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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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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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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.

Mentions: We believe that the change in motor/dynactin–microtubule binding behavior that we observe upon treatment of these molecules with specific kinases or phosphatases reveals the underlying mechanism controlling direction of transport in vivo for the following reasons. (a) The in vitro activities of dynein, dynactin, and kinesin II were each altered by a specific kinase and phosphatase, and not by the other kinase and phosphatases examined, suggesting that specific phosphorylation alters the microtubule binding properties of each protein. (b) The kinases and phosphatases that do alter motor/dynactin behavior do so in a way that correlates with the expected in vivo activity of these proteins. For example, dynein drives aggregation and PP2A is required to induce aggregation; we find that dynein is active when isolated from aggregated cells and that PP2A converts inactive dynein from dispersed cells to active dynein. (c) PKA, PKC, and PP2A have been demonstrated to regulate pigment granule transport, and PP1 has been implicated in other microtubule-dependent movements, suggesting that the alterations in behavior conferred on the motors/dynactin by these enzymes are not an in vitro artifact. (d) These enzymes only alter the behavior of the motors or dynactin from melanophores that have transported pigment in one direction and not the other. For example, PKA inhibits the microtubule binding activity of dynein that has been isolated from aggregated cells. Dynein that is isolated from dispersed cells is already in its inactive state and is not further inhibited by PKA. (e) Finally, when motors are isolated from cells with aggregated or dispersed pigment, one motor is active, whereas the other is not. PKA and PP2A reciprocally reverse the behavior of the motors in vitro and retain this feature. One motor is active, whereas the other is inactive, after treatment with either of these enzymes. These findings suggest that cyclic phosphorylation and dephosphorylation induce dispersion and aggregation in melanophores (Rozdzial and Haimo 1986) by cyclically activating and inhibiting motor/dynactin–microtubule interactions, thereby controlling the direction of transport; this system is modeled in Fig. 7.


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

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

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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Related In: Results  -  Collection

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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.
Mentions: We believe that the change in motor/dynactin–microtubule binding behavior that we observe upon treatment of these molecules with specific kinases or phosphatases reveals the underlying mechanism controlling direction of transport in vivo for the following reasons. (a) The in vitro activities of dynein, dynactin, and kinesin II were each altered by a specific kinase and phosphatase, and not by the other kinase and phosphatases examined, suggesting that specific phosphorylation alters the microtubule binding properties of each protein. (b) The kinases and phosphatases that do alter motor/dynactin behavior do so in a way that correlates with the expected in vivo activity of these proteins. For example, dynein drives aggregation and PP2A is required to induce aggregation; we find that dynein is active when isolated from aggregated cells and that PP2A converts inactive dynein from dispersed cells to active dynein. (c) PKA, PKC, and PP2A have been demonstrated to regulate pigment granule transport, and PP1 has been implicated in other microtubule-dependent movements, suggesting that the alterations in behavior conferred on the motors/dynactin by these enzymes are not an in vitro artifact. (d) These enzymes only alter the behavior of the motors or dynactin from melanophores that have transported pigment in one direction and not the other. For example, PKA inhibits the microtubule binding activity of dynein that has been isolated from aggregated cells. Dynein that is isolated from dispersed cells is already in its inactive state and is not further inhibited by PKA. (e) Finally, when motors are isolated from cells with aggregated or dispersed pigment, one motor is active, whereas the other is not. PKA and PP2A reciprocally reverse the behavior of the motors in vitro and retain this feature. One motor is active, whereas the other is inactive, after treatment with either of these enzymes. These findings suggest that cyclic phosphorylation and dephosphorylation induce dispersion and aggregation in melanophores (Rozdzial and Haimo 1986) by cyclically activating and inhibiting motor/dynactin–microtubule interactions, thereby controlling the direction of transport; this system is modeled in Fig. 7.

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
Related in: MedlinePlus