Regulation of Keystone Predation by Small Changes in Ocean Temperature
ture for 60 min and then centrifuged at 100,000g for 30 min at 4°C. Supernatants and pellets were analyzed by SDS-polyacrylamide gel electrophoresis (PAGE) and the relative amounts of the proteins in each fraction were estimated by scanning densitometry of the stained gels. In the absence of actin, no SipA (at all concentrations used in the assay) was detected in pellets after centrifugation at 100,000g. 13. Samples were loaded onto carbon-coated grids, stained with 1% uranyl acetate, and
... acetate, and visualized under the electron microscope. 14. Actin was isolated was from rabbit skeletal muscle [ J. A. Spudich and S. Watt, J. Biol. Chem. 246, 4866 (1971)] and labeled with pyrene [T. Kouyama and K. Mihashi, Eur. J. Biochem. 114, 33 (1981)]. Pyrene-Gactin (4 M) was polymerized at room temperature for 30 min in actin polymerization buffer (APB) [20 mM Pipes (pH 7.0), 75 mM KCl, 2 mM MgCl 2 , 0.1 mM EGTA, 0.1 mM dithiothreitol, and 0.05 mM ATP] and then diluted to different concentrations in the presence or absence of equimolar concentrations of SipA. After a 4-hour incubation at room temperature, the fluorescence intensity was measured on a fluorescence spectrophotometer (Hitachi F-2000) with excitation wavelength set at 365 nm and emission wavelength at 407 nm. Alternatively, pyrene-G-actin was diluted to various concentrations in the presence and absence of SipA in APB without KCl and MgCl 2 . Polymerization was initiated by adding 75 mM KCl and 2 mM MgCl 2 . After a 4-hour incubation at room temperature, the fluorescence intensity was measured as described above. To examine the effect of SipA on F-actin stability, we diluted 1 M of polymerized actin, in the presence or absence of SipA (1 M), in APB (with or without KCl and MgCl 2 ) to 0.1 M and measured the fluorescence intensity over time. To evaluate the effect of SipA on actin polymerization, we precleared pyrene-G-actin by centrifugation at 100,000g for 4 hours at 4°C after dilution in APB without KCl and MgCl 2 . Actin concentration was adjusted to 1 M in the presence or absence of SipA (1 M). Polymerization was initiated by adjusting the buffer concentration to 75 mM KCl and 2 mM MgCl 2 , and the fluorescence intensity was measured over time. 15. D. Zhou and J. E. Galán, unpublished results. 16. We thank E. Taylor for providing pyrene-labeled actin, and members of the Galán laboratory for critical reading of the manuscript. Supported by NIH grants AI30492 and GM52543 ( J.E.G.) and DK25387 (M.S.M.). Key species interactions that are sensitive to temperature may act as leverage points through which small changes in climate could generate large changes in natural communities. Field and laboratory experiments showed that a slight decrease in water temperature dramatically reduced the effects of a keystone predator, the sea star Pisaster ochraceus, on its principal prey. Ongoing changes in patterns of cold water upwelling, associated with El Niño events and longer term geophysical changes, may thus have far-reaching impacts on the composition and diversity of these rocky intertidal communities.