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#actin filaments

14 public questions tagged with this topic.

Cytoskeleton

Intermediate filaments differ from actin filaments because:

Three cytoskeletal filament systems differ chemically mechanically and kinetically defining cytoplasmic organization. Actin filaments polar 7 nm helical polymers of ATP G actin assembling barbed plus fast pointed minus slow exhibiting treadmilling ATP hydrolysis dependent turnover myosin motors. Microtubules polar 25 nm tubes of GTP tubulin dimers showing dynamic instability and kinesin dynein transport highways plus end tracking. Intermediate filaments 10 nm rope like nonpolar exceptionally stable structures without polarity unique to metazoans. Lack of polarity stems from assembly pathway: parallel dimers form antiparallel tetramers neutralizing polarity tetramers associate laterally into unit length filaments of 60 nm that anneal end to end producing filament without distinct plus minus ends no nucleotide binding required for polymerization. Consequently motor proteins kinesin dynein myosin have no directional track on intermediate filaments for directional walking; instead filaments serve as stable scaffold anchored at desmosomes hemidesmosomes providing tensile strength resisting shear stress up to 300 percent strain. Stability greater than actin or microtubules not more dynamic no motor function no ATP requirement.

Ref: Alberts et al., Molecular Biology of the Cell, Chapter 16: Intermediate filaments lack polarity distinction from actin and microtubules.

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Cytoskeleton

What is the role of Myosin V in intracellular transport?

Myosin V is prototypical class V unconventional myosin that operates on actin filaments not microtubules serving local delivery after long range microtubule transport. The molecule is dimeric with two motor heads each containing six calmodulin or essential light chain IQ motifs forming a 24 nm lever arm, a coiled coil stalk and a globular tail domain that binds cargo adaptors such as Rab11, Rab27, melanophilin and Myo4p binding proteins. It moves processively hand over hand toward barbed plus end near plasma membrane hydrolyzing one ATP per step with coordinated gating that prevents simultaneous detachment. The motor center of mass advances 36 nm per ATP matching actin helical repeat of 13 monomers while each head swings about 72 nm. This architecture allows organelles, secretory vesicles, endoplasmic reticulum tubules, mRNA granules and melanosomes to traverse dense cortical actin networks where kinesin and dynein cannot operate. Regulation involves cargo binding relieving autoinhibition, calcium calmodulin influencing lever stiffness and coincidence detection with Rab GTPases ensuring correct delivery and recycling.

Ref: Vale, Cell 2003; Hammer & Sellers, Nat Rev Mol Cell Biol 2012 – Myosin V processive transport on actin filaments.

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Cytoskeleton

Myosin head movement along actin filaments is driven by:

Myosin motor cycle couples ATP hydrolysis to filament sliding via conformational changes in conserved motor domain. In absence of ATP myosin head strongly bound to actin in rigor state, stereospecific binding between actin helix and myosin cardiomyopathy loop. Binding of ATP to P loop pocket between upper and lower 50 kilodalton subdomains induces opening of actin binding cleft reducing affinity thousand fold leading to detachment. While detached ATP hydrolyzed to ADP Pi mediated by Switch I Switch II closing around gamma phosphate, triggering recovery stroke where lever arm moves about 90 degrees to pre power stroke conformation storing elastic energy. Myosin ADP Pi rebinds actin weakly, phosphate release gates transition to strongly bound state closing cleft, producing power stroke large rotation of converter domain swinging lever arm 5 to 10 nanometer dragging actin. ADP release returns to rigor awaiting new ATP. Thus detachment powered by ATP binding, priming by hydrolysis, force generation by Pi release, not calcium influx, GTP hydrolysis, tail phosphorylation primary driver of movement.

Ref: Alberts et al., Molecular Biology of the Cell, 7th ed., Chapter 16: Myosin ATP Hydrolysis and Movement.

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Cytoskeleton

Which of the following is NOT a function of actin filaments?

Functional specialization within cytoskeleton explains why certain cellular processes are drug sensitive. Actin filaments 7 nanometer double helices participate in cortical tension, formation of lamellipodia filopodia, contractile ring assembly during cytokinesis powered by myosin II, and cytoplasmic streaming in plant Chara driven by myosin XI moving organelles along actin cables at up to 50 micron per second. Microtubules 25 nanometer tubes made of alpha beta tubulin heterodimers provide tracks for chromosome segregation; mitotic spindle assembled from centrosomes, kinetochores attach via Ndc80 complex to dynamic plus ends, and motors kinesin 5 Eg5 generating outward force plus dynein pull separate sister chromatids in anaphase. Vesicle transport using actin myosin occurs short range near cortex but bulk chromosome movement depends on tubulin GTP hydrolysis providing dynamic instability and error correction through Aurora B mediated detachment. Colchicine nocodazole disrupting microtubules block mitosis with metaphase arrest, while cytochalasin blocking actin spares chromosome segregation. Hence chromosome segregation not actin dependent process.

Ref: Lodish et al., Molecular Cell Biology, 9th ed., Chapter 19: Microtubules in Chromosome Segregation vs Actin.

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Cytoskeleton

Which of the following proteins stabilizes actin filaments by binding along their length?

Skeletal muscle thin filaments extend from Z disc to near M line, length precisely regulated 1.0 micrometer in cardiac, 1.1 to 1.3 in skeletal for optimal overlap with thick filaments for force generation. Nebulin, 600 to 900 kilodalton protein encoded by NEB gene, composed of 185 tandem nebulin repeats of 35 amino acids each predicted to contact single actin monomer plus C terminal SH3 domain anchoring at Z disc via interactions with CapZ, titin Z repeats and desmin intermediate filaments. By spanning entire thin filament, nebulin acts as molecular ruler templating polymerization length during myofibrillogenesis and stabilizing filament against lateral sliding and cofilin mediated severing. Structural studies show repeats align along actin helical groove stabilizing interaction with tropomyosin troponin. Human null mutations cause nemaline myopathy with variable thin filament length and weakness. CapZ caps barbed plus end, tropomodulin caps pointed minus end, gelsolin severs, but lateral stabilization along whole shaft best described by nebulin ruler function important for muscle structure.

