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Long-range cooperative binding of kinesin to a microtubule in the presence of ATP.

Muto E, Sakai H, Kaseda K - J. Cell Biol. (2005)

Bottom Line: Relative to the stationary WT/E236A kinesin on a MT, wild-type kinesin bound preferentially in close proximity, but was biased to the plus-end direction.These results suggest that kinesin binding and ATP hydrolysis may cause a long-range state transition in the MT, increasing its affinity for kinesin toward its plus end.Thus, our study highlights the active involvement of MTs in kinesin motility.

View Article: PubMed Central - PubMed

Affiliation: Form and Function Group, PRESTO, JST, Mino, Osaka 562-0035, Japan. emuto@brain.riken.go.jp

ABSTRACT
Interaction of kinesin-coated latex beads with a single microtubule (MT) was directly observed by fluorescence microscopy. In the presence of ATP, binding of a kinesin bead to the MT facilitated the subsequent binding of other kinesin beads to an adjacent region on the MT that extended for micrometers in length. This cooperative binding was not observed in the presence of ADP or 5'-adenylylimidodiphosphate (AMP-PNP), where binding along the MT was random. Cooperative binding also was induced by an engineered, heterodimeric kinesin, WT/E236A, that could hydrolyze ATP, yet remained fixed on the MT in the presence of ATP. Relative to the stationary WT/E236A kinesin on a MT, wild-type kinesin bound preferentially in close proximity, but was biased to the plus-end direction. These results suggest that kinesin binding and ATP hydrolysis may cause a long-range state transition in the MT, increasing its affinity for kinesin toward its plus end. Thus, our study highlights the active involvement of MTs in kinesin motility.

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Directionality of lone binding. (A) Schematic illustration of an representative sequence of binding events. Arrowhead indicates the position of the WT/E236A-bead. New bindings are colored red and turn yellow in subsequent images. Total binding counted in Fig. 3 B may include those that are genuinely induced by the WT/E236A-bead and those that are facilitated by the presence of other test beads. Even if the cooperative binding induced genuinely by the WT/E236A-bead was symmetric, the movements of these bound beads toward the MT plus end might lead to the asymmetric distribution of the subsequent binding. (B) Distribution of the lone bindings summed for 10 MTs with a bound WT/E236A-bead (total 320 bindings). (C) Distribution of the lone bindings summed for 10 MTs with a bound E236A/E236A-bead (total 414 bindings). In both B and C, bin width is 1 μm. With a similar observation time (∼190 min), more lone bindings were counted for MTs with a bound E236A/E236A-bead as compared with the MTs with a bound WT/E236A-bead. This result is not surprising given that less frequent binding means a larger fraction of lone bindings in the total binding count.
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fig4: Directionality of lone binding. (A) Schematic illustration of an representative sequence of binding events. Arrowhead indicates the position of the WT/E236A-bead. New bindings are colored red and turn yellow in subsequent images. Total binding counted in Fig. 3 B may include those that are genuinely induced by the WT/E236A-bead and those that are facilitated by the presence of other test beads. Even if the cooperative binding induced genuinely by the WT/E236A-bead was symmetric, the movements of these bound beads toward the MT plus end might lead to the asymmetric distribution of the subsequent binding. (B) Distribution of the lone bindings summed for 10 MTs with a bound WT/E236A-bead (total 320 bindings). (C) Distribution of the lone bindings summed for 10 MTs with a bound E236A/E236A-bead (total 414 bindings). In both B and C, bin width is 1 μm. With a similar observation time (∼190 min), more lone bindings were counted for MTs with a bound E236A/E236A-bead as compared with the MTs with a bound WT/E236A-bead. This result is not surprising given that less frequent binding means a larger fraction of lone bindings in the total binding count.

Mentions: Now to evaluate the directionality of cooperative binding, based on the same binding data, only those new bindings occurring in the absence of any other beads moving on the MT were counted. Although the experimental conditions were optimized here to increase the chance of lone bindings occurring, the value for total bindings shown in Fig. 3 B may still include those bindings that are facilitated by the presence of other test beads on the MT (Fig. 4 A). The distribution of the lone bindings revealed that for MTs with a bound WT/E236A-bead, the binding was significantly higher in the plus-end than the minus-end direction (205 plus-end events and 115 minus-end events, P < 0.01; chi-square test; Fig. 4 B). In contrast, for MTs with a bound E236A/E236A-bead, distribution of lone bindings was not significantly biased (203 plus-end and 211 minus-end events, P = 0.694; chi-square test; Fig. 4 C). We conclude that cooperative binding induced genuinely by the WT/E236A-bead was biased toward the plus-end direction.


