The second messenger calcium is one of the best studied signaling molecules in biology. It has important roles in nearly all eukaryotic cells and is known to control a wide range of intracellular processes that range from secretion and contraction to differentiation and apoptosis. Calcium can act globally through cell-wide pulses, waves, and persistent increases in cytoplasmic concentration, but it can also act locally in microscale regions as the effective diffusion of calcium is markedly slowed by abundant calcium-binding proteins.
One of the first and best characterized roles of calcium is the triggering of muscle contraction. In resting striated muscle cells, depolarization-induced Ca2+ increases are rapidly and directly transduced into myosin II-mediated cell contraction. In other cell types, calcium signals act less directly to promote contractile forces, often acting through calcium-calmodulin, myosin light-chain kinase, and other effectors to activate myosin motors and increase the availability of myosin binding sites on actin filaments.
Myosin II is known to be important for generating contractile forces in motile cells. Contractile forces are important for breaking adhesive connections to extracellular binding sites at the back of the cell and may be important for pushing the bulk of the cell body forward during migration. Since contraction primarily happens at the back of the cell, one might expect calcium's role in motile cells to occur in the same place. There is evidence for such a role for calcium at the back, but there have also been reports suggesting a role for calcium at the front (Blaser et al., 2006
; Brundage et al., 1991
; Gomez et al., 2001
; Xu et al., 2004
). A series of such reports with often conflicting results has left us with an unclear picture of whether calcium has a single or multiple roles in motile cells.
Two recent papers (Evans and Falke, 2007
; Wei et al., 2008
) show that local calcium signals occur at the leading edge of polarized and migrating cells, supporting a model that calcium can be a critical component of a cell's polarization machinery and/or that it can play a role in steering cells toward a chemoattractant source.
In order to move in a directed fashion, a cell must define a front and a back and segregate a number of activities, such as protrusion to the front and contraction to the back. Evans and Falke (2007)
studied spontaneously polarizing macrophages to investigate an established but incompletely defined positive feedback system that is important for establishing and maintaining this polarity. In this system, phosphatidylinositol 3-kinase (PI3K) acts upstream of the monomeric GTPase Rac to promote localized actin polymerization, and these downstream events feed back in a way that is not yet fully understood to promote further local PI3K activity. Evans and Falke (2007)
hypothesized that calcium signaling may play an unappreciated role in this system. Using intracellular fluorescent reporters, they found that depletion of extracellular calcium disrupts the positive feedback loop, as measured by either PI3K activity or actin polymerization. Furthermore, they found that a fluorescently labeled protein kinase C, a protein whose recruitment to the plasma membrane is triggered by intracellular calcium increases, localizes to the leading edge in polarized cells. This localization disappeared if calcium influx, PI3K activity, or actin polymerization was blocked. These results argue that calcium plays an important role in the positive feedback generating the leading edge in these cells.
The recent study by Wei, Cheng, and collaborators (Wei et al., 2008
) provides evidence that spatially and temporally restricted flickers of calcium signaling are enriched near the leading edge of motile fibroblasts. The authors find that a stretch-activated calcium channel, TRPM7, is required for the flickers and that the flickers do not occur if factors needed for traction force are disrupted. Thus, calcium flickers may provide a mechanism for transmitting information about membrane tension created by actin-generated protrusion at the edge of the cell back to regulate upstream and downstream signaling components. These flickers present an excellent candidate for the calcium signaling proposed to act in the PI3K feedback system. However, Wei et al. (2008)
hypothesize that the flickers have an additional role. They find that the flickers correlate with cellular turning behavior and may play a causal role in steering cell migration. The authors find that cell turning and chemotaxis are impaired when TRPM7 levels are reduced using RNA interference. They also find that the difference in the integrated intensity of calcium flickers on one side of the leading edge versus the other correlates strongly with the cells' turning behavior.
Evidence has been building over several years that the leading edge is not a single uniform compartment but rather a heterogeneous region within which localized signaling and cytoskeletal remodeling events can continually redirect the motion of a cell. Local bursts of calcium signaling now join bursts of PIP3 production (Arrieumerlou and Meyer, 2005
) and bursts of Rac activity (Gardiner et al., 2002
) as key local events at the leading edge. An appealing model is that cells count these spatially-restricted, short-lived events downstream of receptor activation and bias their migration toward the source of the chemoattractant. These local events would interact with self-organizing systems that drive motility through global polarization and local propagating waves of actin nucleation (Weiner et al., 2007
). Cells would then steer by using local receptor signals to increase the efficacy of membrane protrusion and adhesion on the side where chemoattractant concentration is higher.
The combined results from Falke's and Cheng's groups now suggest that calcium flickers are key components of both the local steering and the cell polarity machineries. However, a clear resolution of which factors act upstream and play causal roles in creating changes in the direction of migration, which factors act downstream to transduce these signals into mechanical forces, and whether these activities can be separated at all, remains to be figured out.