Ref: Alberts et al., Molecular Biology of the Cell, 7th ed., Chapter 16: Nebulin and Thin Filament Stabilization.

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Cytoskeleton

Actin filaments interact with which motor protein for intracellular transport?

Two distinct superfamilies of cytoskeletal motors evolved to walk along filaments using ATP hydrolysis. Myosin motors share N terminal head motor domain containing actin binding interface including helix turn helix, cardiomyopathy loop, P loop ATPase, Switch I II for gamma phosphate sensing and lever arm with IQ motifs binding calmodulin light chains. Upon interaction with F actin 7 nanometer helical filament, head undergoes conformational cycle releasing phosphate driving swing of lever arm producing force along filament. Diverse isoforms specialize: myosin II non processive forming bipolar filaments for contraction, myosin V processive dimer for cargo, myosin I single headed tension sensor. In contrast dynein heavy chain AAA plus ring and coiled coil stalk binds microtubule 25 nanometer tubule, kinesin motor domain with tubulin binding loops moves along protofilaments. Nexin is non motor linker between doublet microtubules in axoneme. Therefore actin filament based intracellular transport and contractility specifically require myosin family as dedicated actin associated motor providing directional movement.

Ref: Alberts et al., Molecular Biology of the Cell, 7th ed., Chapter 16: Myosin Motors and Actin Filaments.

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Cytoskeleton

Which cytoskeletal component is responsible for intracellular transport of organelles?

Cells rely on cytoskeletal tracks differentiated by cargo range. Short range cortical movements beneath plasma membrane up to few microns use actin filaments 7 nanometers for myosin driven transport, such as myosin Va carrying melanosomes or myosin VI endocytosis. Long range interphase transport spanning tens microns from centrosome near nucleus to periphery requires rigid persistent tracks with polarity uniform over large distances, fulfilled by microtubules 25 nanometers hollow cylinders of 13 protofilaments built from alpha beta tubulin heterodimers. Microtubules exhibit dynamic instability allowing search and capture but stabilized subsets marked by acetylation detyrosination serve as preferred highways. Kinesin motors, kinesin 1 2 3 families move toward plus end delivering mitochondria, ER to Golgi vesicles, mRNA granules, while cytoplasmic dynein with dynactin adaptor moves retrograde toward minus end returning lysosomes and signaling endosomes. Intermediate filaments lack motors and polarity, myosin filaments contractile not tracks. Therefore intracellular organelle long distance trafficking depends predominantly on microtubule network and associated motors.

Ref: Lodish et al., Molecular Cell Biology, 9th ed., Chapter 19: Microtubules and Organelle Transport.

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Cytoskeleton

What is the function of cofilin in actin filament dynamics?

Disassembly pathways must balance assembly to allow shape change during migration and division. Cofilin, also called ADF, 15 to 19 kilodalton protein conserved from yeast to mammals, directly severs and depolymerizes actin. Binding preference for ADP F actin, prevalent in older filament regions away from ATP cap, induces 5 degree reduction in helical twist per subunit from 167 to 162 degrees, weakening longitudinal contacts and increasing filament compliance. At boundaries between cofilin decorated and bare segments mechanical stress concentrates leading to breakage. Severing multiplies filament number, creating new barbed ends that can elongate if profilin actin available and pointed ends that depolymerize, increasing turnover. Cofilin activity suppressed by phosphorylation at Ser3 mediated by LIMK downstream of Rho ROCK and PAK signaling, reactivated by slingshot and chronophin phosphatases. Mutations preventing phosphorylation cause loss of stress fibers and exaggerated lamellipodia. Unlike nucleation factors Arp2/3 formin nucleating, unlike thymosin sequestering, cofilin primary role severing and increasing filament turnover essential for motility.

Ref: Lodish et al., Molecular Cell Biology, 9th ed., Chapter 18: Cofilin Severing and Actin Turnover.

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Cytoskeleton

The process of treadmilling in actin filaments is accelerated by:

Cellular actin turnover measured by fluorescence recovery after photobleaching half time 30 seconds versus minutes in vitro requires accelerated disassembly and reassembly. Treadmilling rate defined by addition at barbed end balanced by loss at pointed end, but spontaneous off rate modest. Accessory proteins increase both fluxes dramatically. Cofilin family severs ADP rich filament segments cooperatively, generating numerous short filaments exposing many pointed ends that depolymerize rapidly ten fold higher off rate. Cofilin also promotes debranching and filament twisting destabilizing contacts. Resulting ADP G actin released bound to cofilin has low affinity for polymerization. Profilin then catalyzes nucleotide exchange by opening actin nucleotide cleft lowering ADP affinity hundred fold, converting to ATP G actin. Profilin ATP actin complex adds preferentially to barbed end, especially when delivered by formin FH1 polyproline tracts, completing cycle. Together they accelerate subunit flux 50 to 100 fold. Tubulin builds microtubules, dynein microtubule motor, myosin generates contraction not treadmilling, nebulin stabilizes muscle thin filaments, tropomyosin protects from cofilin, none accelerate both ends simultaneously like cofilin profilin pair.

Ref: Alberts et al., Molecular Biology of the Cell, 7th ed., Chapter 17: Actin Treadmilling and Cofilin Profilin Regulation.

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