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Microtubule cross-linking triggers the directional motility of kinesin-5.

Kapitein LC, Kwok BH, Weinger JS, Schmidt CF, Kapoor TM, Peterman EJ - J. Cell Biol. (2008)

Bottom Line: Eg5, the vertebrate kinesin-5, has two modes of motion: an adenosine triphosphate (ATP)-dependent directional mode and a diffusive mode that does not require ATP hydrolysis.In the spindle, this might allow Eg5 to diffuse on single microtubules without hydrolyzing ATP until the motor is activated by binding to another microtubule.This mechanism would increase energy and filament cross-linking efficiency.

View Article: PubMed Central - PubMed

Affiliation: Department of Physics and Astronomy, Vrije Universiteit, 1081 HV Amsterdam, Netherlands.

ABSTRACT
Although assembly of the mitotic spindle is known to be a precisely controlled process, regulation of the key motor proteins involved remains poorly understood. In eukaryotes, homotetrameric kinesin-5 motors are required for bipolar spindle formation. Eg5, the vertebrate kinesin-5, has two modes of motion: an adenosine triphosphate (ATP)-dependent directional mode and a diffusive mode that does not require ATP hydrolysis. We use single-molecule experiments to examine how the switching between these modes is controlled. We find that Eg5 diffuses along individual microtubules without detectable directional bias at close to physiological ionic strength. Eg5's motility becomes directional when bound between two microtubules. Such activation through binding cargo, which, for Eg5, is a second microtubule, is analogous to known mechanisms for other kinesins. In the spindle, this might allow Eg5 to diffuse on single microtubules without hydrolyzing ATP until the motor is activated by binding to another microtubule. This mechanism would increase energy and filament cross-linking efficiency.

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Full-length Eg5 motility on single microtubules depends on ionic strength. (A and B) Frames (A) and kymograph (B) from a time-lapse recording showing single molecules of Eg5-GFP (green) moving directionally along a microtubule (red) in the presence of 70 mM Pipes. (C and D) Frames (C) and kymograph (D) from a time-lapse recording showing single molecules of Eg5-GFP (green) diffusing along a microtubule (red) in the presence of 70 mM Pipes plus 40 mM KCl. (E–H) MD calculated from Eg5-GFP motility in the presence of ATP (black) and ADP (red) in 70 mM Pipes plus 0, 20, 40, or 60 mM KCl. Fits represent MD = vτ. (I–M) MSD calculated from Eg5-GFP motility in the presence of ATP (black) and ADP (red) and at the indicated ionic strengths. Fits represent MD = vτ and MD = v2τ2 + 2Dτ + offset for the ATP data and MD = 2Dτ + offset for the ADP data. All numerical results are listed in Table I. (N) Histogram of the duration of binding events for 0 mM KCl and for 60 mM KCl added. Lines are single exponential fits (exp[−t/tav]) to the data (0 mM: tav = 34 ± 3, n = 212; 60 mM: tav = 16 ± 2, n = 119). Error bars represent SD. Bars, 1 μm.
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fig1: Full-length Eg5 motility on single microtubules depends on ionic strength. (A and B) Frames (A) and kymograph (B) from a time-lapse recording showing single molecules of Eg5-GFP (green) moving directionally along a microtubule (red) in the presence of 70 mM Pipes. (C and D) Frames (C) and kymograph (D) from a time-lapse recording showing single molecules of Eg5-GFP (green) diffusing along a microtubule (red) in the presence of 70 mM Pipes plus 40 mM KCl. (E–H) MD calculated from Eg5-GFP motility in the presence of ATP (black) and ADP (red) in 70 mM Pipes plus 0, 20, 40, or 60 mM KCl. Fits represent MD = vτ. (I–M) MSD calculated from Eg5-GFP motility in the presence of ATP (black) and ADP (red) and at the indicated ionic strengths. Fits represent MD = vτ and MD = v2τ2 + 2Dτ + offset for the ATP data and MD = 2Dτ + offset for the ADP data. All numerical results are listed in Table I. (N) Histogram of the duration of binding events for 0 mM KCl and for 60 mM KCl added. Lines are single exponential fits (exp[−t/tav]) to the data (0 mM: tav = 34 ± 3, n = 212; 60 mM: tav = 16 ± 2, n = 119). Error bars represent SD. Bars, 1 μm.

Mentions: Ionic strength is known to influence motor–microtubule interactions (Okada and Hirokawa, 2000). To explore Eg5 regulation, we used in vitro single-molecule fluorescence motility assays to examine Eg5-GFP motility on individual microtubules in buffers with various ionic strengths. We found that in buffers with relatively low ionic strength (<100 mM potassium), motors moved unidirectionally toward one end of the microtubule with a speed of ∼10–15 nm/s, as we observed previously (Fig. 1, A and B; Kwok et al., 2006). In the presence of additional salt (20–50 mM KCl), Eg5-GFP motility appeared diffusive along the microtubule axis without clear directionality (Fig. 1, C and D). The fluorescence intensities of individual spots were similar under all conditions tested, demonstrating that the motors were in the same oligomeric state (unpublished data).


