#dynein

Cytoskeleton

What would be the expected effect of a mutation that prevents dynein’s interaction with dynactin?

Cytoplasmic dynein requires adaptor complexes to achieve efficient cargo transport because motor alone exhibits low processivity and weak cargo binding. Dynactin is twenty three subunit complex containing short Arp1 filament, beta-spectrin adaptor, p150Glued subunit with CAP-Gly microtubule-binding domain and coiled-coil dimerization region that binds dynein intermediate chain. Interaction via extended CC1 fragment of p150Glued locks dynein-dynactin together, increasing run length from submicron to several microns by coordinating two motor domains and suppressing detachment. Preventing this in

Ref: Schroer Annu Rev Cell Dev Biol; dynactin p150Glued enhances dynein processivity and cargo binding.

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Cytoskeleton

The AAA+ domain in dynein is responsible for:

Structural analysis of dynein heavy chain shows tail responsible for dimerization and cargo adaptor binding, followed by six AAA+ domains arranged as heterohexameric ring with central pore. AAA1 bears conserved Walker A P-loop GXXXXGKT and Walker B hhhhDE motif essential for ATP binding and hydrolysis, AAA2-AAA4 modulate allosteric communication, AAA5-AAA6 stabilize ring and interact with stalk and strut. Upon ATP hydrolysis at AAA1, conformational changes propagate around ring altering interface between AAA5 and stalk buttress, shifting coiled-coil registry and linker docking. This generates

Ref: Carter et al. Nature 2011; AAA+ AAA1 Walker A/B hydrolyzes ATP generating 4-5 pN force.

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Cytoskeleton

How does dynein achieve movement along microtubules?

Dynein stepping mechanism contrasts with kinesin neck-linker model, employing large AAA ring plus linker swing. In absence of nucleotide, microtubule-binding domain tightly bound, linker straight docking at AAA5. ATP binding to AAA1 induces closure of AAA1-AAA2 interface, pulling buttress from AAA5 that shifts stalk coiled-coil registry from alpha to beta, weakening microtubule affinity and detaching motor. Hydrolysis allows linker undocking from AAA5 and migration toward AAA2 site forming pre power stroke bent conformation. Forward diffusion and microtubule rebinding triggers release of phosp

Ref: Roberts Cell 2013; ATP binding detaches dynein, ADP release plus linker rotation powers stroke.

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Cytoskeleton

Dynein movement along microtubules requires:

Dynein mechanochemistry is powered by Mg-ATP, with intracellular concentration typically one to three millimolar far above Km around twenty to fifty micromolar, supporting near maximal velocity in vivo. Cycle involves ATP binding to AAA1 causing microtubule release, hydrolysis triggering primer stroke of linker into bent conformation, microtubule reattachment forward, then phosphate and ADP release driving power stroke returning linker to straight conformation. Without ATP supply motor enters rigor bound to microtubule, transport stalls. Metabolic poisons depleting ATP, such as sodium azide pl

Ref: Shpetner J Cell Biol; dynein ATPase Km ~30μM, continuous Mg-ATP needed for stepping cycle.

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Cytoskeleton

Which experimental evidence would best demonstrate that dynein is required for Golgi positioning?

Perinuclear positioning of Golgi ribbon relies on continuous dynein-dependent transport of Golgi stacks toward microtubule minus ends clustered at centrosome. Direct causality requires selective dynein perturbation while monitoring microtubules. Approach involves inducible expression of p150Glued CC1 fragment that competes for dynein binding to dynactin, siRNA against dynein heavy chain DYNC1H1, or pharmacologic ciliobrevin D that inhibits AAA+ ATPase without depolymerizing tubulin. Outcome assessed by immunofluorescence for cis marker GM130, medial Golgin-84, trans TGN46 shows rapid conversio

Ref: Burkhardt Cell 1997; dynein-dynactin perturbation fragments Golgi, demonstrates retrograde positioning requirement.

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Cytoskeleton

What is the function of the linker domain in dynein?

Linker domain is a four helix bundle lever that spans AAA ring, mechanically linking tail to motor. In post power stroke conformation it is straight, docked near AAA5, positioning cargo forward. ATP binding to AAA1 induces closure of ring and steric clash pushing linker off AAA5 toward AAA2 docking site, forming bent pre power stroke state that primes motor. This repositioning stores elastic strain. Upon microtubule rebinding and ADP release, linker snaps back toward AAA5, executing forceful swing that drags cargo complex relative to microtubule by several nanometers. Because length exceeds 10

Ref: Schmidt Nature 2012; linker as lever converting ATP hydrolysis into mechanical work in dynein.

