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Lecture 11

Biology 2382B Lecture 11: Actin Filaments

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Biology 2382B
Robert Cumming

Lecture 11 – Actin filaments Actin filaments – microfilaments - Actin filaments = microfilaments – smallest - Actin is corticol o Found near the cortex (plasma membrane) - Cortical function o Cortex is near the outside - Actin is found underneath the plasma membrane o Can still extend into the cell - Actin is corticol: supports the plasma membrane - Involved in cell shape and movement because it is in contact with plasma membrane o Actin network forms structures that facilitates movement o Cell that can move or muscle contraction - Responds to extracellular signals o Receptors on the plasma membrane that change the cytoskeleton o Response impacts the actin cytoskeleton – changes dramatically to signals Actin based structures - Actin can form networks and bundles - Unlike tubulin (single rigid structure), actin can form networks and bundles o Can be many shapes and give rise to many structures in the cell - Microvilli: support cell membrane - Adherence belts: forms structures that help hold a cell’s shape - Processes involving movement of the cell membrane - Moving vesicles inside cell involves actin-related structure - Stress fibers: involved in movement (phagocytosis) - Actin does more things than tubulin does o Tubulin can only be linear (straight rod) where things move up and down - Actin – network and bundles – does more things in terms of shape Actin: structure - Microfilaments are made of actin - Different types of actin and different actins have different functions - Vertebrates isoforms – 4  (muscle) (cortex) and  (stress fibres) o Alpha beta and gamma actin - Alpha: found in muscles - Beta: corticol actin found underneath plasma membrane – almost all cells have this - Gamma: stress fibers involved in cell migration - Actin, when translated, forms a 3D globular structure (G-actin) - G-actin (globular) polymerizes into F-actin (filamentous) microfilaments - G-actin has 4 domains and there is a cleft (ATP-binding cleft) o Opening on one side, giving it polarity - G-actin is a monomer but it has polarity because of ATP-binding cleft o Has a + end and a - end - When actin polymerizes, it goes in a distinct orientation where ATP binding cleft is always in the same orientation - When monomer polymerizes, the actin microfilament has polarity o ATP binding cleft is always facing the same direction o One end has a cleft and other doesn’t - All actin microfilaments have the same polarity and orientation - ATP binding cleft is always pointing towards the – end - G-actin can polymerize into F-actin o F-actin microfilament has polarity (there is a + end and a – end) ▪ Has to do with the fact that the ATP binding cleft is always in the same orientation (towards – end) - G-actin (globular) polymerizes into F-actin (filamentous) micofilaments Actin: polarity - Know about – end and + end because of myosin S1 - “Decorated” with myosin S1 o Visualize the polarity of an actin filament with myosin S1 o See the direction of the arrow heads - Made with myosin (motor protein) - If you take an actin filament and add S1 mysoin, it decorates the actin o Forms a distinct pattern looking like arrow heads o Arrow heads show the polarity of actin - Arrow heads always point towards the - end - Actin has polarity and myosin S1 is a tool - Can’t tell the polarity of a regular actin filament by looking at it under a microscope but if it is decorated with myosin S1, distinguish between the + and - end - Actin filament + S1 myosin = can always find the - end - Actin microfilament is stabilized by decorating it with S1 myosin o Actin covered with S1 myosin is stable and can be used as a nucleus where polymerization rates can be observed o Like using flagella nucleus with tubulin dimers to see polymerization and depolymerisation - If actin is covered with S1 myosin = it will not destabilize - Can be used as a nucleating agent - Add G-actin monomers and observe what is needed for polymerization o Need G-actin in the ATP state - Polymerization of actin filaments occurs preferentially at the (+) end o Similar to tubulin - If above the critical concentration, get polymerization o Faster at + end o Slower at the – end - In vitro experiment in a closed system measuring mass: o Add G-actin monomers in the ATP form o As monomers are added, mass of monomers increases o At some point, hit a critical