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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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Directional preference of cooperative binding. (A) Schematic representation of the analysis of lone binding. (B) Distribution of the lone binding summed for 20 MTs. In both A and B, an arrowhead indicates the position of the preexisting, moving kinesin bead. As multiple kinesin beads were simultaneously moving along the MT most of the time, only 2% of the total binding events was counted for this lone-binding analysis.
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fig2: Directional preference of cooperative binding. (A) Schematic representation of the analysis of lone binding. (B) Distribution of the lone binding summed for 20 MTs. In both A and B, an arrowhead indicates the position of the preexisting, moving kinesin bead. As multiple kinesin beads were simultaneously moving along the MT most of the time, only 2% of the total binding events was counted for this lone-binding analysis.

Mentions: We next wished to determine if there is any bias in the direction of cooperative binding. In the analysis of binding frequency described above, directionality was not explicitly considered because in situations where multiple beads were simultaneously moving along a MT, it was difficult to determine whether it was the forward or the rear bead that facilitated the new binding. To simplify the analysis, directionality was examined only in cases where a single kinesin bead was moving on the MT before the analysis (Fig. 2 A). The distribution of subsequent new binding events relative to the position of the moving beads was then examined. To guarantee equal opportunity for new binding in either direction, new bindings were included in the statistics only when the available area of MT exceeded 6 μm in length on both sides of the moving kinesin beads. Although such a “lone” situation was relatively rare, records obtained over a total observation period of ∼4 h (corresponding to 20 MTs) were scrutinized and 76 new bindings were counted (Fig. 2 B). The result revealed a biased distribution in the plus-end direction; within a 6-μm distance, binding in the plus-end direction was 2.2 times higher than the binding in the minus-end direction (52 plus-end events vs. 24 minus-end events). Assuming no particular preference in the direction of cooperative binding, the probability of such an uneven distribution within statistical fluctuation is <0.2% (chi-square test).


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

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

Directional preference of cooperative binding. (A) Schematic representation of the analysis of lone binding. (B) Distribution of the lone binding summed for 20 MTs. In both A and B, an arrowhead indicates the position of the preexisting, moving kinesin bead. As multiple kinesin beads were simultaneously moving along the MT most of the time, only 2% of the total binding events was counted for this lone-binding analysis.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2171822&req=5

fig2: Directional preference of cooperative binding. (A) Schematic representation of the analysis of lone binding. (B) Distribution of the lone binding summed for 20 MTs. In both A and B, an arrowhead indicates the position of the preexisting, moving kinesin bead. As multiple kinesin beads were simultaneously moving along the MT most of the time, only 2% of the total binding events was counted for this lone-binding analysis.
Mentions: We next wished to determine if there is any bias in the direction of cooperative binding. In the analysis of binding frequency described above, directionality was not explicitly considered because in situations where multiple beads were simultaneously moving along a MT, it was difficult to determine whether it was the forward or the rear bead that facilitated the new binding. To simplify the analysis, directionality was examined only in cases where a single kinesin bead was moving on the MT before the analysis (Fig. 2 A). The distribution of subsequent new binding events relative to the position of the moving beads was then examined. To guarantee equal opportunity for new binding in either direction, new bindings were included in the statistics only when the available area of MT exceeded 6 μm in length on both sides of the moving kinesin beads. Although such a “lone” situation was relatively rare, records obtained over a total observation period of ∼4 h (corresponding to 20 MTs) were scrutinized and 76 new bindings were counted (Fig. 2 B). The result revealed a biased distribution in the plus-end direction; within a 6-μm distance, binding in the plus-end direction was 2.2 times higher than the binding in the minus-end direction (52 plus-end events vs. 24 minus-end events). Assuming no particular preference in the direction of cooperative binding, the probability of such an uneven distribution within statistical fluctuation is <0.2% (chi-square test).

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