INTRACELLULAR CYCLIC-AMP, NOT CALCIUM, DETERMINES THE DIRECTION OF VESICLE MOVEMENT IN MELANOPHORES - DIRECT MEASUREMENT BY FLUORESCENCE RATIO IMAGING

Intracellular movement of vesiculated pigment granules in angelfish me lanophores is regulated by a signalling pathway that triggers kinesin and dynein-like microtubule motor proteins. We have tested the relativ e importance of intracellular Ca2+ ([Ca2+]i) vs cAMP ([cAMP]i) in the control of such motility by adrenergic agonists, using fluorescence ra tio imaging and many ways to artificially stimulate or suppress signal s in these pathways. Fura-2 imaging reported a [Ca2+]i elevation accom panying pigment aggregation, but this increase was not essential since movement was not induced with the calcium ionophore, ionomycin, nor w as movement blocked when the increases were suppressed by withdrawal o f extracellular Ca2+ or loading of intracellular BAPTA. The phosphatas e inhibitor, okadaic acid, blocked aggregation and induced dispersion at concentrations that suggested that the protein phosphatase PP-1 or PP-2A was continuously turning phosphate over during intracellular mot ility. cAMP was monitored dynamically in single living cells by microi njecting cAMP-dependent kinase in which the catalytic and regulatory s ubunits were labeled with fluorescein and rhodamine respectively (Adam s et al., 1991. Nature (Lond.). 349: 694-697). Ratio imaging of FlCRhR showed that the alpha-2-adrenergic receptor-mediated aggregation was accompanied by a dose-dependent decrease in [cAMP]i. The decrease in [ cAMP]i was both necessary and sufficient for aggregation, since cAMP a nalogs or microinjected free catalytic subunit of A kinase-blocked agg regation or caused dispersal, whereas the cAMP antagonist R(p)cAMPS or the microinjection of the specific kinase inhibitor PKI5-24 amide ind uced aggregation. Our conclusion that cAMP, not calcium, controls bidi rectional microtubule dependent motility in melanophores might be rele vant to other instances of nonmuscle cell motility.