concentration after which polymerization of the microfilament occurs o All monomers you add above critical concentration goes into a growing filament o There is a set amount of monomers always present in the solution to maintain the critical concentration o Have concentration of monomers that is constant (at the critical concentration) o Anything above the critical is going into the growing microfilament ▪ When go above, the added subunits are added to the growing microfilament o At the critical concentration, there will be monomers present to maintain the concentration so when you add, you are above it - Steady state equilibrium with G-actin monomers: o At steady state, the equilibrium concentration of the pool of the unassembled units is the critical concentration (cc) - Critical concentration: o Above: polymerize if in ATP form o Below: depolymerize ▪ When depolymerize, its in the ADP form - To polymerize again, must re-energize it - To polymerize, G actin has to be in the ATP form - AFTER polymerization, there is hydrolysis to ADP o Similar to tubulin: beta subunit is in GTP form and is hydrolyzed after polymerization - Don’t need to hydrolyze to polymerize, hydrolysis occurs after it - Steady state only works if everything is in ATP form where things can go back in forth o Not something that happens in the cell o Not relevant to a cell, only occurs in vitro - Steady state is an artificial in-vitro system where at critical concentration, if not adding monomers, a monomer can go on and go off o Can add non-hydrolyzable forms of ATP o If they are in the ADP state the system will not work o Will only work if you use a hydrolyzable form - Steady state: run out of monomers Actin assembly - G actin: above critical concentration – polymerize IF IN ATP FORM - When above critical concentration and have monomers, the first thing that happens is the formation of a nucleus (nucleating center) - After formation of a nucl - eating agent, get elongation at a much faster rate o Polymerize + end faster than the – end if above cc - Initial slope can be initially slow or fast o Nucleus – fast o No nucleus – initial lag before elongation - Above critical concentration, the first thing that occurs will be the formation of nuclei resulting in a lag o If there is nuclei already present, start elongating o If there isn’t, need to form them first before elongation occurs - If it grows quickly or slowly depends on presence of nucleating agent - Gradual upspring: nucleating occurring - The more monomers you add, the more it will grow - Steady state: if allow things to use themselves up but will never occur in a cell o Only occurs when you get to the critical concentration and run out of subunits o Situation where adding and subtracting subunits at the – and + end o Will not occur in a cell because they will not be in the ATP form o Not functionally important - In experiment, actin monomers are constantly added o If they are constantly added, will remain above the critical concentration and will get polymerization - AT the critical concentration, get steady state but it rarely occurs in a cell Actin polymerization and critical concentration - Need G-actin bound to ATP for polymerization - - end and + end have DIFFERENT critical concentrations - Critical concentration for the + end is lower than the cc of the - end - + end: 0.12 M of G-actin in ATP form o If amount of free G- actin is above 0.12, will get polymerization at + end o If below 0.12, will depolymerize at + end - - end: 0.60 M o Need to be at a higher concentration of G-actin in ATP form to get polymerization - Polymerization is faster at the + end (10x faster) and it does this at a lower concentration - Because there are 2 different critical concentrations, microfilament can be found in between the 2 critical concentrations o Put it in 0.40 M of G actin in ATP form o Polymerize at + end but depolymerize at the - end - Process called treadmilling: in between 2 critical concentrations o Adding to one end but subtracting from the other end o Looks like microfilament is moving towards the + end o Appears as if blue monomers are moving towards the – end (moving towards the right) - Relative movement because adding to + end and subtracting from – end - Microfilament moves and can push things around the cell when it polymerizes at one end and depolymerizes at the other - Polymerization at the + end is faster than the – end o + end will grow faster than the – end will shrink o Proteins regulate how fast things