Describe actin filament composition & properties.
- Polar G-actin (globular) individual subunits
- Tightly binds ATP/ADP; ATP-binding clefts point towards minus end
- Assembles head-to-tail in a helical 8 nm filaments (F-actin), easily adds to plus end
Describe the importance of actin filaments.
- Provides strength & shape at cell cortex (under PM)
- Forms cell surface projections that can be dynamic or stable
Describe actin nucleation.
Spontaneous binding of two G-actin is unstable, but stabilized by the addition of a third to form a trimer.
The trimer acts as a nucleus for further G-actin addition; the number of G-actin added is proportional to their concentration.
Define critical concentration.
Concentration of free actin at which the rate of G-actin addition equals the rate of its loss.
Typically higher for the minus end than the plus end as monomers are added at the minus end with more difficulty, therefore needing a higher concentration to drive net addition.
Describe G-actin ATP/ADP binding.
G-actin can catalyze ATP hydrolysis; this happens very slowly when free (G-actin usually ATP bound) and accelerated when in a filament.
G-actin-ADP has reduced binding affinity to other G-actin and thus more likely to dissociate.
When new G-actin-ATP are added quickly enough, a stable ATP cap will form.
Describe an advantage of actin treadmilling.
Preserves filament structure and allows flexibility during many processses.
Describe the role of Arp2/3 in actin nucleation.
Arp2 & Arp3 form a heptameric complex activated by a Nucleation Promoting Factor (NPF) which causes a conformational change.
Arp2/3, which are nucleating proteins, resemble plus ends of actin filaments and therefore removes the requirement of further nucleation.
Nucleation is most efficient when Arp2/3 binds to a pre-existing filament, where the new branch grows at a 70º angle from an old branch. NPF then dissociates after formation.
Describe the composition and role of Formins in actin nucleation.
Formins are dimers forming a ring-like complex around the plus end of filaments, remaining associated to this end as the filaments grow.
Through their N-terminal tails, new actin subunits are bound and added. One of these monomers remains bound, one gets displaced which allows subunit addition.
Can nucleate new filaments & accelerate growth of existing ones.
Describe the role of Profilin in actin nucleation.
Binds to plus end of G-actin subunits, promoting binding of G-actin to the plus end of F-actin while inhibiting addition of G-actin to the minus end. Then dissociates, allowing for repetition.
Also bound by other actin-related proteins like Formins & NPF, which often occurs at the plasma membrane.
Describe the role of Thymosin in actin nucleation.
Binds to G-actin, prevents association with both ends of F-actin & lowers concentration of free actin for polymerization.
G-actin cannot bind thymosin & profilin at the same time, and release from thymosin typically means quick binding to profilin.
Describe the process of cell migration in relation to actin assembly.
Utilizing the Arp2/3 pathway, extracellular cues (i.e. growth factors) activate NPF at the leading edge of the cell through GTPases like Rac1 & Cdc42. Spatial cues are provided through PIPs like PI(4,5).
The Arp2/3 complex is activated by NPF, driving plus-end growth and new nucleation events at a 70º angle to pre-existing filaments.
Nucleation at ATP-bound regions of a filament pushes the membrane forwards as a sheet.
Describe the role of CapZ in relation to actin filaments.
Filament capping protein which binds plus ends to prevent subunit addition/loss. The minus end remains unbound, and thus can still gain/lose subunits. The overall rates of filament assembly/disassembly are lower.
Binds plus ends of new filaments at the cell’s leading edge.
Many short branches are better than long filaments. CapZ prevents growth of longer filaments so that short branches can form.
*Concentrates G-actin for Arp2/3-mediated nucleation.
Describe the roles of Fimbrin and α-actinin in relation to actin filaments.
Both are bundling proteins widely distributed in cells.
Fimbrin is a small polypeptide with two actin-binding sites that cross-links actin into tight bundles. Important for filopodia formation.
α-actinin is a homodimer with two actin-binding sites spaced further apart. Cross-links actin more loosely, which leaves room for other proteins. Important for stress fiber formation.
Describe the role of Filamin in relation to actin filaments.
Filamin is a homodimer w/ two actin-binding domains. These domains are spaced by a v-shaped linkage. Cross-links filaments so they are oriented at near-right angles.
Forms a loose & flexible gel, important or lamellopodia formation.
Describe the role of Gelsolin in relation to actin filaments.
Filament severing protein.
Two actin-binding subdomains, one binding on the filaments’ exposed surface and another binding the normally hidden inside surface.
Binds to a side of the filament until a small gap is created (by thermal fluctuation), when inserts into the gap to break the filament.
Once severed, gelsolin remains bound to the end, prevents growth/shrinkage.
Describe the role of Cofilin in relation to actin filaments.
Small globular protein to bind along the side of filaments, causing it to twist more tightly than normal. Stress weakens subunit binding.
Binds better to ADP-actin, so helps to dismantle older filaments. Important for cell migration.
Cofilin activity limits acting branching to the leading edge, in tandem with profilin/thymosin binding to recycled G-actin.
Define the role of myosin.
Actin-associated motor protein dimer (two light, two heavy chains) to generate force and movement.
Uses ATP to walk along actin filaments, always towards the plus end.
Define important aspects of myosin movement.
Tightly bound to actin w/o ATP/ADP.
ATP binds head region, triggers conformation change to reduce actin affinity, cause dissociation.
Hydrolysis of ATP causes displacement/cocking of head.
Rebinding of actin causes release of phosphate, generates power stroke & releasing ADP. Head moves closer to plus end.
Each head region independent.
Describe the role of troponin and tropomyosin in actin binding.
Troponin, activated by action potentials triggering calcium release from the ER, pulls on tropomyosin which originally blocks myosin binding sites on actin filaments.
This then allows actin to bind myosin.
Describe non-muscle myosin movement.
Transiently assembled with myosin. This assembly is regulated by phosphorylation done by myosin light chain kinase.
This allows head regions to bind to actin, releasing tail regions which allow bipolar filament assembly.
Essential for cell movement & shape changes (i.e. cytokinesis).
Describe the role of microtubules.
Intracellular transport by arrays from nucleus to PM; generates the mitotic spindle.
Describe microtubule structure.
Heterodimer subunits of 𝛼-tubulin and β-tubulin; 𝛼-tubulin is GTP-bound, β-tubulin can bind GTP or GDP.
𝛼β-tubulin subunits assemble head-to-tail
into protofilaments, with 𝛼-tubulin towards minus end, β-tubulin towards plus end.
13 protofilaments assemble into a hollow microtubule. β-tubulin binds to 𝛼-tubulin above it, and 𝛼-to-𝛼 and β-to-β binding laterally.
Describe microtubule formation.
Unlikely to happen spontaneously; requires nucleation.
Plus end grows & shrinks more quickly than the minus end.
Differentiate actin and microtubule formation.
Actin uses ATP/ADP, microtubules/tubulin uses GTP/GDP.
Both follow the same trends.
