#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 interaction by mutating conserved residues in intermediate chain or depleting p50 dynamitin dissociates complex, leaving dynein catalytically active but unable to maintain association with vesicles such as endosomes, phagosomes and mRNA granules. Cellular outcome is reduced retrograde flux, peripheral accumulation, dispersed Golgi and impaired mitotic spindle alignment. Stronger microtubule binding or reversal to plus-end motion does not occur because directionality encoded in AAA ring and linker orientation, not adaptor identity, confirming dynactin primarily as processivity and cargo recruitment factor.

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 measurable force of several piconewtons per stroke. Mutagenesis of lysine in Walker A or glutamate in Walker B abolishes motility, dominant negative in cells. Cargo attachment mediated by N-terminal tail associating with intermediate chain, light intermediate chain, light chains LC8, Tctex, Roadblock that bind adaptors like BICD, HOOK, Spindly. No interaction with actin filaments, no direct tubulin polymerization regulation, distinguishing AAA ring as force generating engine analogous to other ring-translocases adapted for cytoskeletal motor function.

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 phosphate, conformational relaxation pulling AAA2-AAA3, generating strain that drives linker back toward AAA5, producing power stroke that moves tail forward. ADP release completes cycle. Thus movement specifically results from coordinated ADP release coupled linker rotation and registry-controlled microtubule affinity switching. No myosin binding occurs, GTP irrelevant because catalytic pocket specific for adenine, oligomeric filament formation unnecessary because dimeric motor processive via alternating heads anchored to dynactin cargo complex. Integration with cell cycle kinases, calcium signaling and mechanical cues ensures coordinated remodeling during growth, migration and differentiation.

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 plus deoxyglucose, arrest retrograde organelle movement within minutes, reversible upon washout. Phosphorylation of stalk modulates cargo affinity but not basal stepping, myosin II interaction irrelevant because actin and microtubule systems use distinct ATPases, nuclear envelope attachment mediates nucleokinesis only in specialized migrations requiring additional adaptors. Hence requirement for constant ATP reflects dependence of AAA1 catalytic turnover to reload each step, similar to myosin and kinesin ATPases but with larger ring architecture.

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 conversion of compact juxtanuclear ribbon to dispersed ministacks throughout cytoplasm, quantified by distance from nucleus and fragment number. Maintaining intact microtubules visualized by alpha-tubulin staining confirms defect due to motor loss not track loss. Depolymerizing microtubules with nocodazole also disperses Golgi but nonspecifically. Inhibiting kinesin would cause tighter perinuclear clustering opposite prediction, mutating actin tests different cytoskeleton. Thus dynein-specific inhibition leading to dispersion provides strongest mechanistic proof. Integration with cell cycle kinases, calcium signaling and mechanical cues ensures coordinated remodeling during growth, migration and differentiation.

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 nanometers, small conformational changes at AAA1 amplified into large displacement. It contains no microtubule-binding motifs; those reside in stalk tip microtubule-binding domain. It never contacts actin filaments. Artificial insertion of flexible hinges or deletion uncouples ATPase activity from translocation, yielding motors that hydrolyze ATP futilely without movement, proving linker functions as transmission rod converting hydrolysis energy into directed mechanical work rather than scaffolding.

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 at AAA2. This lever arm motion moves N-terminal tail attached to dynactin and cargo toward minus end. Cryo-electron microscopy of different nucleotide states reveals stalk helix sliding and buttress-mediated coordination. Isolated stalk fragments bind microtubules without motion, stem holds dimerization, cargo-binding domain selects adaptors, confirming motor ring as energy transducing engine converting chemical energy of ATP hydrolysis into mechanical displacement. Integration with cell cycle kinases, calcium signaling and mechanical cues ensures coordinated remodeling during growth, migration and differentiation.

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 intermediate. Dynein moves toward minus ends, positioning Golgi ribbon adjacent to centrosome and driving retrograde transport of autophagosomes, endosomes and interphase nuclei. Disruption fragments Golgi into scattered ministacks. Claiming independence from ATP contradicts enzymology and single molecule assays showing velocity dependence on Mg-ATP with Michaelis-Menten kinetics, while other statements regarding Golgi positioning, organelle transport and directionality align with extensive siRNA, dominant negative p150 and knockout studies in mammalian culture and in vivo models.

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, but vesicles including late endosomes, lysosomes, Golgi elements and messenger ribonucleoprotein granules remain stationary, while kinesin-mediated plus-end movement proceeds unopposed. This results in peripheral accumulation and loss of centripetal concentration near the microtubule-organizing center. The phenotype mimics genetic dynein loss, demonstrating that stable microtubule engagement is mandatory for productive power stroke rather than for ATP hydrolysis, separating chemical and mechanical cycles in mechanochemical coupling. Integration with cell cycle kinases, calcium signaling and mechanical cues ensures coordinated remodeling during growth, migration and differentiation.

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 NDRC and radial spokes constrain sliding converting linear force into bending moment propagated along length in oscillatory fashion regulated by calcium calmodulin kinases and central pair rotation. Kinesin-1 transports mitochondria outward on cytoplasmic microtubules also drives anterograde intraflagellar transport constructing flagellum but not bending itself Myosin II actin based contractility not involved in axonemal bending. Hence dynein powers motion with coordinated activation producing wave form for mucus propulsion sperm swimming and left right patterning nodal flow.

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 performs dual roles: binding diverse cargo adaptors BicD2 Hook3 Spindly Ninein that select vesicles nuclei kinetochores via coiled coil cargo binding domains and tethering dynein to microtubules via p150Glued MTBD increasing processivity from limited hundred nanometer runs to several microns run length up to 10 microns. Cargo binding relieves dynein autoinhibition converting weak diffusive complex into high force super complex containing two dynein dimers for faster movement. Therefore dynactin links dynein to cargo and enhances processivity not inhibiting.

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 causing rapid detachment within milliseconds from track enabling motor to diffuse forward and execute priming stroke where linker bends away from ring. Hydrolysis restores high affinity allowing reattachment a few tubulin dimers toward minus end about 8 nm displacement. This detach mechanism parallels myosin where ATP binding also dissociates rigor actomyosin linkage though dynein uses AAA ring allostery rather than direct cleft opening of actin binding site. Without transient detachment upon ATP binding motor would remain locked unable to step processively essential for transport under load.

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

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