The AAA3 domain of cytoplasmic dynein acts as a switch to facilitate microtubule release

Dewitt, Mark; Cypranowska, C.A.; Cleary, Frank B.; Belyy, Vladislav; Yıldız, Ahmet

doi:10.1038/nsmb.2930

Cited by 83 publications

(137 citation statements)

References 49 publications

(150 reference statements)

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“…4 A and B), because it has been shown that a dynein construct having only a single active head is still able to move processively as long as the second head retains the ability bind to the MT (46). We used a heterodimeric construct in which the active head was doubly biotinylated and labeled with a QR, whereas the other unlabeled head contained a P-loop (Walker A) K-to-A mutation in AAA1, which renders it unable to bind or hydrolyze ATP (47). This dead-head construct has (A) Probability distribution of step sizes either correlated with an angle change (green) or not correlated with an angle change (uncorrelated, dark green).…”

Section: Resultsmentioning

confidence: 99%

Angular measurements of the dynein ring reveal a stepping mechanism dependent on a flexible stalk

Lippert

Dadosh

Hadden

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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The force-generating mechanism of dynein differs from the forcegenerating mechanisms of other cytoskeletal motors. To examine the structural dynamics of dynein's stepping mechanism in real time, we used polarized total internal reflection fluorescence microscopy with nanometer accuracy localization to track the orientation and position of single motors. By measuring the polarized emission of individual quantum nanorods coupled to the dynein ring, we determined the angular position of the ring and found that it rotates relative to the microtubule (MT) while walking. Surprisingly, the observed rotations were small, averaging only 8.3°, and were only weakly correlated with steps. Measurements at two independent labeling positions on opposite sides of the ring showed similar small rotations. Our results are inconsistent with a classic power-stroke mechanism, and instead support a flexible stalk model in which interhead strain rotates the rings through bending and hinging of the stalk. Mechanical compliances of the stalk and hinge determined based on a 3.3-μs molecular dynamics simulation account for the degree of ring rotation observed experimentally. Together, these observations demonstrate that the stepping mechanism of dynein is fundamentally different from the stepping mechanisms of other well-studied MT motors, because it is characterized by constant small-scale fluctuations of a large but flexible structure fully consistent with the variable stepping pattern observed as dynein moves along the MT.ynein is a molecular motor that walks processively toward the minus end of microtubules (MTs) in an ATP-dependent manner (1-3). Axonemal dyneins drive the motility of eukaryotic cilia and flagella, whereas cytoplasmic dynein is responsible for a wide range of functions within eukaryotic cells, including the retrograde transport of cargo such as autophagosomes in neurons (4), alignment of the mitotic spindle (5, 6), and chromosome segregation during mitosis (7). Disruption of dynein-mediated neuronal transport has been implicated in neurodegeneration (8), and mutations in dynein and dynein-associated proteins can cause a range of diseases, including spinal muscular atrophy (9, 10), lissencephaly (11) and Charcot-Marie-Tooth disease type 2 (reviewed in ref. 12). Despite the importance of dynein function, the mechanism by which dynein walks along the MT is not yet well understood.The motor domains of dynein are formed from six concatenated AAA domains, interrupted by a long, antiparallel, coiled-coil stalk that emerges from AAA4 and terminates in the microtubulebinding domain (MTBD) and the buttress that extends from AAA5 and is thought to stabilize the stalk (13,14). Two motor domains form a dimer via their N-terminal tails (2, 3). ATP binding and hydrolysis drive dynein mechanochemistry; the binding of ATP to AAA1 induces a conformational change leading to dissociation of the MTBD from the MT. Hydrolysis on the dissociated head induces a primed conformation that then rebinds to the MT. The forward step, or power stroke,...

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Section: Resultsmentioning

confidence: 99%

Angular measurements of the dynein ring reveal a stepping mechanism dependent on a flexible stalk

Lippert

Dadosh

Hadden

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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“…AAA1 activity appears not to be strictly synchronized to that of the other AAA domains (4,10,25) and AAA3 hydrolyzes ATP an order of magnitude slower than AAA1 (34). Thus, AAA3 may be ADP bound only at "appropriate" points in the cycle, such as when the head is detached from the MT or when the rear head AAA1 binds ATP (thereby assisting in MT release).…”

Section: Discussionmentioning

confidence: 99%

“…Although AAA3 plays an important role in controlling dynein-MT attachment (22,23,25), the details are just emerging. By probing dynein-MT interactions in the absence of load (9,34) and with force applied to the dynein C terminus (34), DeWitt et al and Bhabha et al concluded that AAA3 must be in a posthydrolysis state for ATP-induced, AAA1-mediated MT release. Our MTBR and C-terminal pulling results support these findings, but if tension is applied via the linker, then AAA1-mediated MT release is allowed when AAA3 enters the ATP state.…”

Section: Discussionmentioning

confidence: 99%

“…Finally, we dissect AAA1-and AAA3-mediated nucleotide-induced modulation of dynein's inherent response to force, identifying (i) a previously undescribed weakening of MT attachment caused by ADP binding at AAA3 and (ii) a novel function for the linker in the AAA3-mediated "gating" of the nucleotide-dependent regulation of dynein-MT binding by AAA1. When tension is absent or applied via dynein's C terminus, ATP binding to AAA1 induces MT release only if AAA3 is in the posthydrolysis state, as described recently (9,34). However, under more physiological conditions in which tension is applied to the linker, ATP binding to AAA3 is sufficient to "open" the regulatory gate.…”

