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Figure 1. Dynein, dynactin, and kinesin II remain on pigment granules during bidirectional transport. Immunoblot analysis of purified pigment granules isolated from equal numbers of aggregated (A) or dispersed (D) melanophores and probed with antibodies to dynein (74.1 antibody against dynein intermediate chain), dynactin (p150 antibody against the p150glued subunit), and kinesin II (K2.4 antibody against the 85-kD subunit).
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Figure 2. Dynein, dynactin and kinesin II display differential microtubule cosedimentation behavior that varies with the direction of pigment transport. (A) Pigment granuleâassociated motors/dynactin differentially bind microtubules. Triton X-100 extracts of pigment granules (pg extract, lanes 1 and 2) isolated from aggregated (A) and dispersed (D) melanophores, and the microtubule pellets (MT pel, lanes 3 and 4) and supernatants (MT sup, lanes 5 and 6) obtained after cosedimentation of microtubules with the extracts were immunoblotted for (a) dynein, (b) dynactin, and (c) kinesin II. Microtubule pellets were simultaneously blotted for dynactin and kinesin II (d). (B) The soluble pool of dynein, dynactin, and kinesin II differentially binds microtubules. Clarified lysates (lanes 1 and 2) from aggregated and dispersed melanophores, and the microtubule pellets (MT pel, lanes 3 and 4) and supernatants (MT sup, lanes 5 and 6) obtained after cosedimentation of microtubules with the lysates were probed for dynein (a) or simultaneously for dynactin and kinesin II (b). Microtubule pellets were also probed for conventional kinesin (c). 150, p150glued subunit of dynactin; 85, 85-kD subunit of kinesin II. Dynein, dynactin, and kinesin II exhibit differential microtubule binding as a function of the direction of pigment granule transport whereas conventional kinesin does not.
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Figure 3. Dynein, dynactin, and kinesin II's microtubule binding activity rises and falls during aggregation and dispersion. Light micrographs of dispersed (disp) melanophores undergoing aggregation (aggr) and then redispersion (redisp) of pigment for the times indicated. The corresponding immunoblots, resulting from cosedimentation of microtubules with soluble motors/dynactin isolated from melanophores at the time points indicated, have been probed for dynein, dynactin, and kinesin II.The pool of active dynein and dynactin increases during aggregation and decreases during dispersion, whereas kinesin II displays the opposite behavior. Bar, 50 μm.
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Figure 4. Microtubule capture by immobilized dynein, dynactin, and kinesin II varies as a function of the direction of pigment transport. (A) Model for microtubule capture assay. Protein A beads with bound antibodies (primary antibodies [1° ab] 74.1, p150, or K2.4) are incubated with aggregated or dispersed Xenopus melanophore lysates to immunoprecipitate the appropriate motor/dynactin. Motor/dynactin antibodyâbead complexes (left) are then challenged to bind microtubules (MTs). Microtubules are captured by active dynein, dynactin, or kinesin II immobilized on the beads (right). (B) Dynein (a), dynactin (b), and kinesin II (c) from aggregated (A) and dispersed (D) melanophore lysates were immunoprecipitated, incubated with taxol-stabilized microtubules, and probed either for motors/dynactin (top) or for tubulin (bottom). Heavy and light chains of the immunoprecipitating antibodies are also detected in the top panels. DIC, intermediate chain of cytoplasmic dynein; 150, p150glued subunit of dynactin; 85, 85-kD motor subunit of kinesin II; tub, tubulin. Dynein and dynactin from aggregated cells and kinesin II from dispersed cells capture microtubules. (C) Microtubule capture by motors is ATP sensitive. Dynein and dynactin immunoprecipitated from aggregated melanophore extracts and kinesin II immunoprecipitated from dispersed extracts were incubated with taxol-stabilized microtubules, washed, and incubated in the presence (+) or absence (â) of 1 mM ATP. Immunoblotting for tubulin reveals that microtubules dissociate from dynein and kinesin II, but not from dynactin, in the presence of ATP.