polymerize and depolymerize - Proteins regulate what is happen at the the 2 ends o The rate differences are not that important - When look at G-actin in different cells (different cells have different amounts) but generally G-actin concentration is about 400 M - Diagram: the blue actin filament shows a particular position o Have microfilament acting as a nucleus and have a concentration in between the 2 critical concentrations o 0.40 M: add at + end and remove from the – end o as + end is polymerizing, the distance from the + end to the blue monomers is INCREASING o Blue actin monomers get further away from the + end at the same time you are removing from the – end o Blue monomers get closer to the – end o Looks like monomers are moving from the + end to the – end (treadmilling) Regulation of actin polymerization - Cellular concentration of G-actin is 400 M, but Cc is 0.12M o Cell has the resources to do a lot of things o Cellular concentration is much higher then Cc of the + end o … so all of it should be polymerized… but it isn’t o All the G-actin is not present in the correct form (ADP) - Mechanisms needs to be in place so it is not always polymerizing - Proteins bind to and sequester actin o Binding to monomers and removing them and regulating what form actin is - Thymosin sequesters actin and provides a reservoir o Abundant protein in the cell o Binds to G actin and removes it from the 400 M concentration o Makes G-actin unavailable o Functional level of G-actin is low because it is bound up by thymosin o If it is bound to G-actin, it is not apart of the Cc equation o When cell needs it, thymosin releases G-actin and it can polymerize and perform a function o Sequesters actin physically (taking it out of cc) - Profilin promotes actin polymerization by charging G-ADP into G-ATP actin o Protein responsible for energizing G-actin o Converts ADP G-actin to ATP G-actin o Regardless of the concentration of actin, if it is in the ADP form, it will not polymerize o Level of profilin regulates the level of functional actin o Energizes ADP actin to ATP actin at the right level to allow polymerization as needed o If don’t want polymerization, inactivate profilin o Need profilin to energize actin in the ATP form to be available for polymerization o Regulates the amount of actin in the ATP form - 2 proteins responsible for keeping the high levels of G-actin out of critical concentration o One by physically binding it o Other by keeping it in low energy form (not ATP) - Cofilin enhances depolymerization o When added to a microfilament, it depolymerizes actin - If have cofilin, even if there is polymerization going on, have battle between the two - 3 proteins regulate the amount of actin that is polymerized in a cell Actin capping proteins - There is A LOT of actin in the cell (way above Cc) and to regulate actin, there are many mechanisms (proteins capping, regulating assembly) to regulate its polymerization - + ends and – ends are almost never free - Like MT, proteins cap the – and the + end of actin and regulate what occurs - Actin capping proteins block assembly and disassembly - When capped, nothing happens at that end - CapZ binds to the (+) end of actin microfilament o Nothing will happen at the + end – will not polymerize or depolymerize o No assembly or disassembly o – end can do work depending on if it is below or above the Cc - Find the critical concentration of an end: o If add CapZ to microfilaments, it will block + end o Any polymerization or depolymerization that occurs will be at the - end o Observe the Cc of the - end - Tropomodulin binds the (-) end, and prevents assembly and disassembly o + end can grow or shrink ▪ Find out what the Cc is - If have both cap proteins, the actin filament will not change size - Capping proteins block what happens at the + and - end Actin-disrupting drugs - Study actin cytoskeletal dynamics - Cytochalasin - depolymerizes actin microfilaments - Phalloidin - stabilizes actin microfilaments o Binds and does not allow them to change size o Allows you to visualize actin because it is actin-specific o Make it fluorescent, add it to a cell and it only binds the actin Assembly (and branching) - Actin found at high levels in the cell so + end is highly regulated o + end is almost never free to polymerize because it will do it very quickly - Formins act as nucleating proteins and assemble unbranched filaments o Always found at + end ▪ Regulates how fast + end polymerizes and depolymerizes o Starts process of assembly and