How Cells Find Their Way

Organisms need to sense their environment. By sensing, they can develop, heal wounds, protect against invaders, and create blood vessels. Chemotaxis, or directional sensing, allows cells to detect chemicals with exquisite sensitivity. Some chemotactic cells can sense chemical gradients that differ by only a few percent from a cell's front to its back. Although discovery of the molecule types involved in chemotaxis, as with other kinds of cell signaling events, has mounted, the details of how this dynamic process works is somewhat shrouded in mystery. But in the last few years, researchers have applied sophisticated imaging technologies to dissect this process, and now the details are coming to light. The phenomenology itself is decades old. Neutrophil migration was observed in the early part of the 20th century, and was captured on film 50 years ago by the late David Rogers of Vanderbilt University. (Rogers' film can be viewed on the Web at: expmed.bwh.harvard.edu/projects/motility/neutrophil.html.) This film shows in dramatic fashion a neutrophil chasing a bacterium that is laying down a chemoattractant gradient--most likely the bacterial peptide f-MLP, which is induced by elements of the host immune system. A neutrophil tracks down the chemoattractant's source, moving in a polar fashion, leading with one end, retracting the other, and quickly changing course as the bacterium itself changes direction; all features of the chemotactic response that researchers are trying to get a handle on. Darling of Developmental Biologists Some of the earliest insights into eukaryotic chemotaxis came from the lowly slime mold, Dictyostelium discoideum, or Dicty, the darling of developmental biologists. Slime mold, or soil amoeba, which Dicty researchers prefer, offers numerous research advantages, its primary benefit being that its genetic system is easily amenable to experimentation. It also can be grown in large quantities and induced to differentiate synchronously. Moreover, this simple system has many of the same elements as neutrophils for sensing and tracking down a chemoattractant, though it does so for different reasons. Neutrophils seek out invaders, while slime molds use the signaling pathway for finding food, for intercellular communication, and for starting its differentiation from free-floating amoebae to a multicellular organism. Genetic dissection of the protein pathways involved in slime mold chemotaxis is more advanced than neutrophils, since knocking out genes is relatively straightforward. Research on slime mold, for example, first showed that the cAMP receptor, a member of the G-protein coupled receptor (GPCR) family, was important in chemotaxis, a finding that foreshadowed a similar finding in leukocytes.1 Says long-time Dicty researcher Rick Firtel, cell and developmental biology chairman, University of California, San Diego, Dicty represents "our best shot at truly understanding [chemotaxis]." The chemotactic response in Dictyostelium is dramatic as well as dynamic. When starving, a few amoebae form an aggregation center that emanates pulses of cAMP, which are then relayed from cell to cell. Time-lapse videos reveal waves of cAMP in the form of spirals or concentric rings, coursing through the aggregating cells. The amoebae move, chemotactically, toward the higher cAMP concentration. And while molecular genetic dissection of this progression has identified dozens of proteins involved, this approach alone cannot completely describe this process, which changes in both time and space. Live and In Color Researchers have turned to live cell-imaging techniques to further investigate chemotactic responses. Peter Devreotes, cell biology and anatomy director, Johns Hopkins University School of Medicine, was the first to apply green fluorescent protein (GFP)-tagging to the study of GPCR molecules in live cells. Looking at the distribution of some known receptors on a Dicty amoeba's surface, he found that the receptors are uniformly distributed, a finding that was surprising because these cells normally have fluid membranes.2 This result eliminated the simplest possibility that chemoattractant receptors clustering at the leading edge generated polarity through asymmetric signaling. Henry Bourne, a cellular and molecular pharmacology professor, University of California, San Francisco, got the same result while working with cultured myeloid leukemic cells, which can be induced to behave like neutrophils: GFP-tagged C5a receptor molecules do not move to the cells' front, but remain uniformly distributed along the