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Dynein, dynactin, and kinesin II's interaction with microtubules is regulated during bidirectional organelle transport.

Reese EL, Haimo LT - J. Cell Biol. (2000)

Bottom Line: Dynein and dynactin bind to microtubules when obtained from cells with aggregated pigment, whereas kinesin II binds to microtubules when obtained from cells with dispersed pigment.Moreover, the microtubule binding activity of these motors/dynactin can be reversed in vitro by the kinases and phosphatase that regulate the direction of pigment granule transport in vivo.These findings suggest that phosphorylation controls the direction of pigment granule transport by altering the ability of dynein, dynactin, and kinesin II to interact with microtubules.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of California at Riverside, Riverside, California 92521, USA.

ABSTRACT
The microtubule motors, cytoplasmic dynein and kinesin II, drive pigmented organelles in opposite directions in Xenopus melanophores, but the mechanism by which these or other motors are regulated to control the direction of organelle transport has not been previously elucidated. We find that cytoplasmic dynein, dynactin, and kinesin II remain on pigment granules during aggregation and dispersion in melanophores, indicating that control of direction is not mediated by a cyclic association of motors with these organelles. However, the ability of dynein, dynactin, and kinesin II to bind to microtubules varies as a function of the state of aggregation or dispersion of the pigment in the cells from which these molecules are isolated. Dynein and dynactin bind to microtubules when obtained from cells with aggregated pigment, whereas kinesin II binds to microtubules when obtained from cells with dispersed pigment. Moreover, the microtubule binding activity of these motors/dynactin can be reversed in vitro by the kinases and phosphatase that regulate the direction of pigment granule transport in vivo. These findings suggest that phosphorylation controls the direction of pigment granule transport by altering the ability of dynein, dynactin, and kinesin II to interact with microtubules.

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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 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.

Mentions: Pigment granules saltate during both directions of transport in Xenopus melanophores, and isolated pigment granules show biased rather than unidirectional movements on microtubules (Rogers et al. 1997). These observations suggest that dynein and kinesin II might be active during both directions of movement. To determine if there is opposing motor activity during aggregation or dispersion, we examined the microtubule binding behavior of dynein, dynactin, and kinesin II obtained from melanophores in the process of aggregating and dispersing pigment. Cells with dispersed pigment were induced to undergo pigment aggregation and redispersion (Fig. 3). At various times during transport, cells were lysed, and the motors/dynactin were examined for their microtubule binding activity. During aggregation, the pool of active dynein and dynactin increases, whereas that of kinesin II decreases (Fig. 3, compare lane 2 with lane 1). In cells with fully aggregated pigment, the size of the active pool of dynein and dynactin is at a maximum whereas no active kinesin II is detected (Fig. 3, lane 3). During redispersion of pigment, the pool of active dynein and dynactin falls as that of kinesin II rises (Fig. 3, lane 4) until, in fully redispersed cells, the size of the active pool of kinesin II is at a maximum, whereas no active dynein or dynactin is detected (Fig. 3, lane 5). These observations suggest that some opposing motor is active during each direction of transport, a finding that may explain why pigment granules saltate during aggregation and dispersion. Therefore, change in the net direction of pigment granule transport likely occurs when the ratio of active dynein to active kinesin II reaches some critical threshold.


Dynein, dynactin, and kinesin II's interaction with microtubules is regulated during bidirectional organelle transport.

Reese EL, Haimo LT - J. Cell Biol. (2000)

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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Related In: Results  -  Collection

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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.
Mentions: Pigment granules saltate during both directions of transport in Xenopus melanophores, and isolated pigment granules show biased rather than unidirectional movements on microtubules (Rogers et al. 1997). These observations suggest that dynein and kinesin II might be active during both directions of movement. To determine if there is opposing motor activity during aggregation or dispersion, we examined the microtubule binding behavior of dynein, dynactin, and kinesin II obtained from melanophores in the process of aggregating and dispersing pigment. Cells with dispersed pigment were induced to undergo pigment aggregation and redispersion (Fig. 3). At various times during transport, cells were lysed, and the motors/dynactin were examined for their microtubule binding activity. During aggregation, the pool of active dynein and dynactin increases, whereas that of kinesin II decreases (Fig. 3, compare lane 2 with lane 1). In cells with fully aggregated pigment, the size of the active pool of dynein and dynactin is at a maximum whereas no active kinesin II is detected (Fig. 3, lane 3). During redispersion of pigment, the pool of active dynein and dynactin falls as that of kinesin II rises (Fig. 3, lane 4) until, in fully redispersed cells, the size of the active pool of kinesin II is at a maximum, whereas no active dynein or dynactin is detected (Fig. 3, lane 5). These observations suggest that some opposing motor is active during each direction of transport, a finding that may explain why pigment granules saltate during aggregation and dispersion. Therefore, change in the net direction of pigment granule transport likely occurs when the ratio of active dynein to active kinesin II reaches some critical threshold.

Bottom Line: Dynein and dynactin bind to microtubules when obtained from cells with aggregated pigment, whereas kinesin II binds to microtubules when obtained from cells with dispersed pigment.Moreover, the microtubule binding activity of these motors/dynactin can be reversed in vitro by the kinases and phosphatase that regulate the direction of pigment granule transport in vivo.These findings suggest that phosphorylation controls the direction of pigment granule transport by altering the ability of dynein, dynactin, and kinesin II to interact with microtubules.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of California at Riverside, Riverside, California 92521, USA.

ABSTRACT
The microtubule motors, cytoplasmic dynein and kinesin II, drive pigmented organelles in opposite directions in Xenopus melanophores, but the mechanism by which these or other motors are regulated to control the direction of organelle transport has not been previously elucidated. We find that cytoplasmic dynein, dynactin, and kinesin II remain on pigment granules during aggregation and dispersion in melanophores, indicating that control of direction is not mediated by a cyclic association of motors with these organelles. However, the ability of dynein, dynactin, and kinesin II to bind to microtubules varies as a function of the state of aggregation or dispersion of the pigment in the cells from which these molecules are isolated. Dynein and dynactin bind to microtubules when obtained from cells with aggregated pigment, whereas kinesin II binds to microtubules when obtained from cells with dispersed pigment. Moreover, the microtubule binding activity of these motors/dynactin can be reversed in vitro by the kinases and phosphatase that regulate the direction of pigment granule transport in vivo. These findings suggest that phosphorylation controls the direction of pigment granule transport by altering the ability of dynein, dynactin, and kinesin II to interact with microtubules.

Show MeSH