Sorting the Messages

For this article, Steve Bunk interviewed Richard S. Lewis, associate professor of molecular and cellular physiology, Stanford University School of Medicine. Data from the Web of Science (ISI, Philadelphia) show that Hot Papers are cited 50 to 100 times more often than the average paper of the same type and age. R.E. Dolmetsch, K. Xu, and R.S. Lewis, "Calcium oscillations increase the efficiency and specificity of gene expression," Nature, 392:933-6, April 30, 1998. (Cited in about 170 papers since publication) Just about everything binds in a cell, but some things bind more than others. Take calcium. As a messenger it's multipotent, capable of sending signals along numerous transcriptional pathways. When free calcium in the cytosol increases beyond normal levels, many events can occur. In such a condition, how does the cell determine which pathways to activate? Different frequencies of cytosolic calcium oscillations long have been thought to contribute to signaling specificity in cells, but until this paper appeared, no one ever had tested the hypothesis. "What held up progress in the area was not having a good system for working this out," notes senior author Richard S. Lewis, associate professor of molecular and cellular physiology at Stanford University School of Medicine. His team developed a method for creating controlled oscillations of cytosolic calcium in vitro that bypasses the normal antigen-to-receptor binding. Their "calcium clamp" technique treats T cells with a compound called thapsigargin that depletes the cells' internal calcium stores and activates calcium channels in the plasma membrane. The cytosolic calcium levels then can be controlled by varying the concentration of extracellular calcium. Another research group, led by chemistry and biochemistry professor Roger Y. Tsien at the University of California, San Diego, developed a different method that was described in a frequently cited paper published alongside this one.1 Their team synthesized a derivative of the second messenger inositol 1,4,5-triphosphate (InsP3). The analogue molecule, designed to be activated by flashes of ultraviolet light, also generates spikes of cytosolic calcium. The calcium rise activates several transcriptional factors, including NF-AT (nuclear factor of activated T cells). NF-AT is crucial to expression of the interleukin-2 (IL-2) gene, which encodes an essential growth factor for T cells. Tsien's group showed that InsP3's efficiency in activating NF-AT is maximized when oscillations in the concentration of intracellular calcium occur at about one minute intervals. Lewis and colleagues compared the responses of three calcium-activated transcription factors, including NF-AT and NF-kB, to oscillations of differing frequencies. They found that NF-AT responses required frequent oscillations (more than one per 400 seconds), whereas NF-kB was activated at high and low frequencies (down to one per 30 minutes). NF-kB influences expression of the gene that encodes interleukin-8 (IL-8), an immune system chemokine. Richard S. Lewis "More than the technical approach we used, I think the idea that the temporal dynamics of calcium response may convey information to the cell is a big idea," he suggests. The clamp technique simplifies the system enough to vary only one parameter, avoiding effects on other pathways. "Now, the challenge is to take this result from an in vitro system and work back, to what actually happens in the body." For this next step, his lab has constructed a two-photon microscope. The laser scanning device, often employed to observe single neurons in vivo, uses relatively low-scattering and long-wavelength infrared light to excite flourescent-dye molecules. Only the cells in the focal plane are illuminated, allowing noninvasive, extended viewing without "bleaching out" the sample. "The basic idea is to look at cells undergoing some kind of defined developmental decision, or carrying out a defined effector function, and to ask, 'Is there a particular kind of calcium signal associated with these events?'" Lewis explains. Although potential therapeutic applications of such research involve complications that go far beyond those of pharmacology, he says, "If you could decode what the language is that the cell is using to carry out these responses, that would give you the opportunity to either silence or enhance them." Steve Bunk (sbunk@uswest.net) is a contributing editor for The Scientist. 1. W.-H. Li et al., "Cell-permeant caged InsP3 ester shows that Ca2+ spike frequency can optimize gene expression," Nature, 392:936-41, 1998. (Cited in more than 125 papers since publication)

Sorting the Messages

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