In vitro reconstitution of fish melanophore pigment aggregation

Movement and positioning of melanophore pigment organelles depend on microt ubule- and actin-dependent motors, but the regulation of these forces are p oorly understood. Here, we describe a cell free and fixed time motility ass ay for the study of the regulation of microtubule-dependent pigment organel le positioning in vitro. The assay involves introduction of microtubule-ast ers assembled in clam oocyte lysates into lysates prepared from Fundulus he teroclitus melanophores with either aggregated or dispersed pigment. When a sters were introduced in lysates prepared from melanophores with dispersed pigment, pigment organelles bound astral microtubules and were evenly distr ibuted throughout the aster. In contrast, when asters were introduced into lysates prepared from melanophores with aggregated pigment, pigment organel les accumulated around the centrosome, mimicking a pigment aggregate. The a ddition of anti-dynein intermediate chain antibody (m74-1), previously show n to interfere with binding of dynactin to dynein and thereby causing detac hment of dynein from organelles, blocked the ATP-dependent aggregation of p igment in vitro and induced a depletion of pigment from the centrosomal are a. The results show that dynein is essential for pigment aggregation and in volved in maintenance of evenly dispersed pigment in vitro, analogous to ce llular evidence, and suggest a possible role for dynactin in these processe s as well. Cell Motil. Cytoskeleton 48:1-10, 2001. (C) 2001 Wiley-Liss, Inc .