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Figure 5. Microtubule capture behavior by dynein and kinesin II is reversed by PKA and PP2A. Dynein (a), dynactin (b), and kinesin II (c) were immunoprecipitated from aggregated (A) and dispersed (D) melanophore lysates and then incubated with either ATP alone (control, lanes 1 and 2), with ATP and the catalytic subunit of PKA (lanes 3 and 4), or with PP2A (lanes 5 and 6). Immunoprecipitants were subsequently washed free of ATP and kinase or phosphatase, incubated with taxol-stabilized microtubules, and then assessed for the presence of bound microtubules by immunoblotting for tubulin. The preparations were also blotted for dynein, dynactin, or kinesin II. DIC, dynein intermediate chain; 150, p150glued subunit of dynactin; 85, 85-kD subunit of kinesin II; tub, tubulin. PKA inhibits microtubule (MT) capture by dynein and enhances microtubule capture by kinesin II from aggregated cells, whereas PP2A enhances microtubule capture by dynein and inhibits microtubule capture by kinesin II from dispersed cells. The ability of dynactin to capture microtubules is unaffected by PKA or PP2A treatment. Note that tubulin captured by kinesin II appears as a doublet (c; and see also in Fig. 4 and Fig. 6), suggesting that tubulin may be modified by the kinesin II immunoprecipitate.
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Figure 6. Microtubule capture by dynactin is inhibited by PKC and enhanced by PP1. Dynein (a), dynactin (b), and kinesin II (c) were immunoprecipitated from aggregated (A) and dispersed (D) melanophore lysates and then incubated with either ATP alone (control, lanes 1 and 2) or with ATP and the catalytic subunit of PKC (lanes 3 and 4). Other immunoprecipitants were either incubated in Ca2+/calmodulin (control, lanes 5 and 6) or incubated with Ca2+/calmodulin and PP2B (PP2B, lanes 7 and 8) or were untreated (control, lanes 9 and 10) or incubated with the catalytic subunit of PP1 (lanes 11 and 12). Immunoprecipitants were subsequently washed, incubated with taxol-stabilized microtubules, and assessed for the presence of bound microtubules by immunoblotting and probing for tubulin. PKC inhibits microtubule (MT) capture by dynactin immunoprecipitated from melanophores with aggregated pigment, but has marginal effect on the ability of dynein or kinesin II to capture microtubules. The ability of dynein, dynactin, and kinesin II to capture microtubules is unaffected by PP2B treatment. PP1 treatment enhances microtubule capture by dynactin immunoprecipitated from dispersed melanophore lysates, whereas dynein and kinesin II are unaffected.
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Figure 7. Model for the bidirectional transport of pigment granules along microtubules. Protein phosphorylation and dephosphorylation control the direction of transport in melanophores. A pigment granule at the minus end of a microtubule (left) is bound to the microtubule by active dynein and dynactin (filled heads), whereas inactive kinesin II (clear heads) is unable to bind to the microtubule. PKA and PKC, activated upon stimulation of melanophores with MSH, convert dynein and dynactin to their inactive forms (clear heads), whereas PKA converts kinesin II to its active form (filled heads), allowing it to bind to microtubules. Active kinesin II transports the pigment granule towards the plus end of the microtubule, the direction of pigment granule transport corresponding to dispersion in vivo. Active kinesin II (filled heads) on a pigment granule at the plus end of the microtubule (right) is converted to its inactive form (clear heads) by PP2A when PKA activity is depressed upon stimulation of melanophores with melatonin. Simultaneous modification by PP2A of dynein activates it (filled heads) to its microtubule binding form. Active dynein transports the pigment granule towards the minus end of the microtubule. Activation of dynactin to its microtubule binding form (filled heads) by PP1 may enhance dynein-mediated transport or may anchor pigment granules at the minus ends of microtubules after their transport.
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