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Section 3

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Western University
Biology 2382B
Sashko Damjanovski

SECTION 3 Actin Filaments – Microfilaments • Cortical function • Cell shape and movement • Tubulin is the thickest cytoskeletal component, microfilaments and actin are the thinnest • Tubulin forms long tubes that are unbranched and straight • Actin is different in that it can form branches • Actin has a cortical function – found at the cortex (cortex = outside) • Centrosome (main MTOC) is close to the nucleus o Forms MTs that go from the center towards the outside of the cell o These MTs take things from the center to the surface o Once it gets to the surface, it is actin that is underlying the membrane • Actin vs. microtubules o Actin can form networks and bundles o MTs can somewhat form bundles (e.g., in nerve axons, MAPs), but they usually do not o Actin bundles are quite common (e.g., microvilli) o Actin can branch out into networks Actin Based Structures • Epithelial cells – skin cells, trachea cells lining esophagus o Have to form a barrier and have to transport things across them o Have to have different structures • Migrating cells – e.g., filopodia, lamellopodium – associated with stress fibers • Muscle – actin and myosin (motor proteins) contract, move • Non-muscle functions – phagocytosis, moving things around (endocytosis and exocytosis), contractile ring during cytokinesis Actin: Structure • Several isoforms of actin: a, b and g o Though for our purposes, these are all the same o All found in globular/G-actin form o Basic unit is a monomer – not found in dimers o Monomer is called globular actin or G- actin • G-actin (globular) polymerizes into F-actin (filamentous) microfilaments • Has four lobes o Has a cleft that gives it polarity, cleft is an ATP-binding site o Has a “top” and a “bottom” – one side is different from another • When actin polymerizes, all of the actin molecules polymerize in the same orientation o Cleft is always facing the same direction in that filament o The filament has polarity • Recall that tubulin is always a-b, it is polar because one side is always a and the other side is always b • Actin always has a on one side, cleft on the other side – when these molecules polymerize, cleft is always in the same position • Polymerized actin is a double helix (goes from monomer to filament) and has polarity • (-) end where the cleft is present, (+) end where the cleft is not present Actin: Polarity • Polarity allows for independent regulation of actin assembly or disassembly (“decorated” with myosin S1) • Adding S1 myosin can bind to and give it a distinct, arrowhead pattern (“decoration”) • S1 is a part of actin’s motor protein • Decorating actin with S1 myosin stabilizes actin – less likely to polymerize and depolymerize Polymerization of actin filaments occurs preferentially at the (+) end • Faster polymerization at (+) side • As with microtubules, same idea with critical concentration – need to be above certain critical concentration to get polymerization • Recall identical graph with tubulin • In vitro system, start adding subunits (in this case, actin monomers); concentration of monomers goes up until you reach critical concentration, where you get polymerization ActinAssembly The polymerization of actin filaments occurs in three phases, starting with a pool ofATP-bound G-actin monomers. During the first phase, the G-actin monomer is slowly aggregated until a stable nucleus is formed. In the second phase, the nucleus rapidly elongates by addition of monomers to both of its ends. The (+) end elongates faster than the (-) end. The result F-actin filament reaches the third phase, when its ends are in steady state equilibrium with the G-actin monomers.At steady phase, the equilibrium concentrate of the pool of unassembled units is called the critical concentration, or cc. adding more G-actin raises the monomer concentration above cc, favoring further polymerization.At monomer concentrations below cc, F-actin will depolymerize. • Once above the cc, the presence of some sort of nucleus helps polymerization • G-actin monomer has to be inATP form to polymerize • As they polymerize, there is polarity (cleft is always in the same position) • First have to form nucleus – it is on this nucleus that things will grow • If above the cc, first thing to occur will be to form nuclei o If they start off with nuclei and add monomers, elongation will occur quickly o If they start off with monomers, need to form nuclei first; will take longer to reach elongation phase Actin Polymerization and Critical Concentration • Need G-actin bound toATP for polymerization • The (-) and (+) ends of actin have different cc o If above both of their cc, polymerization at both ends o If below both of their cc, depolymerization at both ends • (+) end cc is 0.12 μm • (-) end cc is 0.6 μm • Need 5 times as many subunits to polymerize at the (-) end • If cc is between (e.g., 0.4 μm of subunits available) – polymerize at (+) end, depolymerize at (-) end o Tread milling phenomenon occurs when concentration is between both critical concentrations o Distance from monomers to (+) end is increasing while distance from monomers to (-) is decreasing o While monomers aren’t necessarily moving, they appear to be doing so (appear to be on a “treadmill”) • Treadmilling can occur in tubulin; like actin, tubulin has different cc at (+) and (-) ends o Can, but does not, occur because (-) end is almost always capped in MTOC o Almost no cases where tubulin treadmilling occurs Regulation ofActin Polymerization • Cellular concentration of G-actin is 400 mM, but Cc is 0.12mM • All should be polymerized, but isn’t – cell regulates that, it wants a pool of ready actin so it cannot polymerize • Thymosin sequesters actin and provides a reservoir o When G-actin binds to thymosin, it is removed from the high concentration of free G-actin o When bound to thymosin, G-actin is not free; it is not available for polymerization, not part of that concentration o By regulated thymosin, cells can regulate polymerization o Most of the free G-actin in the cell is bound to