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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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Cooperative binding induced by heterodimeric kinesin, WT/E236A. (A) Schematic representation of binding assay using heterodimeric kinesin. (B) Three representative example distributions of binding frequency for a MT with a bound WT/E236A-bead. Total binding number and observation time is (from top to bottom) 324, 1,174 s; 158, 1,200 s; 292, 1,193 s. (Video 2). (C) Three example distributions for a MT with a bound E236A/E236A-bead. Total binding number and observation time is (from top to bottom) 99, 1,026 s; 78, 1,200 s; 119, 1,200 s. In both B and C, binding was suppressed in the immediate vicinity of the reference bead due to physical obstruction of the binding site by the reference bead. Length of the MT on either side of the reference bead, measured from the center of the bead, is indicated in the graph. Bin size = 1 μm. (D) Distribution of the binding frequency averaged for 10 MTs within a 6-μm distance either side of the reference bead. The red line is for MTs with a bound WT/E236A-bead (1,429 bindings were counted over a total observation period of 187 min) and the blue line is for MTs with a bound E236A/E236A-bead (699 bindings were counted over a total observation period of 196 min). Asterisks indicate that the difference in mean binding frequencies between the two groups was significant (P < 0.01; t test). Error bar indicates SEM. Bin size = 1 μm. Note that the absolute value of the binding frequency in this experiment cannot be directly compared with the binding frequency shown in Fig. 1 C, because the conditions of the wild-type–kinesin beads were different between the two experiments (see Materials and methods).
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fig3: Cooperative binding induced by heterodimeric kinesin, WT/E236A. (A) Schematic representation of binding assay using heterodimeric kinesin. (B) Three representative example distributions of binding frequency for a MT with a bound WT/E236A-bead. Total binding number and observation time is (from top to bottom) 324, 1,174 s; 158, 1,200 s; 292, 1,193 s. (Video 2). (C) Three example distributions for a MT with a bound E236A/E236A-bead. Total binding number and observation time is (from top to bottom) 99, 1,026 s; 78, 1,200 s; 119, 1,200 s. In both B and C, binding was suppressed in the immediate vicinity of the reference bead due to physical obstruction of the binding site by the reference bead. Length of the MT on either side of the reference bead, measured from the center of the bead, is indicated in the graph. Bin size = 1 μm. (D) Distribution of the binding frequency averaged for 10 MTs within a 6-μm distance either side of the reference bead. The red line is for MTs with a bound WT/E236A-bead (1,429 bindings were counted over a total observation period of 187 min) and the blue line is for MTs with a bound E236A/E236A-bead (699 bindings were counted over a total observation period of 196 min). Asterisks indicate that the difference in mean binding frequencies between the two groups was significant (P < 0.01; t test). Error bar indicates SEM. Bin size = 1 μm. Note that the absolute value of the binding frequency in this experiment cannot be directly compared with the binding frequency shown in Fig. 1 C, because the conditions of the wild-type–kinesin beads were different between the two experiments (see Materials and methods).

Mentions: Directional, cooperative binding was also confirmed in another experiment where a kinesin bead was first fixed on the MT and then the subsequent binding of other kinesin beads was examined (Fig. 3 A). This experiment was designed to more efficiently analyze the directionality of cooperative binding. To make a kinesin bead stationary on the MT in the presence of ATP, we used a previously constructed heterodimeric kinesin, WT/E236A, which can hydrolyze ATP, and yet remain fixed on the MT (Table S1, available at http://www.jcb.org/cgi/content/full/jcb.200409035/DC1; Kaseda et al., 2002). In addition to this refinement, compared with the previous experiment shown in Figs. 1 and 2, we lowered both the kinesin density on the beads and the concentration of beads used in the binding assay, thereby increasing the chance of lone bindings (see Materials and methods for details).


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

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

Cooperative binding induced by heterodimeric kinesin, WT/E236A. (A) Schematic representation of binding assay using heterodimeric kinesin. (B) Three representative example distributions of binding frequency for a MT with a bound WT/E236A-bead. Total binding number and observation time is (from top to bottom) 324, 1,174 s; 158, 1,200 s; 292, 1,193 s. (Video 2). (C) Three example distributions for a MT with a bound E236A/E236A-bead. Total binding number and observation time is (from top to bottom) 99, 1,026 s; 78, 1,200 s; 119, 1,200 s. In both B and C, binding was suppressed in the immediate vicinity of the reference bead due to physical obstruction of the binding site by the reference bead. Length of the MT on either side of the reference bead, measured from the center of the bead, is indicated in the graph. Bin size = 1 μm. (D) Distribution of the binding frequency averaged for 10 MTs within a 6-μm distance either side of the reference bead. The red line is for MTs with a bound WT/E236A-bead (1,429 bindings were counted over a total observation period of 187 min) and the blue line is for MTs with a bound E236A/E236A-bead (699 bindings were counted over a total observation period of 196 min). Asterisks indicate that the difference in mean binding frequencies between the two groups was significant (P < 0.01; t test). Error bar indicates SEM. Bin size = 1 μm. Note that the absolute value of the binding frequency in this experiment cannot be directly compared with the binding frequency shown in Fig. 1 C, because the conditions of the wild-type–kinesin beads were different between the two experiments (see Materials and methods).
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Related In: Results  -  Collection

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

fig3: Cooperative binding induced by heterodimeric kinesin, WT/E236A. (A) Schematic representation of binding assay using heterodimeric kinesin. (B) Three representative example distributions of binding frequency for a MT with a bound WT/E236A-bead. Total binding number and observation time is (from top to bottom) 324, 1,174 s; 158, 1,200 s; 292, 1,193 s. (Video 2). (C) Three example distributions for a MT with a bound E236A/E236A-bead. Total binding number and observation time is (from top to bottom) 99, 1,026 s; 78, 1,200 s; 119, 1,200 s. In both B and C, binding was suppressed in the immediate vicinity of the reference bead due to physical obstruction of the binding site by the reference bead. Length of the MT on either side of the reference bead, measured from the center of the bead, is indicated in the graph. Bin size = 1 μm. (D) Distribution of the binding frequency averaged for 10 MTs within a 6-μm distance either side of the reference bead. The red line is for MTs with a bound WT/E236A-bead (1,429 bindings were counted over a total observation period of 187 min) and the blue line is for MTs with a bound E236A/E236A-bead (699 bindings were counted over a total observation period of 196 min). Asterisks indicate that the difference in mean binding frequencies between the two groups was significant (P < 0.01; t test). Error bar indicates SEM. Bin size = 1 μm. Note that the absolute value of the binding frequency in this experiment cannot be directly compared with the binding frequency shown in Fig. 1 C, because the conditions of the wild-type–kinesin beads were different between the two experiments (see Materials and methods).
Mentions: Directional, cooperative binding was also confirmed in another experiment where a kinesin bead was first fixed on the MT and then the subsequent binding of other kinesin beads was examined (Fig. 3 A). This experiment was designed to more efficiently analyze the directionality of cooperative binding. To make a kinesin bead stationary on the MT in the presence of ATP, we used a previously constructed heterodimeric kinesin, WT/E236A, which can hydrolyze ATP, and yet remain fixed on the MT (Table S1, available at http://www.jcb.org/cgi/content/full/jcb.200409035/DC1; Kaseda et al., 2002). In addition to this refinement, compared with the previous experiment shown in Figs. 1 and 2, we lowered both the kinesin density on the beads and the concentration of beads used in the binding assay, thereby increasing the chance of lone bindings (see Materials and methods for details).

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