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

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.

Figure 1.

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.

Figure 2.

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.

Figure 3.

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).

Figure 4.

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.