thymosin, it serves as a reservoir • Profilin promotes actin polymerization by charging G-ADP into G-ATP actin o Energizes the monomer o NeedATP form for polymerization • Cofilin enhances depolymerisation o Usually at the (-) end Actin Capping Proteins – BlockAssembly and Disassembly • Can also regulate polymerization by capping the ends of the growing filament o Block assembly or disassembly by capping the (+) end, (-) end, or both o Two natural proteins/molecules involved here: CapZ and Tropomodulin • CapZ binds to the (+) end and prevents growth o Leaves the (-) end free, which can grow or shrink, depending on concentration of free monomers • Tropomodulin binds the (-) end and prevents disassembly o Leaves the (+) end free, which can also grow or shrink, depending on concentration of free monomers • Actin-disrupting drugs o Cytochalasin - depolymerizes actin filaments and affects anything that requires actin o Phalloidin - stabilizes actin filaments  Can also be used as a label since it specifically binds to actin Assembly and Branching • Formins assemble unbranched filaments • Formins are at the (+) end and regulate the growth of disassembly of (+) end, prevents capping of the (+) end • By regulating formin, cell can regulate assembly and disassembly • Formin has a number of different domains, such as FH2 domain that sits at (+) end and, most importantly, rho-binding domain • Formin itself is regulated by rho-GTPase • Rho in GTP form binds to formin, can turn formin on, cause assembly at (+) end Activated Arp2/3 Mediated Filament Branching • Actin is unique because it forms branches viaArp2/3 • Arp2/3 forms a branch off an existing filament • Binds to side of (+) end of existing filament, allows polymerization from that spot • New (+) end grows from whereArp2/3 is bound • Arp2/3 regulates branching; it itself is also regulated by nucleation promoting factor (NPF) • NPF activatesArp2/3, allows it to facilitate branching • Several NPFs, WASP protein is the most important o WASP needed in active form to activateArp2/3 o WASP itself needs to be activated by Cdc42-GTPase • Cdc42 in GTP forms binds to WASP, activates it; active WASP can then bind to and activateArp2/3 = BRANCHING Arp2/3-DependentActinAssembly during Endocytosis • Branching and actin polymerization is used in endocytosis and in phagocytosis • Endocytosis – receptor, forms vesicle, specific things in PM recognize cargo, pinching off, vesicle moves in • Lots of proteins involved, a few can involve actin polymerization; NPFs such as WASP present • Once endocytosis begins to occur, WASP is activated and causes actin polymerization and actin branching • Actin branching occurs in such a way that it is pulling PM into the cell; continued actin polymerization is actively pulling PM in the cell/cytoplasm • Eventually, vesicle is formed and continued actin branching pushes the vesicle deeper into the cytoplasm/cell • Microtubule can take it further down (retrograde) • Arp2/3 activated by WASP during endocytosis to help formation of endocytic vesicle and transport it into the cell Phagocytosis andActin Dynamics • Phagocytosis – engulfing a pathogen, making a much bigger vesicle; often involves immune system • Antibody recognizes and binds to bacteria • Leukocyte (white blood cell) undergoes phagocytosis of bacteria • Antibodies are recognized by cell-surface receptors on leukocytes and once those antibodies bind to the leukocyte cell- surface, receptor binding will activate actin polymerization • Actin polymerization will push the PM around this bacteria • Endocytosis = pulling a membrane inward, phagocytosis = pushing membrane outward • Eventually, entire pathogen is encircled in vesicle and lysosomes can be linked to it • Actin polymerization can be used during endocytosis and phagocytosis because actin polymerization can move membrane • Can create a vesicle either by pulling in or pushing out the membrane Actin-Binding Proteins and Cellular Structures • Bundles – fimbrin and actinin link parallel, unbranched actin filaments to form bundles (stronger than filaments) • Networks – spectrin and filamin help actin form networks • Support to membrane – dystrophin link the cytoskeleton of the PM • Microvilli are thick projections that stick out of epithelial cells o Intestinal cells contain microvilli; intestinal cells absorb things, want a large surface area o Intestinal epithelial cells have hundreds of microvilli on flat surface to increase surface area • Microvilli vs. cilia o Microvilli: bundles of actin pushing up out of the membrane to increase surface area; are actin, are non-motile o Cilia: are microtubules, are motile • Bundles within microvilli are held together by things like fimbrin and actinin • Red blood cells depend on actin binding proteins to support the cell membrane.As do Microvilli, and muscles. • Red blood cells – ankyrin links cytoskeleton to membrane • Microvilli – ezrin links cytoskeleton to membrane • Muscle cells – dystrophin links cytoskeleton to membrane Myosin:Actin’s Motor Protein • Myosin II (muscle) most abundant o Heavy and light chains o Head is anATPase – binds to actin and moves it along usingATP o Neck binds light chain  Light chains are not involved in cargo binding  Light chains regulate function of myosin, regulate movement o Tail binds “cargo” o Ca important • Heavy chain binds to actin and cargo, and light chain regulates movement • Function of muscle cells – contract and move things • In most joints, muscle cell will contract/pull on a tendon, that tendon will pull on a bone • Tendon is made up of ECM (extracellular matrix – proteins outside of cells) • S1 is a single-head domain which can bind to actin which stabilizes actin o S1 is a degradation product or proteolytic cleavage product of myosin; that fragment of it forms specific shape and coats actin Myosin Classes • 3 main classes of myosin to remember (although there are many others, you only have to know I, II, and V) • 3 main functions – membrane association, contraction, transport • (+) end directed • Myosin I o Only one that is found as a monomer, only one heavy chain o Binds membranes during endocytos
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