regulates what happens at the + end - Formins act as nucleating agents – brings together several actin monomers o Forms a nucleus where other monomers can be added - If things are polymerizing or depolymerizing, formin will regulate what is happening at the + end - Speed of polymerization/depolymerization occurs is regulated by formin - Formin has to be regulated – it has to be active to work o GTP-dependent protein o Regulated by rho-GTP o Rho-GTP is required to activate formin - Formin can regulate polymerization only when it is active - Actin polymerization is so powerful that it has to be regulated by many levels o Formin: regulates the + end ▪ Formin needs to be regulated – rho GTP ▪ Rho must be in the GTP form to activate formin ▪ Rho in the GDP form will not activate - If formin is not active, it will not nucleate and it will not allow actin to grow or shrink if it is bound to the + end - Formin in inactive state if bound to actin will cap it permanently – stopping it from polymerizing or depolymerizing - + end can polymerize quickly and + end polymerizes and low critical concentration ∴ Rho activates formin which then allows actin to polymerize or depolymerize, regulating the speed of things at the + end depending on the critical concentration - Rho regulates how fast formin will work - Formin: nucleating agent and regulates what happens at the + end of an unbranched filament o Nucleating factors and formin only regulate the speed of things and whether things happen at all - Have to be above cc to polymerize, and once you are above, whether you polymerize or not and how fast can be regulated by formin - Even if above critical concentration, it regulates how fast polymerization will occur - Below critical concentration and formin is active = controlled disassembly - Sits at + end and regulates how fast polymerization or depolymerisation occurs depending if above or below Cc - Polymerization occurs at the + end - Movement is RELATIVE - Putting monomers at + end: something has to move o Either microfilament is pushed downwards or membrane is pushed upwards - Different things can happen depending on how things are attached - RELATIVE MOVEMENT: either actin is still and it polymerizes and pushes something or actin itself is moving and pushing again something that will not move - Activated Arp2/3 mediated filament branching o Regulates branching at the + end - Actin can branch unlike MT o Can have an existing microfilament and get a branch (another microfilament growing off) - Actin can form complex networks while tubulin cannot - Branches depends on Arp2/3 o Looks like 2 monomers of G-actin that is stuck onto existing microfilament and allows growth in new direction o Growth is always at + end - When growing a branch, it grows at the + end away from the branch point - Require nucleation promoting factor (NPF) such as WASp or WAVE – both of which have to be activated (by Cdc42 and Rac respectively) - Nucleating promoting factor: something that is actually required for nucleation o Activation molecule – cdc42 and Rac - Arp2/3 acts like a nucleating point but needs nucleation promoting factor - Nucleation promoting factor: o WASP: regulated by cdc 42 o WAVE: regulated by Rac - Arp2/3 is regulated by cdc42 and Rac – both need to be in the GTP form - Have a microfilament and want to form a branch point o Need Arp2/3 to form the branch point o Arp2/3 needs a nucleation promoting factor – WASP or WAVE - To form a branch: o Cdc42 in GTP form will activate WASP o Wasp activates arp2/3 and allow branching o MUST be above the Cc - Regulating what is happening with respect to another branch growing o To get another branch growing, need nucleation which is provided by Arp2/3 o Arp requires the help of other nucleation promoting factors (WASP + WAVE) - Regulation is required because branching and active polymerization is very powerful o Actin polymerizes very quickly o Cell or cell organelles can move o Cell regulates polymerization through formin, WASP, WAVE, Arp2/3 ▪ Rho, cdc42, Rac, Rho regulating these fings - If don’t regulate can have unwanted actin growth - Listeria (parasite): uses unwanted actin growth that the cell does not want o When Listeria gets into cells, it tries to spread from cell-to-cell by using actin network to push it into the next cell - Listeria has protein surface - ActA o ActA works like a nucleating promoting factor o Polymerizes actin rapidly and activates Arp2/3 o Get rapidly polymerizing actin network behind the listeria - Polymerizing actin pushes listeria throughout the cell o Happens very quickly with a lot of force o Pushes listeria into the plasma membrane and shoves it into the next well - Listeria gets from one cell to another by using actin polymerization and making them move so fast that they go into the next cell - Actin polymerization is driven artificially by listeria molecules to move around so fast that they smack into plasma membrane and punch through o Can break into the next cell and replicate and repeat - Actin polymerization is unregulated by listeria parasites o Move around with actin tails behind them that are polymerizing and pushing it along Arp2/3-dependent actin assembly during endocytosis - Actin polymerization moving things is the cell uses actin (phagocytosis and endocytosis) - Movement is RELATIVE - Actin polymerizes somewhere – have nucleating agent (Arp2/3) o What is going to move when actin polymerizes is with respect to what depends on how things are attached to each other Endocytosis: - Actin polymerization can be used to facilitate endocytosis o Actin is staying still and moving the vesicle - There is a piece on the plasma membrane with a signal to undergo endocytosis - Actin polymerization can be used to drive endocytosis: how moving vesicles around - Grab membrane and as you polymerize actin and attach the membrane to the actin network o As actin network is growing away from the plasma membrane, pull away part of the plasma membrane - Start by polymerizing actin and as you get more and more actin, it will polymerize into the cytoplasm - As actin is polymerized into the cytoplasm, if it is attached to the plasma membrane, it will pull the plasma membrane into the cytoplasm o Bud off the vesicle - As you polymerize the actin deeper into the cytoplasm, can move the vesicle into the cytoplasm - Polymerizing at + end and pushing things in the opposite direction Phagocytosis and actin dynamics - Actin polymerizing is pushing the plasma membrane OUT, away from the cell surface - Have plasma membrane and if polymerize actin against it, the + end of the actin push against it o If the actin is supported and it is not moving, it will push the plasma membrane outwards - Plasma membrane can form a phagocytic vesicle and engulf whatever is out in front of the cell - Actin polymerization can move little membrane or big membranes o ITS RELATIVE to whether actin is moving or whether the membrane is moving - Polymerization is regulated o Formin: unbranched polymerization ▪ Rho GTP o Arp2/3: branched polymerization ▪ Cdc42 for WASP and Rac for WAVE to activate arp2/3 - Molecules are regulated by proteins that must be in GTP form Actin-binding proteins and cellular structures - 3 classes: bundles, network and support - There are actin-binding proteins that regulate actin structure and how it looks - Proteins bundle actin, form network and link actin to the plasma membrane - Bundles: if have parallel actin microfilaments with short small proteins in between them, can form bundles o Fimbrin and alpha actin – proteins that support bundles - Parallel actin bundles: found in microvilli o Projections in cells (intestinal cells to increase surface area) o Made of actin bundles that are supported by fimbrin (for microvilli) - Fimbrin and alpha actin bind actin filaments together - Microvilli is not the same as cilia o Cilia are projections that became MTs and they move o Microvilli are projections of actin – they do not move - Networks: proteins can take actin and form networks o Actin can branch (ability to form complex structures) o Spectrin and filamin can bind to actin and cause even more cross bridging and cross links - Spectrin and filament crosslink actin networks - Form a complex network of actin molecules - Actin underneath the plasma membrane looks very complex like a meshwork o Meshwork of actin is in the cortex of most cells o It supports the plasma membrane - Support: proteins link actin cytoskeleton to the plasma membrane - Actin is found at the cortex and regulates cell shape o Can push or pull on the plasma membrane o To do that, it needs to be connected to the plasma membrane - Proteins exist that link the actin cytoskeleton to membrane proteins (plasma membrane) o Dystrophin - Proteins support the actin network by linking it to a plasma membrane protein - The link of the corticol-actin cytoskeleton to the plasma membrane is very important - Red blood cells depend on actin binding proteins to support the cell membrane o Biconcave shape o They