Section: Significancementioning

confidence: 99%

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Cytoplasmic dynein regulates its attachment to microtubules via nucleotide state-switched mechanosensing at multiple AAA domains

Nicholas

Berger

Rao³

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

111

212

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Cytoplasmic dynein is a homodimeric microtubule (MT) motor protein responsible for most MT minus-end-directed motility. Dynein contains four AAA+ ATPases (AAA: ATPase associated with various cellular activities) per motor domain (AAA1-4). The main site of ATP hydrolysis, AAA1, is the only site considered by most dynein motility models. However, it remains unclear how ATPase activity and MT binding are coordinated within and between dynein's motor domains. Using optical tweezers, we characterize the MT-binding strength of recombinant dynein monomers as a function of mechanical tension and nucleotide state. Dynein responds anisotropically to tension, binding tighter to MTs when pulled toward the MT plus end. We provide evidence that this behavior results from an asymmetrical bond that acts as a slip bond under forward tension and a slip-ideal bond under backward tension. ATP weakens MT binding and reduces bond strength anisotropy, and unexpectedly, so does ADP. Using nucleotide binding and hydrolysis mutants, we show that, although ATP exerts its effects via binding AAA1, ADP effects are mediated by AAA3. Finally, we demonstrate "gating" of AAA1 function by AAA3. When tension is absent or applied via dynein's C terminus, ATP binding to AAA1 induces MT release only if AAA3 is in the posthydrolysis state. However, when tension is applied to the linker, ATP binding to AAA3 is sufficient to "open" the gate. These results elucidate the mechanisms of dynein-MT interactions, identify regulatory roles for AAA3, and help define the interplay between mechanical tension and nucleotide state in regulating dynein motility.cytoplasmic dynein | mechanosensing | optical tweezers | AAA+ ATPases | microtubules N umerous eukaryotic cellular processes require motion and force generated by cytoskeletal motor proteins, among which cytoplasmic dynein (hereinafter, "dynein") is unique for its size, complexity, and versatility. As a homodimeric, divergent AAA+ ATPase (AAA: ATPase associated with various cellular activities), dynein drives the majority of microtubule (MT) minusend-directed motility in most eukaryotes (1). The motor functions as a massive protein complex (2), but its catalytic core consists of two identical heavy chains, each with six AAA modules (AAA1-6) linked in tandem to form a ring (Fig. 1A). AAA1-4 bind nucleotides, whereas AAA5 and -6 are structural (3, 4). A ∼15-nm "stalk" emerging from AAA4 (3, 4) separates the AAA modules from the MT-binding domain (MTBD). The stalk configuration influences both MT affinity and ATPase activity (5) and thereby mediates bidirectional allosteric communication between the AAA ring and the MTBD (3, 6). Finally, a ∼10-nm "linker" also emerges from the ring and undergoes cyclic reorientations that generate force and displacement (7-9).For dynein to "walk," one motor domain ("head") must remain MT-bound while the other moves (10-13), thus requiring coordination of the "internal" cycles of both heads. Dynein may use allosteric mechanosensing (possibly through the stalk) to differentiate be...

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“…The nucleotide state at AAA3 regulates dynein’s motility by acting on its mechanochemical cycle (DeWitt et al, 2014; Nicholas et al, 2015): stepping dynein contains ADP at AAA3, while ATP or no nucleotide (“apo”) at this site results in a slower dynein that is tightly bound to MTs (DeWitt et al, 2014). This AAA3-mediated inhibition occurs even when ATP binds to AAA1, which normally triggers MT release.…”

Section: Introductionmentioning

confidence: 99%

Lis1 Has Two Opposing Modes of Regulating Cytoplasmic Dynein

DeSantis

Cianfrocco

Htet

et al. 2017

Cell

129

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Summary Regulation is central to the functional versatility of cytoplasmic dynein, a motor involved in intracellular transport, cell division, and neurodevelopment. Previous work established that Lis1, a conserved regulator of dynein, binds to its motor domain and induces a tight microtubule-binding state in dynein. The work we present here—a combination of biochemistry, single-molecule assays, and cryo-electron microscopy—led to the surprising discovery that Lis1 has two opposing modes of regulating dynein, being capable of inducing both low and high affinity for the microtubule. We show that these opposing modes depend on the stoichiometry of Lis1 binding to dynein and that this stoichiometry is regulated by the nucleotide state of dynein’s AAA3 domain. The low affinity state requires Lis1 binding to dynein at a novel conserved site, mutation of which disrupts Lis1’s function in vivo. We propose a new model for the regulation of dynein by Lis1.

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The AAA3 domain of cytoplasmic dynein acts as a switch to facilitate microtubule release

Cited by 83 publications

References 49 publications

Angular measurements of the dynein ring reveal a stepping mechanism dependent on a flexible stalk

Angular measurements of the dynein ring reveal a stepping mechanism dependent on a flexible stalk

Cytoplasmic dynein regulates its attachment to microtubules via nucleotide state-switched mechanosensing at multiple AAA domains

Lis1 Has Two Opposing Modes of Regulating Cytoplasmic Dynein

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