XB-ART-36949Eur Biophys J July 1, 2007; 36 (6): 675-81.
Load-dependent release limits the processive stepping of the tetrameric Eg5 motor.
Tetrameric motor proteins of the Kinesin-5 family are essential for eukaryotic cell division. The microscopic mechanism by which Eg5, the vertebrate Kinesin-5, drives bipolar mitotic spindle formation remains unknown. Here we show in optical trapping experiments that full-length Eg5 moves processively and stepwise along microtubule bundles. Interestingly, the force produced by individual Eg5 motors typically reached only approximately 2 pN, one-third of the stall force of Kinesin-1. Eg5 typically detached from microtubules before stalling. This behavior may reflect a regulatory mechanism important for the role of Eg5 in the mitotic spindle.
PubMed ID: 17333163
PMC ID: PMC1914257
Article link: Eur Biophys J
Genes referenced: kif11 pdf
Article Images: [+] show captions
|Fig. 1. Motor attachment, experimental setup and bead traces. a–d, f Possible Eg5-motor attachments to beads (silica, 0.5 μm diameter) via the genetically encoded N-terminal His-tag of Eg5. a All four motor domains are bound, preventing motility. b One motor domain is unbound, likely allowing only non-processive motility. c Two motor domains are free, one at each end, likely allowing only non-processive motility. d One motor domain is bound, leaving a dimeric motor end free to interact with the microtubule. e Traces of bead motility generated by individual Eg5 (green) and Kinesin-1 (grey) motors. The averaged (15-point) and median-filtered (0.3 s (Eg5) and 0.05 s (Kinesin-1) sliding windows; rank 10) signal is overlaid in red over both traces. The trap stiffnesses were 0.03 pN/nm (Kinesin-1) and 0.013 pN/nm (Eg5). f One dimer is bound, one dimer is free; sketch of a silica-sphere with motor held in the laser-trap, such that it interacts with a surface-attached microtubule track|
|Fig. 2. Motor-bead attachment. Fractions of beads not interacting with a microtubule over 2 min (grey), moving (red), attached for less than 2 min (without motion) (purple), irreversibly stuck (blue). a Constant high average number of antibodies per bead (6,950), variation of motor concentration. b Constant concentration of motors in solution (corresponding to 2,410 per bead), variation of antibody density in order to obtain optimal motor/antibody-ratio (r-value) to favor species d and f (Fig. 1). c Systematic reduction of the absolute number of motor/antibody-complexes at constant r-values between 1 and 2 revealed a clear concentration dependence of motile events. Data were collected typically over periods of minutes. The number of beads that was used at each concentration is shown under the histograms|
|Fig. 3. Steps of Kinesin-1/control. Kinesin-1 motility using the same attachment chemistry to immobilize individual motors on 0.5 μm silica spheres. Stepwise displacement (uncalibrated axis) is evident before (grey) and after filtering the data (red, 4,096 Hz sampling rate, 15-point averaging and median-filtering: 0.05 s sliding window, rank 10)|