Microtubule cross-linking triggers the directional motility of kinesin-5.

Kapitein LC, Kwok BH, Weinger JS, Schmidt CF, Kapoor TM, Peterman EJ - J. Cell Biol. (2008)

Full-length Eg5 motility on single microtubules depends on ionic strength. (A and B) Frames (A) and kymograph (B) from a time-lapse recording showing single molecules of Eg5-GFP (green) moving directionally along a microtubule (red) in the presence of 70 mM Pipes. (C and D) Frames (C) and kymograph (D) from a time-lapse recording showing single molecules of Eg5-GFP (green) diffusing along a microtubule (red) in the presence of 70 mM Pipes plus 40 mM KCl. (E–H) MD calculated from Eg5-GFP motility in the presence of ATP (black) and ADP (red) in 70 mM Pipes plus 0, 20, 40, or 60 mM KCl. Fits represent MD = vτ. (I–M) MSD calculated from Eg5-GFP motility in the presence of ATP (black) and ADP (red) and at the indicated ionic strengths. Fits represent MD = vτ and MD = v2τ2 + 2Dτ + offset for the ATP data and MD = 2Dτ + offset for the ADP data. All numerical results are listed in Table I. (N) Histogram of the duration of binding events for 0 mM KCl and for 60 mM KCl added. Lines are single exponential fits (exp[−t/tav]) to the data (0 mM: tav = 34 ± 3, n = 212; 60 mM: tav = 16 ± 2, n = 119). Error bars represent SD. Bars, 1 μm.
© Copyright Policy
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fig1: Full-length Eg5 motility on single microtubules depends on ionic strength. (A and B) Frames (A) and kymograph (B) from a time-lapse recording showing single molecules of Eg5-GFP (green) moving directionally along a microtubule (red) in the presence of 70 mM Pipes. (C and D) Frames (C) and kymograph (D) from a time-lapse recording showing single molecules of Eg5-GFP (green) diffusing along a microtubule (red) in the presence of 70 mM Pipes plus 40 mM KCl. (E–H) MD calculated from Eg5-GFP motility in the presence of ATP (black) and ADP (red) in 70 mM Pipes plus 0, 20, 40, or 60 mM KCl. Fits represent MD = vτ. (I–M) MSD calculated from Eg5-GFP motility in the presence of ATP (black) and ADP (red) and at the indicated ionic strengths. Fits represent MD = vτ and MD = v2τ2 + 2Dτ + offset for the ATP data and MD = 2Dτ + offset for the ADP data. All numerical results are listed in Table I. (N) Histogram of the duration of binding events for 0 mM KCl and for 60 mM KCl added. Lines are single exponential fits (exp[−t/tav]) to the data (0 mM: tav = 34 ± 3, n = 212; 60 mM: tav = 16 ± 2, n = 119). Error bars represent SD. Bars, 1 μm.
Mentions: Ionic strength is known to influence motor–microtubule interactions (Okada and Hirokawa, 2000). To explore Eg5 regulation, we used in vitro single-molecule fluorescence motility assays to examine Eg5-GFP motility on individual microtubules in buffers with various ionic strengths. We found that in buffers with relatively low ionic strength (<100 mM potassium), motors moved unidirectionally toward one end of the microtubule with a speed of ∼10–15 nm/s, as we observed previously (Fig. 1, A and B; Kwok et al., 2006). In the presence of additional salt (20–50 mM KCl), Eg5-GFP motility appeared diffusive along the microtubule axis without clear directionality (Fig. 1, C and D). The fluorescence intensities of individual spots were similar under all conditions tested, demonstrating that the motors were in the same oligomeric state (unpublished data).

Bottom Line: Eg5, the vertebrate kinesin-5, has two modes of motion: an adenosine triphosphate (ATP)-dependent directional mode and a diffusive mode that does not require ATP hydrolysis.In the spindle, this might allow Eg5 to diffuse on single microtubules without hydrolyzing ATP until the motor is activated by binding to another microtubule.This mechanism would increase energy and filament cross-linking efficiency.

View Article: PubMed Central - PubMed

Affiliation: Department of Physics and Astronomy, Vrije Universiteit, 1081 HV Amsterdam, Netherlands.

ABSTRACT
Although assembly of the mitotic spindle is known to be a precisely controlled process, regulation of the key motor proteins involved remains poorly understood. In eukaryotes, homotetrameric kinesin-5 motors are required for bipolar spindle formation. Eg5, the vertebrate kinesin-5, has two modes of motion: an adenosine triphosphate (ATP)-dependent directional mode and a diffusive mode that does not require ATP hydrolysis. We use single-molecule experiments to examine how the switching between these modes is controlled. We find that Eg5 diffuses along individual microtubules without detectable directional bias at close to physiological ionic strength. Eg5's motility becomes directional when bound between two microtubules. Such activation through binding cargo, which, for Eg5, is a second microtubule, is analogous to known mechanisms for other kinesins. In the spindle, this might allow Eg5 to diffuse on single microtubules without hydrolyzing ATP until the motor is activated by binding to another microtubule. This mechanism would increase energy and filament cross-linking efficiency.

Show MeSH
Related in: MedlinePlus