cell membrane.3 These results show that cells are sensitive to chemoattractant uniformly located around their perimeters, which leaves the question unanswered of how do the cells know where the attractant is and in what direction to go. Experiments on both Dicty and neutrophils point the finger to membrane organization itself, as the lipid phosphatidylinositol (3,4,5)-trisphosphate (PIP3) rapidly accumulates at the edge with the highest attractant concentration following stimulation. Again, imaging experiments led the charge. Following GFP-tagged, PH-domain-containing proteins, (PH-domain-containing proteins can be used as probes for PIP3, as they bind to activated PIP3), the Devreotes lab and later the Firtel and Bourne labs showed that the PH domains translocate to the leading edge within seconds of stimulation.4-6 In fact, the PH domains fall off the membrane almost as fast as they jump on: 12 seconds and it's come and gone. The processes' dynamic nature is likely what gives these cells the ability to sense and integrate both spatial and temporal changes in its environment. As evidence from imaging studies accumulates that PIP3 is the leading edge's organizing principle, more classical kinds of genetic experiments back up this idea. When PIP3 accumulation at the cell's front is impaired, which is accomplished by knocking out PI3 kinase activity, (the PIP3 activator), either genetically or with inhibitors, both Dicty cells and neutrophils loose their polarity and have trouble moving directionally.7 The cells "wander around, looking very much like a dog that's smelling something but doesn't know where it is," says Bourne. Some think that although PIP3 is a key component of a cell's ability to sense direction, it might not be the sole determining factor. There is a bevy of kinases and GTPases, all with a demonstrated role in chemotaxis, and many of which concentrate in the leading edge following stimulation. The kinase Akt/PKB, cytosolic regulator of adenyl cyclase (CRAC), and a new Ph-domain containing protein, PhdA, all have been caught on camera, translocating to the leading edge. In more recent experiments from the Devreotes lab, fluorescence resonance energy transfer (FRET) is being used to pick apart some of the earliest events and interactions in the Dicty chemotactic response. In work published earlier this year in Science, Devreotes watched in real time as the subunits of G protein split apart following GPCR activation, an event that unleashes the G protein on downstream effector molecules.8 The next step in this chemotaxis cascade will likely be revealed in similar fashion. A look at the videos of an aggregating Dicty or a neutrophil chasing its prey shows why this process has captivated scientists. Viewing them now, albeit with fancy machines, reveals the molecular mechanism behind the behavior. Firtel reminisces about a time when his genetic experiments left him unsatisfied. "We had these developmental phenotypes we really didn't understand," he says. "We decided to get into imaging. It was crystal clear what the defect in the cells was once we did the movie--absolutely perfectly clear." Laura DeFrancesco (defrancesco1@earthlink.net) is a freelance writer in Pasadena, Calif. References 1. P.S. Klein et al., "A chemoattractant receptor controls development in Dictyostelium discoideum," Science, 241:1467-72, 1988. 2. Z. Xiao et al., "Dynamic distribution of chemoattractant receptors in living cells during chemotaxis and persistent stimulation," Journal of Cell Biology, 139:365-74, 1997. 3. G. Servant et al., "Dynamics of a chemoattractant receptor in living neutrophils during chemotaxis," Molecular Biology of the Cell, 10:1163-78, 1999. 4. C. Parent et al., "G protein signaling events are activated at the leading edge of chemotactic cells," Cell, 95:81-91, 1998. 5. R. Meili et al., "Chemoattractant-mediated transient activation and membrane localization of Akt/PKB is required for efficient chemotaxis to cAMP in Dictyostelium," EMBO Journal, 18:2092-105, 1999. 6. G. Servant et al., "Polarization of chemoattractant receptor signaling during neutrophil chemotaxis," Science, 287:1037-40, 2000. 7. S. Funamoto et al., "Role of phosphatidylinositol 3' kinase and a downstream plectrin homology domain-containing protein in controlling chemotaxis in Dictyostelium," The Journal of Cell Biology 153:795-809, May 14, 2001. 8.C. Janetopoulos et al., "Receptor-mediated activation of heterotrimeric G-proteins in living cells," Science 291:2408-11, March 23, 2001.

How Cells Find Their Way

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