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CRACking Down on the Signals Leading to Directed Cell Migration
To establish whether CRAC is important for chemotaxis, we analyzed the chemotactic behavior of cells lacking CRAC (crac– ) and found them to be highly impaired. These results establish that CRAC has at least two functions: its previously described role in the activation of ACA and a critical role in regulating chemotaxis (Figure 1, part A). This observation raises an apparent paradox: How can a protein recruited at the front of cells to regulate chemotaxis also regulate the activity of ACA at the back of cells? To gain insight into this, we tested a series of C-terminal CRAC deletion mutants and found that expression of any of these mutants in crac– cells restores their ability to migrate directionally, but not the ability to activate ACA. These data show that distinct domains of CRAC independently regulate ACA and chemotaxis. To assess the role of PI3K signaling in CRAC function, we analyzed a series of CRAC PH domain–containing mutants. Deletion of the entire PH domain (ΔPH-CRAC) abolishes all of CRAC’s functions. A point mutant of CRAC (R42C-CRAC) that no longer binds the products of PI3K is not recruited to the leading edge of cells, fails to support chemotaxis, and confers only modest ACA activation, demonstrating that PI3K products play a critical role in all of CRAC’s functions. Overexpression of CRAC and various CRAC mutants in wild-type cells had no significant effect on chemotaxis, but they distinctively altered ACA activation, again supporting the notion that CRAC independently regulates these two processes. We conclude from these results that chemoattractant-mediated activation of PI3K is important for the CRAC-dependent regulation of both chemotaxis and ACA activation. Although we have yet to determine the exact mechanism by which CRAC controls these two processes, we envision that a CRAC-mediated activation event at the leading edge nucleates components of the chemotaxis machinery and these signals at the front are subsequently transmitted to ACA at the back to facilitate signal relay (Figure 1, part B). This relay of intracellular signals is not uncommon during chemotaxis, which requires the coordinated regulation of distinct events in the front and back of cells. Figure 1. A) Cytosolic regulator of adenylyl cyclase (CRAC) regulates chemotaxis and adenylyl cyclase (ACA) activation. Cartoon depicts the series of events leading to the chemoattractant-mediated activation of CRAC in Dictyostelium. B) Streaming during Dictyostelium chemotaxis. Left panel, a picture of Dictyostelium cells as they migrate by aligning in a head-to-tail fashion. Right panel, a model depicting the proposed localized secretion of the chemoattractant cyclic AMP (cAMP), which attracts cells to the back of the cell in front of them. The pictures represent the cellular distribution of CRAC and ACA during this process. PH, pleckstrin homology; GFP, green fluorescent protein; YFP, yellow fluorescent protein. Our study demonstrates that CRAC acts as a central regulator by integrating two interrelated aspects of chemotaxis: directed migration and signal relay. Indeed, signal relay greatly amplifies chemotaxis by enabling the recruitment of neighboring cells. As leukocytes are also known to secrete chemoattractants in response to chemoattractant stimulation, it is enticing to speculate that such a central regulator of chemotaxis also exists in higher eukaryotes.
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hemotaxis, the fundamental process by which cells detect and migrate up an external chemical gradient, is important in a wide range of biological processes, including wound repair, angiogenesis, and axon guidance, as well as for the survival of many lower organisms. The mechanisms that govern how cells sense and respond to chemoattractants are remarkably conserved from the social amoebae Dictyostelium discoideum to mammalian leukocytes, where chemotactic signals are transduced via G protein–coupled receptor signaling pathways. Chemotactic sensing mechanisms are extremely robust but are also highly sensitive. For example, Dictyostelium cells can accurately migrate toward chemoattractant sources that can vary by more than 4 orders of magnitude in concentration, and yet at the same time, these cells can accurately respond to very shallow gradients in which the front of the cell experiences a receptor occupancy that differs by only 1% of that at its back. This exquisite sensitivity requires cells to be able to integrate and accurately transduce signals in a spatiotemporal fashion. 