Reactivated melanophore motility: differential regulation and nucleotide requirements of bidirectional pigment granule transport.

Abstract

Abstract. To study the molecular basis for organized pigment granule transport, procedures were developed to lyse melanophores of Tilapia mossambica under conditions in which pigment granule movements could be reactivated. Gentle lysis of the melanophores resulted in a permeabilized cell model, which, in the absence of exogenous ATP, could undergo multiple rounds of pigment granule aggregation and dispersion when sequentially challenged with epinephrine and cAMP. Both directions of transport required ATP, since aggregation or dispersion in melanophores depleted of nucleotides could be reactivated only upon addition of MgATP or MgATP plus cAMP, respectively. Differences between the nucleotide sensitivities for aggregation and dispersion were demonstrated by observations that aggregation had a lower apparent Km for ATP than did dispersion and could be initiated at a lower ATP concentration. Moreover, aggregation could be initiated by ADP, but only dispersion could be reactivated by the thiophosphate ATP analog, ATPTS. The direction of pigment transport was determined solely by cAMP, since pigment granules undergoing dispersion reaggregated when cAMP was removed, and those undergoing aggregation dispersed when cAMP was added. These results provide evidence that pigment granule motility may be based on two distinct mechanisms that are differentially activated and regulated to produce bidirectional movements.

Regulation of organelle transport in melanophores by calcineurin.

“…The proteolyzed enzyme is in an activated state, has lost its Ca2+/CaM sensitivity, but can be further stimulated by Ca 2+ via the intact B subunit (Manalan and Klee, 1983;Tallant et al, 1988;Hubbard and Klee, 1989). Proteolysis of endogenous CN in the lysed melanophores could explain why no Ca 2÷ dependence of aggregation was observed in these or earlier studies (Rozdzial and Haimo, 1986a); proteolyzed CN is active in the absence of Ca z+. Removal of cAMP, required by lysed cells to maintain pigment in the dispersed state (Rozdzial and Haimo, 1986a), would thus result in net dephosphorylation via the activated CN, and hence, pigment aggregation.…”

“…Proteolysis of endogenous CN in the lysed melanophores could explain why no Ca 2÷ dependence of aggregation was observed in these or earlier studies (Rozdzial and Haimo, 1986a); proteolyzed CN is active in the absence of Ca z+. Removal of cAMP, required by lysed cells to maintain pigment in the dispersed state (Rozdzial and Haimo, 1986a), would thus result in net dephosphorylation via the activated CN, and hence, pigment aggregation. Samples of scales incubated exactly as they would be before lysis and then used for immunoblotting show that some CN is proteolyzed to a 40-kD fragment in the intact melanophores (data not shown).…”

“…Lysed melanophores normally respond to cAMP and ATP by dispersing pigment throughout the cell (Rozdzial and Haimo, 1986a). Dispersion can be assayed visually and allows a rapid determination of cell lysis.…”

“…tion of pigment granule dispersion was induced by placing or agitating the lysed scale pieces in lysis buffer containing 0.5 mM ATP and 1 mM cAMP. Since cAMP will only induce dispersion in lysed melanophores (Rozdzial and Haimo, 1986a), this experimental design includes a simple, rapid vistud assay (pigment dispersion) to ensure that cells were indeed lysed before further experimental manipulations were undertaken. Reactivation of pigment granule aggregation was carried out by placing scale pieces in lysis buffer containing 0.5 mM ATE Gentle vs. Extensive Lysis.…”

Regulated bidirectional motility of melanophore pigment granules along microtubules in vitro

“…As with most cell types, the microtubules are oriented with their minus ends associated with a perinuclear microtubule-organizing center and their plus ends extending out to the cell periphery (7). Melanophores regulate the direction of pigment transport by modulating the intracellular second messenger, cAMP (8,9). Upon stimulation of the cells with the appropriate hormonal stimulus, adenylate cyclase activity is up-regulated, thereby increasing cytosolic cAMP levels.…”