Patterns of elevated free calcium and calmodulin activation in living cells

Abstract

The temporal and spatial dynamics of intracellular signals and protein effectors are being defined as a result of imaging using fluorescent reagents within living cells. We have described a new class of fluorescent analogues termed optical biosensors, which sense chemical or molecular events through their effects on protein transducers. One example of this new class of indicators is MeroCaM, an environmentally sensitive fluorophore which when it is attached to calmodulin reflects the activation of calmodulin by calcium in vitro. We report here that the rise in free calcium and MeroCaM activation occur in the same period during serum stimulation of quiescent fibroblasts. MeroCaM activation also correlates with the spatial pattern of increased free calcium and the contraction of transverse fibres during wound healing. Finally, migrating fibroblasts in the later stages of wound-healing exhibit an increasing gradient of free calcium and MeroCaM activation from the front to the rear.

Migration-associated secretion of melanoma inhibitory activity at the cell rear is supported by KCa3.1 potassium channels

“…In addition to setting the cell volume, intracellular Ca 2+ modulates the turnover of actin filaments; an elevation of Ca 2+ promotes actin depolymerization, whereas a low Ca 2+ concentration favors actin polymerization. The regulation of the cytoskeletal migration machinery is supported by the intracellular gradient of Ca 2+ with the higher concentration at the cell rear [14][15][16]. Another relevant factor for the motility of cells is the Ca 2+ -controlled function of calpains in the release of cell-extracellular matrix contacts [17] at the cell rear.…”

“…It is generally accepted that migrating cells are polarized along their axis of movement and establish an intracellular Ca 2+ gradient with a lower [Ca 2+ ] at the cell npg front [14][15][16]. By mediating salt efflux or influx, followed by osmotic water flow, ion channels and transporters play a pivotal role in regulating cell volume during migration.…”

“…By modulating turnover of actin filaments [14][15][16], activation of calpain [17] and recycling of integrins, Ca 2+ coordinates different components of the cellular migration machinery. Another player linking intracellular Ca 2+ ions and cell migration is the calcium-activated potassium channel KCa3.1.…”

Identification of Channels Promoting Calcium Spikes and Waves in HT1080 Tumor Cells

“…Ca 2ϩ transients have been observed in migrating neurons, neutrophils, fibroblasts, eosinophils, tumor cells, and other cell types (3)(4)(5)(6)(7)(8)(9)(10). These oscillations are thought to participate in coordinating the cyclic temporal features of cell migration, such as pseudopod extension, actin assembly, integrin regulation, the phosphorylation-mediated regulation of focal adhesion formation, and pericellular proteolysis (1)(2)(3)(4)(5)(6)(7)(8)(9)(10). Thus, repetitive Ca 2ϩ signals likely play an important role in the repetitive structural and functional changes required for cell movement.…”

Mediation of chemoattractant-induced changes in [Ca2+]i and cell shape, polarity, and locomotion by InsP3, DAG, and protein kinase C in newt eosinophils.

“…An in vitro contraction model consisting of a gel of myosin II and actin mixed with filamin and gelsolin contracts away from the high concentration end of a [Ca2+]i gradient (Janson et al, 1991). The spatial distribution of the Ca 2+-calmodulin complex, which likely reflects the distribution of its activated target proteins, including those that activate myosin II, is similar to that of [Ca2÷]i in migrating fibroblasts, as demonstrated with a fluorescent analog of calmodulin (Hahn et al, 1992). Ca 2+ also seems to play a role, via integrins, in regulating the attachment of human neutrophils to the substratum (Marks et al, 1991).…”