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Tension on the linker gates the ATP-dependent release of dynein from microtubules.

Cleary FB, Dewitt MA, Bilyard T, Htet ZM, Belyy V, Chan DD, Chang AY, Yildiz A - Nat Commun (2014)

Bottom Line: Here, by engineering the mechanical and catalytic properties of the motor, we show that dynein processivity minimally requires a single active head and a second inert MT-binding domain.Processivity arises from a high ratio of MT-bound to unbound time, and not from interhead communication.In addition, nucleotide-dependent microtubule release is gated by tension on the linker domain.

View Article: PubMed Central - PubMed

Affiliation: Biophysics Graduate Group, University of California, Berkeley, California 94720, USA.

ABSTRACT
Cytoplasmic dynein is a dimeric motor that transports intracellular cargoes towards the minus end of microtubules (MTs). In contrast to other processive motors, stepping of the dynein motor domains (heads) is not precisely coordinated. Therefore, the mechanism of dynein processivity remains unclear. Here, by engineering the mechanical and catalytic properties of the motor, we show that dynein processivity minimally requires a single active head and a second inert MT-binding domain. Processivity arises from a high ratio of MT-bound to unbound time, and not from interhead communication. In addition, nucleotide-dependent microtubule release is gated by tension on the linker domain. Intramolecular tension sensing is observed in dynein's stepping motion at high interhead separations. On the basis of these results, we propose a quantitative model for the stepping characteristics of dynein and its response to chemical and mechanical perturbation.

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The linker provides force to drive the motility of a dynein dimer(a) Dimerization of the C-terminal ring of one head to the N-terminal tail of the other results in a dimer of a free-linker head (FLH) and a bound-linker head (BLH). (b) Kymograph showing that the FLH/BLH heterodimer is capable of processive motility. (c) Run length histogram of FLH/BLH at 2 mM ATP, with maximum likelihood fit (± 95% CI). (d) The average velocities (± SEM) of FLH/BLH constructs carrying an ATPase mutation in either the AAA1 or AAA3 site in one head (ND: motility not detected). (e) Kymographs of ATPase mutants of the FLH or BLH at 2 mM ATP. The AAA1K/A mutation on BLH abolishes directional motility, whereas AAA1E/Q mutation leads to non-directional diffusion along the MT. The same mutations on FLH do not stop motility, indicating that BLH monomer is responsible for FLH/BLH motility.
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Figure 3: The linker provides force to drive the motility of a dynein dimer(a) Dimerization of the C-terminal ring of one head to the N-terminal tail of the other results in a dimer of a free-linker head (FLH) and a bound-linker head (BLH). (b) Kymograph showing that the FLH/BLH heterodimer is capable of processive motility. (c) Run length histogram of FLH/BLH at 2 mM ATP, with maximum likelihood fit (± 95% CI). (d) The average velocities (± SEM) of FLH/BLH constructs carrying an ATPase mutation in either the AAA1 or AAA3 site in one head (ND: motility not detected). (e) Kymographs of ATPase mutants of the FLH or BLH at 2 mM ATP. The AAA1K/A mutation on BLH abolishes directional motility, whereas AAA1E/Q mutation leads to non-directional diffusion along the MT. The same mutations on FLH do not stop motility, indicating that BLH monomer is responsible for FLH/BLH motility.

Mentions: We next tested the mechanism by which the active head can drag its inactive partner forward, presumably via forces generated by the powerstroke of its linker. Although evidence that the linker can power motility was observed in truncated monomeric dyneins32,36, the role of linker in processive motility and force generation remains unclear. The powerstroke model9,11 suggests that a head that generates force must be attached to the other head through its linker domain, either to push against the other head or to pull it forward. We tested this hypothesis by engineering a heterodimer composed of two catalytically identical but mechanically distinct monomers. The N-terminal linker of one monomer was attached to the C-terminus of the other monomer, resulting in a heterodimer of a bound-linker head (BLH) and a free-linker head (FLH) (Fig. 3a). According to the powerstroke model, inhibiting the linker swing in the BLH would be predicted to abolish motility, while motors with an inhibited FLH would remain motile.


