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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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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 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.

Mentions: PKA induces complete dispersion in Xenopus melanophores (Reilein et al. 1998). To determine if this kinase can alter motor/dynactin–microtubule interactions, we treated the immunoprecipitated motors or dynactin with PKA before addition of microtubules. PKA-treated dynein from aggregated cells exhibits a significantly reduced ability to capture microtubules, compared with untreated dynein from aggregated cells (Fig. 5 a, compare tubulin in lane 3 with lane 1). In contrast, PKA-treated kinesin II from aggregated cells exhibits significantly enhanced ability to capture microtubules compared with untreated kinesin II from aggregated cells (Fig. 5 c, compare tubulin in lane 3 with lane 1). In both cases, the amount of microtubules captured by the PKA-treated motors from aggregated cells is equivalent to that captured by untreated motors from dispersed cells (Fig. 5, a and c, compare tubulin in lane 3 with lane 2). The behavior of dynein and kinesin II from dispersed cells is unaltered by PKA treatment (Fig. 5, a and c, compare tubulin in lane 4 with lane 2), as expected if these motors have already been affected by PKA during dispersion in vivo. Unlike dynein and kinesin II, dynactin's ability to capture microtubules is unchanged by treatment with PKA (Fig. 5 b, compare tubulin in lanes 3 and 4 to lane 1 with lane 2). These findings suggest that phosphorylation mediated by PKA induces dispersion in vivo by inactivating dynein– and activating kinesin II–microtubule interactions so that plus end–directed microtubule transport is favored.


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

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

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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Related In: Results  -  Collection

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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.
Mentions: PKA induces complete dispersion in Xenopus melanophores (Reilein et al. 1998). To determine if this kinase can alter motor/dynactin–microtubule interactions, we treated the immunoprecipitated motors or dynactin with PKA before addition of microtubules. PKA-treated dynein from aggregated cells exhibits a significantly reduced ability to capture microtubules, compared with untreated dynein from aggregated cells (Fig. 5 a, compare tubulin in lane 3 with lane 1). In contrast, PKA-treated kinesin II from aggregated cells exhibits significantly enhanced ability to capture microtubules compared with untreated kinesin II from aggregated cells (Fig. 5 c, compare tubulin in lane 3 with lane 1). In both cases, the amount of microtubules captured by the PKA-treated motors from aggregated cells is equivalent to that captured by untreated motors from dispersed cells (Fig. 5, a and c, compare tubulin in lane 3 with lane 2). The behavior of dynein and kinesin II from dispersed cells is unaltered by PKA treatment (Fig. 5, a and c, compare tubulin in lane 4 with lane 2), as expected if these motors have already been affected by PKA during dispersion in vivo. Unlike dynein and kinesin II, dynactin's ability to capture microtubules is unchanged by treatment with PKA (Fig. 5 b, compare tubulin in lanes 3 and 4 to lane 1 with lane 2). These findings suggest that phosphorylation mediated by PKA induces dispersion in vivo by inactivating dynein– and activating kinesin II–microtubule interactions so that plus end–directed microtubule transport is favored.

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