Long-range cooperative binding of kinesin to a microtubule in the presence of ATP.

Muto E, Sakai H, Kaseda K - J. Cell Biol. (2005)

Directionality of lone binding. (A) Schematic illustration of an representative sequence of binding events. Arrowhead indicates the position of the WT/E236A-bead. New bindings are colored red and turn yellow in subsequent images. Total binding counted in Fig. 3 B may include those that are genuinely induced by the WT/E236A-bead and those that are facilitated by the presence of other test beads. Even if the cooperative binding induced genuinely by the WT/E236A-bead was symmetric, the movements of these bound beads toward the MT plus end might lead to the asymmetric distribution of the subsequent binding. (B) Distribution of the lone bindings summed for 10 MTs with a bound WT/E236A-bead (total 320 bindings). (C) Distribution of the lone bindings summed for 10 MTs with a bound E236A/E236A-bead (total 414 bindings). In both B and C, bin width is 1 μm. With a similar observation time (∼190 min), more lone bindings were counted for MTs with a bound E236A/E236A-bead as compared with the MTs with a bound WT/E236A-bead. This result is not surprising given that less frequent binding means a larger fraction of lone bindings in the total binding count.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171822&req=5

fig4: Directionality of lone binding. (A) Schematic illustration of an representative sequence of binding events. Arrowhead indicates the position of the WT/E236A-bead. New bindings are colored red and turn yellow in subsequent images. Total binding counted in Fig. 3 B may include those that are genuinely induced by the WT/E236A-bead and those that are facilitated by the presence of other test beads. Even if the cooperative binding induced genuinely by the WT/E236A-bead was symmetric, the movements of these bound beads toward the MT plus end might lead to the asymmetric distribution of the subsequent binding. (B) Distribution of the lone bindings summed for 10 MTs with a bound WT/E236A-bead (total 320 bindings). (C) Distribution of the lone bindings summed for 10 MTs with a bound E236A/E236A-bead (total 414 bindings). In both B and C, bin width is 1 μm. With a similar observation time (∼190 min), more lone bindings were counted for MTs with a bound E236A/E236A-bead as compared with the MTs with a bound WT/E236A-bead. This result is not surprising given that less frequent binding means a larger fraction of lone bindings in the total binding count.
Mentions: Now to evaluate the directionality of cooperative binding, based on the same binding data, only those new bindings occurring in the absence of any other beads moving on the MT were counted. Although the experimental conditions were optimized here to increase the chance of lone bindings occurring, the value for total bindings shown in Fig. 3 B may still include those bindings that are facilitated by the presence of other test beads on the MT (Fig. 4 A). The distribution of the lone bindings revealed that for MTs with a bound WT/E236A-bead, the binding was significantly higher in the plus-end than the minus-end direction (205 plus-end events and 115 minus-end events, P < 0.01; chi-square test; Fig. 4 B). In contrast, for MTs with a bound E236A/E236A-bead, distribution of lone bindings was not significantly biased (203 plus-end and 211 minus-end events, P = 0.694; chi-square test; Fig. 4 C). We conclude that cooperative binding induced genuinely by the WT/E236A-bead was biased toward the plus-end direction.

Bottom Line: Relative to the stationary WT/E236A kinesin on a MT, wild-type kinesin bound preferentially in close proximity, but was biased to the plus-end direction.These results suggest that kinesin binding and ATP hydrolysis may cause a long-range state transition in the MT, increasing its affinity for kinesin toward its plus end.Thus, our study highlights the active involvement of MTs in kinesin motility.

View Article: PubMed Central - PubMed

Affiliation: Form and Function Group, PRESTO, JST, Mino, Osaka 562-0035, Japan. emuto@brain.riken.go.jp

ABSTRACT
Interaction of kinesin-coated latex beads with a single microtubule (MT) was directly observed by fluorescence microscopy. In the presence of ATP, binding of a kinesin bead to the MT facilitated the subsequent binding of other kinesin beads to an adjacent region on the MT that extended for micrometers in length. This cooperative binding was not observed in the presence of ADP or 5'-adenylylimidodiphosphate (AMP-PNP), where binding along the MT was random. Cooperative binding also was induced by an engineered, heterodimeric kinesin, WT/E236A, that could hydrolyze ATP, yet remained fixed on the MT in the presence of ATP. Relative to the stationary WT/E236A kinesin on a MT, wild-type kinesin bound preferentially in close proximity, but was biased to the plus-end direction. These results suggest that kinesin binding and ATP hydrolysis may cause a long-range state transition in the MT, increasing its affinity for kinesin toward its plus end. Thus, our study highlights the active involvement of MTs in kinesin motility.

Show MeSH