are this shape because their underlying actin corticol cytoskeleton is that shape o Shape provides squishability o RBCs can squeeze and expand o Shape allows changes to occur in the structure without damage - Actin cytoskeleton needs to be linked tighly to the RBCs plasma membrane and pulls it into that shape - Cytoskeleton is attached to the RBCs plasma membrane and pulls it into that shape o RBC has its shape because of the actin cytoskeleton - Actin is crosslinked into a network by spectrin o Spectrin: helps actin form networks - Need proteins to link network to plasma membrane o Band 4.1 links actin to transmembrane proteins on plasma membrane - Band 4.1 binds actin cytoskeleton to transmembrane protein on plasma membrane of RBC o Supports the RBC - Ankyrin binds spectrin to the plasma membrane o Anchors actin cytoskeleton to plasma membrane - Electron micrograph: o Elements (could be actin or spectrin – can’t see what is what) o Looks like cytoskeleton is linked to the plasma membrane and there are a variety of mechanisms that cause this ▪ Band 4.1 and ankryin Microvilli - Microvillis have bundles of actin (actin bundled by fimbrin) o Bundles extend the cell surface o Increase cell surface area - Bundles are held together but want a hold on the plasma membrane - Want things to be stable – link actin cytoskeleton to plasma membrane - Protein - ezrin: links actin cytoskeleton to a transmembrane protein o Provides stability to microvilli o Phosphate group exists on ezyrin o Process is regulated by phosphorylation ▪ When it is phosphorylated, it is bound to the plasma membrane - Anything you are bound to the plasma membrane, may not want to be in the future o Proteins have the ability to be bound to the plasma membrane in one state but not in another - Interactions with actin cytoskeleton and the plasma membrane have to be regulated - May not want actin cytoskeleton there (example: mitosis) may want to let go of the plasma membrane o Support proteins have a mechanism to release them Muscles - Dystrophin: o Important role in muscle cells o Allows actin and myosin to contr o Links actin cytoskeleton and membrane protein - Cytoskeleton is muscle cells is different - Function of actin and myosin in muscle: contract o When muscle contracts, it pulls at a tendon and then tendon pulls at a bone o Bone causes movement o Trying to link actin in muscle to the extracellular matrix (tendon) - Dystrophin provides a link between actin cytoskeleton to membrane protein that links to the extracellular matrix - If dystrophin does not function, every time the muscle contracts, it is not connected to the extracellular matrix o When it contracts, it uses a lot of force to move the extracellular matrix but it can’t o No link between actin cytoskeleton and the outside – no dystrophin - Muscular dystrophy: o Different types that allow a trouble in linking actin cytoskeleton to the extracellular matrix o When muscle contracts, it does so poorly and damages itself o After years of contraction, muscle cells die  progressive disease - Dystrophin is crucial in linking in muscle cells’ actin cytoskeleton to extracellular matrix - Have proteins that form bundles, networks and have to link networks and bundles to the plasma membrane with proteins Myosin: actin’s motor protein - Actin must provide transport for myosin - Myosin II (muscle) most abundant - Myosin is actin’s motor protein o + end directed protein o No – end directed motor - Myosin: moves to the + end - Myosin is similar to kinesin o Has 2 heavy chains – head, neck and tail domain ▪ Similar to kinesin o Head domain – actin binding site – binds to microfilament ▪ Head domain binds to actin ▪ Head is an ATPase - activity that allows movement ▪ In kinesin, head domain binds to the MT o Uses ATP o Neck domain bends with ATP hydrolysis o Tail domain binds to cargo - Myosin light chains: regulate the movement o 2 types: essential and regulatory light chains o Both regulate the step size and the speed of the steps - Beck binds light chains - Can isolate pure myosin from muscles o In muscle, myosin II forms dipolar structure ▪ Like kinesin 5: 2 kinesins come together to form bipolar structure - Mysoin II: many dimers that come together and all tail regions bind in the middle o Head domains on one side and on the other o Myosin II in muscle forms a thick filament - Can isolate myosin and through biochemical digestions, can cleave off different parts of myosin to see what it does o Tail domain: binds cargo or other tail