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 rem

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 in

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-in

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

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

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

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

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 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

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

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

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