Tension on the linker gates the ATP-dependent release of dynein from microtubules.

Cleary FB, Dewitt MA, Bilyard T, Htet ZM, Belyy V, Chan DD, Chang AY, Yildiz A - Nat Commun (2014)

The linker provides force to drive the motility of a dynein dimer(a) Dimerization of the C-terminal ring of one head to the N-terminal tail of the other results in a dimer of a free-linker head (FLH) and a bound-linker head (BLH). (b) Kymograph showing that the FLH/BLH heterodimer is capable of processive motility. (c) Run length histogram of FLH/BLH at 2 mM ATP, with maximum likelihood fit (± 95% CI). (d) The average velocities (± SEM) of FLH/BLH constructs carrying an ATPase mutation in either the AAA1 or AAA3 site in one head (ND: motility not detected). (e) Kymographs of ATPase mutants of the FLH or BLH at 2 mM ATP. The AAA1K/A mutation on BLH abolishes directional motility, whereas AAA1E/Q mutation leads to non-directional diffusion along the MT. The same mutations on FLH do not stop motility, indicating that BLH monomer is responsible for FLH/BLH motility.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4129465&req=5

Figure 3: The linker provides force to drive the motility of a dynein dimer(a) Dimerization of the C-terminal ring of one head to the N-terminal tail of the other results in a dimer of a free-linker head (FLH) and a bound-linker head (BLH). (b) Kymograph showing that the FLH/BLH heterodimer is capable of processive motility. (c) Run length histogram of FLH/BLH at 2 mM ATP, with maximum likelihood fit (± 95% CI). (d) The average velocities (± SEM) of FLH/BLH constructs carrying an ATPase mutation in either the AAA1 or AAA3 site in one head (ND: motility not detected). (e) Kymographs of ATPase mutants of the FLH or BLH at 2 mM ATP. The AAA1K/A mutation on BLH abolishes directional motility, whereas AAA1E/Q mutation leads to non-directional diffusion along the MT. The same mutations on FLH do not stop motility, indicating that BLH monomer is responsible for FLH/BLH motility.
Mentions: We next tested the mechanism by which the active head can drag its inactive partner forward, presumably via forces generated by the powerstroke of its linker. Although evidence that the linker can power motility was observed in truncated monomeric dyneins32,36, the role of linker in processive motility and force generation remains unclear. The powerstroke model9,11 suggests that a head that generates force must be attached to the other head through its linker domain, either to push against the other head or to pull it forward. We tested this hypothesis by engineering a heterodimer composed of two catalytically identical but mechanically distinct monomers. The N-terminal linker of one monomer was attached to the C-terminus of the other monomer, resulting in a heterodimer of a bound-linker head (BLH) and a free-linker head (FLH) (Fig. 3a). According to the powerstroke model, inhibiting the linker swing in the BLH would be predicted to abolish motility, while motors with an inhibited FLH would remain motile.

Bottom Line: Here, by engineering the mechanical and catalytic properties of the motor, we show that dynein processivity minimally requires a single active head and a second inert MT-binding domain.Processivity arises from a high ratio of MT-bound to unbound time, and not from interhead communication.In addition, nucleotide-dependent microtubule release is gated by tension on the linker domain.

View Article: PubMed Central - PubMed

Affiliation: Biophysics Graduate Group, University of California, Berkeley, California 94720, USA.

ABSTRACT
Cytoplasmic dynein is a dimeric motor that transports intracellular cargoes towards the minus end of microtubules (MTs). In contrast to other processive motors, stepping of the dynein motor domains (heads) is not precisely coordinated. Therefore, the mechanism of dynein processivity remains unclear. Here, by engineering the mechanical and catalytic properties of the motor, we show that dynein processivity minimally requires a single active head and a second inert MT-binding domain. Processivity arises from a high ratio of MT-bound to unbound time, and not from interhead communication. In addition, nucleotide-dependent microtubule release is gated by tension on the linker domain. Intramolecular tension sensing is observed in dynein's stepping motion at high interhead separations. On the basis of these results, we propose a quantitative model for the stepping characteristics of dynein and its response to chemical and mechanical perturbation.

Show MeSH
Related in: MedlinePlus