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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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Related in: MedlinePlus

Interaction of kinesin-coated beads with a MT in various nucleotide conditions. (A) Sequential images of kinesin beads interacting with a MT in the presence of 1 mM ATP, taken at 1-s intervals. Bar, 5 μm. (Video 1). (B) Diagrams illustrating how the windows (represented by rectangles of various colors) were defined for the statistical analysis of the binding frequency. The example is taken from the images shown in A. Starting from the position of the preexisting kinesin beads (yellow dots), a window of 1 μm was shifted along a MT in both plus- and minus-end directions until the entire length of MT was covered. (C) The binding frequency calculated as a function of distance from the preexisting kinesin beads. Binding was measured in the presence of 1 mM ATP (blue dot), AMP-PNP (gray dot), and ADP (green dot) at a kinesin-bead concentration of 90 pM. Total number of binding events, number of MTs, and total observation time were 934 binding events, 5 MTs (length = 12.90–18.55 μm), 61 min for ATP; 734 binding events, 65 MTs (length = 11.94–18.10 μm), 114 min for ADP; and 669 binding events, 72 MTs (length = 12.18–18.87 μm) and 115 min for AMP-PNP, respectively. When the position of the kinesin bead was recorded at a higher temporal resolution (10 frame/s), the binding frequency was not affected, indicating that the temporal resolution of 3 frames/s is adequate.
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fig1: Interaction of kinesin-coated beads with a MT in various nucleotide conditions. (A) Sequential images of kinesin beads interacting with a MT in the presence of 1 mM ATP, taken at 1-s intervals. Bar, 5 μm. (Video 1). (B) Diagrams illustrating how the windows (represented by rectangles of various colors) were defined for the statistical analysis of the binding frequency. The example is taken from the images shown in A. Starting from the position of the preexisting kinesin beads (yellow dots), a window of 1 μm was shifted along a MT in both plus- and minus-end directions until the entire length of MT was covered. (C) The binding frequency calculated as a function of distance from the preexisting kinesin beads. Binding was measured in the presence of 1 mM ATP (blue dot), AMP-PNP (gray dot), and ADP (green dot) at a kinesin-bead concentration of 90 pM. Total number of binding events, number of MTs, and total observation time were 934 binding events, 5 MTs (length = 12.90–18.55 μm), 61 min for ATP; 734 binding events, 65 MTs (length = 11.94–18.10 μm), 114 min for ADP; and 669 binding events, 72 MTs (length = 12.18–18.87 μm) and 115 min for AMP-PNP, respectively. When the position of the kinesin bead was recorded at a higher temporal resolution (10 frame/s), the binding frequency was not affected, indicating that the temporal resolution of 3 frames/s is adequate.

Mentions: Fig. 1 A shows sequential images of kinesin beads moving on a single MT, taken at 1-s intervals. New bindings are represented by red dots with their color turning yellow in subsequent images. Quite unexpectedly, the binding of kinesin beads was not completely random, but occurred preferentially in the vicinity of beads that were already moving along the MT. Consequently, a cluster of kinesin beads was formed over several micrometers of MT filament, and longer observation times revealed new clusters being repeatedly formed (Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200409035/DC1).


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

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

Interaction of kinesin-coated beads with a MT in various nucleotide conditions. (A) Sequential images of kinesin beads interacting with a MT in the presence of 1 mM ATP, taken at 1-s intervals. Bar, 5 μm. (Video 1). (B) Diagrams illustrating how the windows (represented by rectangles of various colors) were defined for the statistical analysis of the binding frequency. The example is taken from the images shown in A. Starting from the position of the preexisting kinesin beads (yellow dots), a window of 1 μm was shifted along a MT in both plus- and minus-end directions until the entire length of MT was covered. (C) The binding frequency calculated as a function of distance from the preexisting kinesin beads. Binding was measured in the presence of 1 mM ATP (blue dot), AMP-PNP (gray dot), and ADP (green dot) at a kinesin-bead concentration of 90 pM. Total number of binding events, number of MTs, and total observation time were 934 binding events, 5 MTs (length = 12.90–18.55 μm), 61 min for ATP; 734 binding events, 65 MTs (length = 11.94–18.10 μm), 114 min for ADP; and 669 binding events, 72 MTs (length = 12.18–18.87 μm) and 115 min for AMP-PNP, respectively. When the position of the kinesin bead was recorded at a higher temporal resolution (10 frame/s), the binding frequency was not affected, indicating that the temporal resolution of 3 frames/s is adequate.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171822&req=5

fig1: Interaction of kinesin-coated beads with a MT in various nucleotide conditions. (A) Sequential images of kinesin beads interacting with a MT in the presence of 1 mM ATP, taken at 1-s intervals. Bar, 5 μm. (Video 1). (B) Diagrams illustrating how the windows (represented by rectangles of various colors) were defined for the statistical analysis of the binding frequency. The example is taken from the images shown in A. Starting from the position of the preexisting kinesin beads (yellow dots), a window of 1 μm was shifted along a MT in both plus- and minus-end directions until the entire length of MT was covered. (C) The binding frequency calculated as a function of distance from the preexisting kinesin beads. Binding was measured in the presence of 1 mM ATP (blue dot), AMP-PNP (gray dot), and ADP (green dot) at a kinesin-bead concentration of 90 pM. Total number of binding events, number of MTs, and total observation time were 934 binding events, 5 MTs (length = 12.90–18.55 μm), 61 min for ATP; 734 binding events, 65 MTs (length = 11.94–18.10 μm), 114 min for ADP; and 669 binding events, 72 MTs (length = 12.18–18.87 μm) and 115 min for AMP-PNP, respectively. When the position of the kinesin bead was recorded at a higher temporal resolution (10 frame/s), the binding frequency was not affected, indicating that the temporal resolution of 3 frames/s is adequate.
Mentions: Fig. 1 A shows sequential images of kinesin beads moving on a single MT, taken at 1-s intervals. New bindings are represented by red dots with their color turning yellow in subsequent images. Quite unexpectedly, the binding of kinesin beads was not completely random, but occurred preferentially in the vicinity of beads that were already moving along the MT. Consequently, a cluster of kinesin beads was formed over several micrometers of MT filament, and longer observation times revealed new clusters being repeatedly formed (Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200409035/DC1).

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
Related in: MedlinePlus