little creatures' survival in oxygenless water is acidification of their cells. This triggers an almost complete metabolic shutdown and stops an embryo's development in its tracks, conserving precious energy until it's safe to reverse the acidification and kick-start development again. But the tough shell that provides such effective refuge makes it almost impossible to study an embryo's insides, so the mechanism that causes this acidification has proved to be frustratingly elusive, baffling researchers for 20 years. Joseph Covi and Steven Hand set out to explain the reversible acidification that's crucial for anoxia tolerance( and ).
Covi explains that brine shrimp embryo cells don't yet have fully formed cell membranes, so an embryo is essentially one big cell. Suspended inside it are organelles like lysosomes and yolk platelets. Covi and Hand suspected that when embryos are afloat in comfortable oxygen levels, a common proton pump called V-ATPase pumps protons into these organelles, turning them into proton storage units. But when oxygen levels suddenly plummet, `the embryo's ATP levels crash, the ATP-dependent V-ATPase stops working, and the organelles leak their protons into the embryo's cytoplasm,' Covi says. Could this explain the intracellular acidification seen in anoxic embryos?
To show that this explanation is plausible, Covi and Hand first had to establish that brine shrimp embryos have V-ATPase. Scooping floating cysts out of Utah's Great Salt Lake, they took the embryos back to the lab. They compared brine shrimp embryos' cDNA with known sequences of V-ATPases from other animals. Sure enough, they found that the embryos possess V-ATPase. They also noticed that it's expressed differentially as embryos develop, suggesting that it plays a role during development. To show that V-ATPase is positioned to sequester protons in the organelles, Covi and Hand used an antibody to locate the proton pump in isolated cell fractions. They discovered that V-ATPase is distributed in various membranes, including those of organelles.
But does V-ATPase set up a proton gradient between an embryo's organelles and its cytoplasm? To find out, Covi and Hand tried to stop the proton pump by incubating dechorionated embryos with bafilomycin, a V-ATPase inhibitor. It was a long shot; nobody had breached the cysts' chitin layer before. To their amazement, the embryos stopped developing. `I dropped everything else I was working on,' Covi recalls. He called in Dale Treleaven, an expert in 31P-NMR, a noninvasive technique to measure intracellular pH. Monitoring embryos' pH as they recovered from anoxia, Covi saw that the cytoplasm of bafilomycin-treated embryos remained acidic. So the pumping of protons from the cytoplasm into organelles by V-ATPase is crucial to the reversal of acidification, and is therefore a key factor in an embryo's recovery from anoxia. Finally, to confirm that the release of protons stored in the organelles causes intracellular acidification, Covi incubated embryos in oxygenated seawater with CCCP, a chemical that makes membranes leaky to protons. Sure enough, the cytoplasm acidified, just as it does in anoxic embryos. `The ability to dissipate internal proton gradients under anoxia appears to set brine shrimp embryos apart from other animals,' Covi concludes.
