Cytoskeleton

Cytoskeleton

The structural integrity of epithelial tissues is maintained primarily by:

Epithelial barrier maintenance depends on keratin-based intermediate filament network that spans cytoplasm from perinuclear region to desmosomes connecting adjacent cells and hemidesmosomes anchoring basal cells to underlying basement membrane via integrin alpha6beta4. Keratins K5/K14 heterodimers in stratified epithelia and K8/K18 in simple epithelia polymerize into tonofilaments bundled by plectin and desmoplakin, providing high tensile strength resisting mechanical rupture during stretch, compression and abrasion. Phosphorylation by PKC and p38 under stress increases solubility to allow remodeling during wound healing. While actin concentrated at adherens junctions supports adhesion and contractility via myosin II, and microtubules direct vesicular traffic to apical surfaces, neither provides long-term load bearing like intermediate filaments. Genetic evidence shows missense keratin mutations cause epidermolysis bullosa simplex with cytolysis upon mild trauma, whereas keratin knockout mice display tissue frailty. Therefore intermediate filaments serve as principal determinants of epithelial structural integrity through junction-linked networks. Integration with cell cycle kinases, calcium signaling and mechanical cues ensures coordinated remodeling during growth, migration and differentiation.

Ref: Fuchs Annu Rev Biochem; keratin IF at desmosomes hemidesmosomes provides epithelial mechanical resilience.

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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

Which of the following is an intermediate filament protein found in neurons?

Neuronal cytoskeleton requires robust ten nanometer filaments to stabilize long processes subjected to bending and tension during growth and circuitry formation. Neurofilament proteins comprise heteropolymers of NF-L light sixty one kDa, NF-M medium one hundred two kDa and NF-H heavy one hundred twelve kDa subunits, each with conserved central rod domain forming coiled-coil dimers and variable C-terminal tails enriched in Lys-Ser-Pro repeats heavily phosphorylated to extend as sidearms regulating interfilament spacing and axonal diameter, which directly influences conduction velocity. Alpha-internexin and peripherin coassemble in developing and peripheral neurons. Lamin A is nuclear lamina present in differentiated somatic cells, not neuronal process specific; vimentin expressed in progenitors then replaced; actin forms seven nanometer microfilaments mediating growth cone motility. Immunostaining for neurofilaments distinguishes neuronal tumors and marks axonal injury where accumulation forms spheroids. Mutations in NF genes or associated kinases perturb axonal transport and are linked to amyotrophic lateral sclerosis, Charcot-Marie-Tooth and Parkinson pathologies emphasizing functional specialization.

Ref: Liem J Cell Biol; neurofilaments NF-L/M/H heteropolymers regulate axon caliber, neuron-specific IF.

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Cytoskeleton

What is a major function of intermediate filaments in cells?

Intermediate filaments constitute one of three major cytoskeletal polymers alongside actin microfilaments and microtubules, classified by ten nanometer diameter and apolar rope-like assembly through parallel coiled-coil dimers antiparallel tetramers laterally associating into filaments without polarity. Major classes include keratins in epithelia, vimentin in mesenchyme, desmin in muscle, glial fibrillary acidic protein in astrocytes, neurofilaments in neurons, lamins in nucleus. Their primary mechanical role is tensile resistance to shear and stretching, absorbing deformation through flexible networks anchored at cell-cell desmosomes and cell-matrix hemidesmosomes linked by plectin and desmoplakin. This protects tissues experiencing repetitive strain, as seen in blistering diseases upon keratin mutation. They do not generate ATP, actively transport vesicles, or directly regulate microtubule growth, though they cross-talk via plectin. Knockout studies show fragility rather than motility defects, indicating specialization toward structural support enabling tissue cohesion, nuclear scaffolding for chromatin organization and integration of mechanical signals over long timescales beyond dynamic actin and microtubule remodeling.

Ref: Herrmann J Cell Sci; IF 10nm tensile strength, anchored at desmosomes hemidesmosomes, no polarity.

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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

In cilia, what keeps the doublet microtubules from sliding past each other completely?

Motile cilia contain nine plus two axoneme where outer doublets composed of A and B tubules slide relative to each other when axonemal dynein arms anchored on A tubule bind and walk toward minus end of neighboring B tubule, activated in coordinated wave from base to tip. Unrestrained sliding would cause doublets to telescopically extend apart, disrupting structure. Restraint provided by nexin links now recognized as nexin-dynein regulatory complex, elastic proteinaceous bridges composed of DRC1-4 and associated proteins connecting adjacent doublets circumferentially every ninety six nanometers. They limit sliding amplitude to about sixty to seventy nanometers per beat cycle and store elastic energy that recoils to produce bending. Additional resistance from radial spokes transmitting signals from central pair regulates dynein activity. Electron tomography shows N-DRC as hook-like structures. Protease digestion generating sliding disintegration assay causes ATP-induced complete doublet separation, confirming structural role. Kinesin does not reside in axoneme, actin absent, phosphorylation tunes waveform not tethering.

Ref: Porter & Sale J Cell Biol; nexin-DRC elastic links limit sliding, convert sliding to ciliary bending.

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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

What happens to microtubule-based transport if dynein function is inhibited?

Intracellular transport operates through antagonistic plus-end kinesins and minus-end dynein along same microtubule tracks. Under normal conditions kinesin-1 and kinesin-3 carry cargo outward toward cell periphery, dynein-dynactin returns cargo toward centrosome where microtubule minus ends converge. Acute inhibition by overexpression of dynamitin CC1 fragment dissociating dynactin, treatment with ciliobrevin D blocking AAA1 ATPase, or inducible degron of dynein heavy chain abolishes retrograde motion while anterograde motion persists. Consequences include dispersion of Golgi, accumulation of endosomes and lysosomes at tips of processes, impaired autophagosome-lysosome fusion and increased peripheral residence of signaling endosomes. Microtubules remain polymerized because dynein inhibition does not alter tubulin GTP cap dynamics. Actin compensates only for short-range delivery via myosin V, insufficient for long-range concentration. Consequently phenotype is peripheral clustering rather than enhanced centripetal movement, illustrating tug-of-war principle governing organelle positioning and balance of opposing motors. Integration with cell cycle kinases, calcium signaling and mechanical cues ensures coordinated remodeling during growth, migration and differentiation.

Ref: Schroer Annu Rev Cell Biol; dynein inhibition causes peripheral cargo accumulation, Golgi dispersion.

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