Actin, actin binding proteins, and myosin Flashcards
How does the cell use the actin cytoskeleton?
- provide structure to the cell
- provide protrusive forces at periphery of the cell (by polymerizaton of itself)
- provide mechanical attachments between adhesions and the rest of the cell
- provide contractile forces with myosin (actin is the rail which myosin walks along)
List the actin isoforms found in humans.
Humans have five actin isoforms. Listed below are several of them:
- muscle actins (skeletal, smooth muscle, cardiac)
- cytoplasmic actins (beta and gamma actins)
- muscle to cytoplasmic actins
Describe the structure of the actin monomer.
unusual subdomain structure:
- 1-3 cleft
- partly disordered subdomain 2
- nucleotide binding cleft which hydrolyzes and exchanges ATP very slowly
Describe the structure of the actin filament.
- actin monomers combine into polarized filaments
- subdomain 2 fits into the 1-3 cleft of the next actin monomer (blocking the cleft therefore prevents polymerization)
- once in the filament, ATP hydrolysis is much faster than in the free monomer
Describe in vitro actin filament assembly kinetics.
- nucleation is very slow, because four monomers must come together to initiate assembly. Nucleation is therefore concentration dependent
- elongation is very fast
- the barbed end (1-3 cleft) is the plus end. The pointed end (subdomain 2) is the minus end
- elongation can occur at either end, but is faster and more stable at plus end
- ATP hydrolysis is triggered in the filament.
Are actin filaments stable at equilibrium?
it depends on the concentration of free actin subunits. [actin]eq is a fixed value, also know as the critical concentration, and is different for either end of the actin filament.
How can we use actin polymerization to do work?
- at cellular concentrations, actin filaments are more stable that free monomers, so polymerization can be used to do mechanical work.
- mechanism of this work is brownian ratchet
- this considers fluctuations in filament bending, or fluctuations in membrane bending. allows for membranes to be moved
Explain how there is very little free actin monomers in the cell.
- actin monomers are buffered in cells
- actin binds to beta-thymosin, which buffers it, or prevents it from being added to actin filament
- actin is also bound to profilin, which is the actin nucleotide exchange factor
- rather than binding to actin itself, many other factors recruit actin by binding profilin.
How does beta-thymosin and profilin differentially affect actin filament growth?
- beta thymosin binds both ends of actin monomer and therefore prevents any actin filament growth
- profilin only binds the pointed end (subdomain 2) and therefore barbed end (1-3 cleft) addition is still possible.
- almost all elongation in cells occurs at the barbed end, or plus end.
How do beta-thymosin and profilin prevent actin filament nucleation?
- the same interfaces bound by these proteins which are needed for elongation are also needed for nucleation
- almost all cellular monomers are bound to one of these proteins, so nucleation is prevented in cells.
Describe the actin filament nucleation factors.
- nucleation factors allow the cell to initiate new actin filaments at the right place and time
- formins: rides on growing barbed end. also elongation factor
- arp2/3 complex: creates a new barbed end at a branch
- tandem WH2’s
- severing of capped filaments: cofilin, gelsolin. make two filaments from one, making new barbed and pointed ends
Describe the control of actin filament elongation.
- capping proteins (tropomodulin and arp2/3 at pointed end, capZ at barbed end) prevent elongation and depolymerization.
- elongation factors such as formins and ENA/VASP proteins control actin delivery and are anti-capping proteins, by competing with capping proteins for end-binding
- formins have profilin domains, making them good at delivering actin to growing filaments
Describe actin filament disassembly.
- filament is destabilized by ATP hydrolysis, which dominates away from the barbed end
- ADP-actin sites are possible breakage and severing sites, as proteins such as cofilin and gelsolin prefer ADP-actin
- mechanical strain from myosin can also cause breaks in the filament
- when capping ends arrest growth at the barbed end of a filament, ADP-actin begins to dominate the filament and marks actin as old and ready for recycling. The uncapped pointed end is depolymerized.
List some of the higher order actin filament assemblies.
- branched networks: lamellipodia and ruffles
- parallel bundles: filopodia, focal adhesions
- antiparallel bundles: stress fibers
Describe the branched arrays of actin filaments.
- lamellipodia are broad, flat rapidly polymerizing protrusions seen in cells and 2D environments
- they are rich in densely branched actin for which arp2/3 is responsible
- ruffles are seen in 3D environments
Describe the assembly and disassembly of lamellipodia.
- actin filaments are nucleated at the cell membrane by arp2/3
- WASp/Scar proteins are the Arp2/3 complex activators. these integrate signals to control when and where arp2/3 is activated
- growing filaments push the rest of the mesh away from the membrane and into the cell
- aging filaments are enriched in ADP-actin and are targeted for recycling by cofilin
- depolymerized actin is quickly bound by profilin, recharged with ATP, and ready to be added to the leading edge of new filaments.
Describe parallel bundled arrays of actin.
- filopodia are fast growing parallel bundles of actin that originate from branched meshes
- contain bundling factors such as fascin along their lengths that act like cross-linkers
- they have elongation factors at their tips
Describe how actin cross-linking proteins can form distinct actin assembly structures.
- different bunling proteins result in different structures.
- spacing, rigidity, and orientation of bundling surfaces matter
- parallel bundling factors are fascin, villin, and fimbrin (bind closely-spaced filaments)
- anti-parallel bundiing factor is alpha-actinin. binds to widely separated filaments
- filamin is gel-forming protein
- spectrin forms membrane skeleton
- ERM proteins bind actin filaments to cell membrane
Describe the myosin force generation cycle.
- nucleotide is not bound to myosin. it is attached to actin in rigor
- ATP binds myosin and the head is released from actin
- ATP is hydrolyzed to ADP, and the myosin head is cocked forward
- inorganic phosphate is released from myosin head, and it is able to bind actin again (force-generating)
- ADP is released and myosin head is again in rigor.
Describe the construction of stress fibers.
actin, myosin, and alpha-actinin (acts as a cross linker) rich structures which stretch across the cell and are straight, indicating they are under a lot of tension.
Describe skeletal muscle contraction.
- sarcomere: appearance of regular light and dark bands upon electron microscope imaging. called striated muscle
- similar in structure and function to stress fibers
- contain capz and tropomodulin capping proteins so that actin filaments are stable
- alpha-actinin is in Z band, cross linking
How is skeletal muscle contraction regulated?
- unregulated contraction is prevented by troponin positioning tropomyosin in the way of myosin binding sites on actin
- when calcium is stimulated and available, troponin binds calcium ions and causes tropomyosin to relax, so that myosin can bind actin and contraction can proceed.
Describe the triggering of skeletal muscle contraction.
- nerve impulse reaches T tubules, a membrane structure of muscle cells
- t tubules are closely associated with sarcoplasmic reticulum, which itself is closely associated with the whole myofibril
- an action potential in the t tubule causes calcium release from the sarcoplasmic reticulum, triggering contraction