domains (in the case of myosin II in bipolar structure) - S1 myosin: head domain and part of the neck domain o Used to study how myosin works o Can bind to actin ▪ ATPase activity o Decorates actin to point to the – end - S1 myosin: fragment of myosin molecule used to understand how things work Myosin classes - (+) end directed - 3 main functions – membrane association, contraction, transport - 3 types: myosin 1, 2, 5 - Most myosin are found in dimers or larger structures - Myosin 1: atypical o Short monomer with a short tail o Can bind to the plasma membrane o Plays a role in endocytosis ▪ Actin polymerization pulls on plasma membrane to get endocytosis going ▪ Actin polymerization occurs if it is linked to the plasma membrane but something needs to link it o Links actin network to plasma membrane during endocytosis o When actin polymerizes, it will pull the membrane and myosin 1 is a + end directed motor o Mysoin 1 moving can move the plasma membrane in the correct direction o Myosin 1 binds to membranes (small monomer) o Not involved in most organelle trafficking - Myosin 5: o Similar to kinesin o 2 heavy chains, a head domain that bind actin o Tail that bind to different cargo (variability at the end of the molecule with different family members that bind to different vesicles and move it to the + end) o Many light chains o Involved in organelle transport - Myosin 2: o Involved in muscle contraction o Forms thick filament o Bipolar structure found in between actin filaments - Step size (how big steps are that go along actin) and speed (how fast myosin moves) depend on the size of the NECK o Shorter the neck, slow it moves, smaller the step size - Actin polymerization pulls on plasma membrane and requires something - Sliding filament assay can be used to detect myosin-powered movement Perform experiment - Take myosin and bind it to a glass plate and add fluorescent actin (phalloidin) o Different lengths of myosin and different neck domains to see what is needed - Look what happens to actin as it is moved by myosin - Fluorescent actin molecules in a time frame - Myosin bound to glass slide - Myosin will move actin because it will try to move to the + end - By putting actin on myosin and adding necessary reagents (ATP), actin filament moves at a certain speed - Speed is dependent on how long the neck is o Longer the neck, more light chains associated with it o Longer the neck, longer step size, faster the transport - Length of the neck regulates the SPEED - Myosin 5 has the longest neck, the most light chains and largest step size - Longer the step, the longer the step size Conformational changes of myosin - Myosin uses ATP hydrolysis to move to the + end - Cyclic – myosin moving to + end is a cycle – no beginning or end - Start in (a) rigor state (diagram starts in rigor state) - Have myosin thick filaments over actin filaments o Thick filaments has many head domains and some are bound to the actin o Myosin head is bound to actin - In the absence of ATP, myosin stays bound to actin o Rigor: myosin bound to actin - In the presence of ATP, when ATP binds to head domain, the head domain lets go of the actin o ATP binding causes a change in conformation in the head domain of myosin o Myosin head releases the actin o Shape change and let go - ATP is hydrolyzed o With ATP hydrolysis, have a change of shape such that the head domain moves towards the + end o Binds somewhere down the + end o Myosin changes shape with ATP hydrolysis - ATP hydrolysis: hydrolyze to have an ADP + phosphate - Haven’t changed relative position of actin and myosin o Have myosin head on actin filament - Myosin thick filament did not move yet, the myosin just changed its position - Hydrolysis caused bind to something at the + end but there is no relative motion of the thick filament and actin - To get movement, power stroke will release the phosphate o Change of shape with the release of phosphate o Head domain bends to move the actin filament with respect to the myosin thick filament - Left with myosin head that has an ADP bound to it - Release ADP, back to rigor state, waiting for ATP to bind again - Power stroke: relative movement between actin and myosin and only happens with phosphate release - If ATP doesn’t bind, it stays in rigor - When ATP binds, it lets go - When ATP is hydrolyzed, it binds towards the + end o Have not released phosphate yet, it is just hydrolyzed - When you release the ADP and phosphate, back in rig
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