Chapter 18: MICROFILAMENTS
Actin properties:
· The most abundant protein in eukaryotic cells; highly conserved.
· Types – G-actin is soluble monomers; F-actin are polymerized actin filaments; ATP and ADP binf to G and F actin at the ATPase fold and hold the lobes of the protein together (18-2a).
· G-actin – assembles into F-actin polymers; cations must be present for polymerization; ATP increases the rate of polymerization, but is not required for polymerization.
· F-actin – helical polymer of G-actin (18-2c); helix is polar; myosin decoration experiments show that the pointed (-) end is where the ATP binding site is exposed and the barbed (+) end contacts the neighboring actin subunit(18-3).
Actin structures:
· Bundles and networks; bundles are actin filaments closely packed in parallel arrays (18-5a); networks are loosely packed 2 or 3 dimensional structures (18-5b); bundles and networks are held together by specific actin crosslinking proteins (T18-1).
· Erythrocyte cytoskeleton is comprised of short (~14 subunit) actin filaments, spectrin and associated protein that interact with glycophirin in the membrane (18-7).
· Platelets, muscle and epithelia use long microfilaments in the cytoskeleton (18-8); Platelets use filamin to bind actin to the Gp-1b-IX clotting factor receptor in the membrane; musle cells use dystrophin to bind microfilaments to the glycoprotein complex in the membrane; epithelia use ezrin and EB50 to bind microfilaments to the CFTR receptor in the membrane.
· Actin bundles at membranes – support projections such as microvilli and filopodia (18-10); microvilli increase cell surface as in brush border epithelia; filopodia attach to solid surfaces.
Actin assembly:
· In vitro assembly occurs in there phases – lag, elongation and steady state (18-11); G-actin concentration (Cc+ and Cc-) and ATP determine elongation ability and rate (18-12); elongation at + (barbed) end is 5-10X faster than the – (pointed) end; capping proteins specifically block the + or – ends (18-13b); treadmilling occurs when the G-actin concentration is between the Cc+ and the Cc- (18-13c).
· Toxins stabilize or destabilize actin filaments; cytochalasin binds the + end, blocks polymerization and tilts the equilibrium towards depolymerization; phalloidin binds to the junctions between polymerized actin subunits and stabilizes microfilaments.
· Regulation of actin polymerization in vivo is controlled by G-actin binding proteins (18-15); thymosin b4 (Tb4) sequesters G-actin, enabling G-actin to be present a > Cc-, thus inhibiting actin assembly; profilin promotes filament assembly by binding G-actin and assisting in polymerization; profilin is sequestered by a membrane protein, and released upon external signals to promote actin assembly; filament lengths are controlled by severing and capping proteins (T18-2); severing proteins break the filament, bind to the + end and inhibit addition; capping proteins stabilize actin filaments by binding to the end and inhibiting depolymerization.
· Cell movements – acrosome reaction (18-17); cell movement (18-19a).
Myosin:
· Types and structure – myosin I, II, V and their functions (18-20a); tail domain involved in assembly (18-21); head involved in actin binding, ATP hydrolysis and movement (18-22); conformational changes driven by ATP hydrolysis mediate movement (18-25).
Muscle contraction:
· Muscle types and structure – striated and smooth muscle (18-26); organization of actin and myosin in the sarcomere (18-27); actin capping proteins in the sarcomere (18-28); sliding filament model of contraction (18-29); titin and nebulin stabilize the sarcomere (18-30).
· Skeletal muscle contraction – sarcoplasmic reticulum structure (18-31a); Ca2+ triggers muscle contraction (18-31b,c,d); mechanism of skeletal and smooth muscle contraction via Ca2+, troponin and tropomyosin (18-32a,18-33 ); myosin dependent mechanisms for regulating muscle contraction (18-34).
Actin and myosin in non-muscle cells:
· Epithelial cells (18-35); function during cytokinesis (18-37a); ctoplasmic streaming (18-40).
· Cell movement – steps during movement (18-41); molecular events at the leading edge of cell movement (18-42).