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Cytoskeleton

Which component of dynein is primarily responsible for generating movement?

Generation of force in dynein occurs in motor domain comprising six AAA+ modules organized in a closed ring with large linker arching over it. AAA1 through AAA4 can bind nucleotide, but only AAA1 and AAA3 hydrolyze productively to drive movement. In ATP-bound state AAA1-AAA2 closure pulls AAA2 away from AAA3, propagating conformational change through AAA4-AAA6 to stalk coiled-coil, shifting registry and lowering affinity for microtubule. Subsequent phosphate release closes ring further, driving linker domain rotation of approximately eight nanometers from straight to bent conformation docked a

Ref: Carter Nature 2011; AAA+ ring AAA1 hydrolyzes ATP driving linker swing, motor core force.

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Cytoskeleton

Which statement about dynein function is FALSE?

Dynein heavy chains are among the largest AAA+ machines, containing six concatenated AAA modules forming an asymmetric ring. AAA1 is primary catalytic site where ATP binding closes interface with AAA2, straightening linker and detaching stalk from microtubule. Hydrolysis and phosphate release trigger linker swing from pre to post power stroke, pulling tail forward while microtubule-binding domain reattaches downstream. This cycle strictly depends on ATP concentration and hydrolysis; nonhydrolyzable analogs such as AMP-PNP arrest motility in strong binding state, vanadate traps ADP-Pi intermedi

Ref: Alberts Ch 16 Motor proteins; dynein AAA+ requires ATP hydrolysis, minus-end directed transport.

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Cytoskeleton

What happens when dynein loses its ability to bind microtubules?

Cytoplasmic dynein generates minus-end directed transport using a microtubule-binding domain at the tip of an antiparallel coiled-coil stalk that extends from the AAA ring. The binding interface undergoes ATP-dependent registry shift between strong and weak states. When mutations, deletion or antibody blockade abolishes microtubule attachment, the motor continues to bind ATP and hydrolyze it via AAA1, but force cannot be transmitted to the filament, uncoupling ATPase activity from movement. Cargo adaptors such as BICD2, HOOK and dynactin still recruit dynein via tail and intermediate chains, b

Ref: Vale 2003 Cell; Roberts Cell 2013 dynein stalk MTBD binding essential for cargo translocation.

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Cytoskeleton

Which motor protein moves cilia and flagella?

Cilia and flagella motility requires active sliding filament machine within conserved axoneme evolutionary ancient. Structure nine outer doublet microtubules surrounding central pair retains dynein motors as exclusive driver of beating: outer dynein arms contain two or three heavy chains per arm inner arms more heterogeneous but also axonemal dynein isoforms both anchored to A tubule via docking complex and reaching to B tubule of neighboring doublet executing ATP hydrolysis cycle. Power stroke toward minus end attempts to slide doublets relative each other but nexin dynein regulatory complex

Ref: King, J Cell Sci 2012 – Dynein motor protein moves cilia and flagella beating machinery.

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Cytoskeleton

What is the function of the dynactin complex in dynein activity?

Although dynein can bind microtubules and hydrolyze ATP alone in vitro motility assays in vivo most physiological functions require dynactin 1 MDa multiprotein complex discovered as activator of vesicle transport from squid axoplasm. Structure comprises Arp1 actin related protein filament 8 copies forming short filament 37 nm rod barbed end capped by CapZ pointed end by Arp11 p62 p25 p27 complex shoulder arm containing p150Glued DCTN1 with N terminal CAP Gly plus basic microtubule binding domains and long coiled coil dimer interacting with dynein intermediate chain via CC1 box. Dynactin perfor

Ref: Schroer, Annu Rev Cell Dev Biol 2004 – Dynactin function linking dynein to cargo and enhancing processivity.

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Cytoskeleton

What happens to dynein when ATP binds to its AAA1 domain?

Nucleotide dependent affinity switching allows dynein stepping without dragging cargo backward and enables high force production. In high affinity state stalk MTBD binds tightly to microtubule lattice resisting detachment under piconewton loads up to 7 pN corresponding to apo or ADP bound heavy chain state with stalk helices registry CC1 CC2 in specific alignment. When ATP enters AAA1 pocket AAA ring closure pulls AAA5 and buttress subdomains sliding stalk coiled coils by half heptad altering MTBD helices arrangement converting to low affinity conformation reducing binding energy about tenfold

Ref: Imamula et al., Cell 2007 – Detachment upon ATP binding to AAA1 causes dynein release.

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