#microtubules

Basics of Cell Biology

Which cellular structure plays a role in intracellular transport and is part of the cytoskeleton?

Cytoskeleton provides scaffold and track system built from protein polymers regulated by nucleation factors, GTPases, and post-translational modifications. Actin filaments, 7 nanometer diameter polarized structures polymerizing from ATP-G-actin mediated by Arp2/3 complex branching at 70 degrees and formins nucleating straight cables for filopodia, associate with myosin II for contractility at cortex and myosin V for short-range vesicle transport near cell periphery, driving lamellipodia extension and endocytic invagination. Microtubules, 25 nanometer hollow tubes of alpha-beta tubulin dimers w

Ref: Alberts et al., Molecular Biology of the Cell, 7th ed., Chapter 16: Cytoskeleton and Intracellular Transport.

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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 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 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 protein links intermediate filaments to actin filaments and microtubules?

Plectin is a giant cytolinker of the plakin family that simultaneously recognizes actin, microtubules and intermediate filaments, integrating cytoskeletal networks into a cohesive mechanical continuum. Its structure includes an N-terminal actin-binding domain composed of two calponin-homology motifs, a central 200-nm coiled-coil rod dimerization domain, and a C-terminal repeat domain that binds vimentin, keratin, desmin and glial fibrillary acidic protein with distinct isoform specificity. Alternative splicing of first exons targets isoforms to hemidesmosomes via integrin beta4, to focal adhes

Ref: Alberts Ch 16; Fuchs & Cleveland, Science 1998 plakin plectin crosslinks IF, actin, microtubules.

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Cytoskeleton

What is the primary function of dynein in cellular transport?

Cytoplasmic dynein 1 is sole minus end directed microtubule motor powering retrograde intracellular transport complementary to kinesin superfamily anterograde transport systems establishing bidirectional highways. Structural features include two identical 530 kDa heavy chains dimerized at tail each with six concatenated AAA ATPase domains forming asymmetric ring linker that swings across ring coiled coil stalk ending in microtubule binding domain MTBD and cargo binding tail associated with intermediate light intermediate and light chains LC8 Tctex. Motor moves toward microtubule minus ends anc

Ref: Roberts et al., Nat Rev Mol Cell Biol 2011 – Cytoplasmic dynein minus end directed transport primary function.

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Cytoskeleton

How does kinesin-1 move along microtubules?

Kinesin-1 conventional motor transports cargo over long distances toward plus ends at velocities up to 1 micron per second. Heavy chain dimerization produces two motor heads coordinated via neck linker docking mechanism. Motility described as processive sliding or walking because motor maintains continuous association via at least one head bound at any moment preventing diffusion away from track essential for long range transport. ATP binding to leading head causes neck linker zipping toward plus end throwing lagging head forward to next binding site 8 nm ahead matching tubulin dimer repeat an

Ref: Vale & Fletterick, Annu Rev Cell Dev Biol – Kinesin-1 sliding walking mechanism along microtubules processivity.

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Cytoskeleton

In neurons, microtubule organization differs because:

Neuronal microtubules show compartment specific organization supporting unique transport needs required for polarity and synaptic plasticity. In axon nearly all microtubules oriented with plus ends distal toward growth cone forming uniform polarity highway kinesins deliver efficiently outward dynein returns inward without directional ambiguity allowing fast axonal transport of synaptic vesicles mitochondria. Uniformity maintained by selective transport of microtubules plus organized bundling via TRIM46 at axon initial segment plus patronin minus capping and augmin driven polarity sorting. In d

Ref: Baas et al., J Cell Sci 2016 – Neuron microtubule polarity mixed in dendrites vs uniform in axons.

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Cytoskeleton

The difference between singlet, doublet, and triplet microtubules is:

Microtubule scaffolds appear structurally specialized for distinct cytoskeletal machines requiring different mechanical properties. Singlet microtubules with 13 protofilaments forming 25 nm hollow tube make up cytoplasmic tracks for kinesin dynein transport and mitotic spindle dynamic network supporting search capture. Doublet microtubules hallmark of cilia flagella axoneme outer fibers comprise complete A tubule with 13 protofilaments and incomplete B tubule with 10 protofilaments sharing part of A wall forming fused structure. Triplet microtubules in centrioles and basal bodies add incomplet

Ref: Alberts et al., Molecular Biology of the Cell, Chapter 14 – Singlet doublet triplet difference protofilament number.

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Cytoskeleton

Which component of microtubules is responsible for its dynamic instability?

Microtubule dynamic instability reflects differential nucleotide state of tubulin subunits within polymer lattice storing strain energy. Each heterodimer contains alpha tubulin with nonexchangeable GTP permanently trapped at N site maintaining structural integrity and longitudinal interface and beta tubulin with exchangeable E site that binds GTP in soluble pool. Upon incorporation at growing plus end beta GTP forms GTP cap of one to three dimer layers that prefers straight protofilament conformation and strong lateral bonds mediated by M loop. Soon after incorporation beta GTP hydrolyzed to G

Ref: Alberts et al., Molecular Biology of the Cell 7th ed., Chapter 14: GTP beta tubulin role in dynamic instability cap model.

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