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Assembling Molecular Shuttles Powered by Reversibly Attached Kinesins
Published on: January 26, 2019
Single molecule FRET observation of kinesin-1's head-tail interaction on microtubule
Takahiro Aoki1, Michio Tomishige1, Takayuki Ariga1
1Department of Applied Physics, School of Engineering, the University of Tokyo, Tokyo, 113-8656, Japan.
Abstract:
Kinesin-1 (conventional kinesin) is a molecular motor that transports various cargo such as endoplasmic reticulum and mitochondria in cells. Its two head domains walk along microtubule by hydrolyzing ATP, while the tail domains at the end of the long stalk bind to the cargo. When a kinesin is not carrying cargo, its motility and ATPase activity is inhibited by direct interactions between the tail and head. However, the mechanism of this tail regulation is not well understood. Here, we apply single molecule fluorescence resonance energy transfer (smFRET) to observe this interaction in stalk-truncated kinesin. We found that kinesin with two tails forms a folding conformation and dissociates from microtubules, whereas kinesin with one tail remains bound to the micro-tubule and is immobile even in the presence of ATP. We further investigated the head-tail interaction as well as head-head coordination on the microtubule at various nucleotide conditions. From these results, we propose a two-step inhibition model for kinesin motility.
Insights
Kinesin-1
Area of Science:
- Molecular biology
- Cellular transport mechanisms
- Protein dynamics
Background:
- Kinesin-1 is a motor protein essential for intracellular transport.
- Cargo binding regulates kinesin-1 activity, but the inhibition mechanism is unclear.
- Tail-head interactions are known to inhibit motility.
Purpose of the Study:
- To investigate the mechanism of tail-mediated inhibition of kinesin-1 motility.
- To elucidate the role of tail number in kinesin-1 regulation.
- To understand head-tail and head-head interactions under different nucleotide conditions.
Main Methods:
- Single-molecule Förster Resonance Energy Transfer (smFRET) was used.
- Stalk-truncated kinesin constructs with varying tail numbers were analyzed.
- Kinesin-microtubule interactions were observed under different nucleotide conditions.
Main Results:
- Kinesin with two tails adopts a folded conformation, dissociates from microtubules, and becomes inactive.
- Kinesin with one tail remains bound to microtubules and is immobile, even with ATP.
- Head-tail and head-head interactions were characterized at various nucleotide states.
Conclusions:
- A two-step inhibition model for kinesin-1 motility is proposed.
- The number of tails significantly influences kinesin-1's interaction with microtubules and its activity.
- Understanding this regulation provides insights into intracellular transport control.
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