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Lecture 11

Biology 2290F/G Lecture 11: Actin Filaments

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Department
Biology
Course
Biology 2290F/G
Professor
Ray Zabulionis
Semester
Winter

Description
Section 3: Actin Filaments Actin Filaments – Microfilaments - Cortical function, cell shape and movement - Cortical: near the corticle, which is near the outside of the cell - Actin is involved in cell shape and movement because it's in contact with the PM - Actin responds to extracellular signals o Has lots of receptors on it that change the cytoskeleton, impacting the actin cytoskeleton according to signals Actin based structures - Epithelial cells, migrating cells, muscle and non-muscle functions - All of these structures are formed because actin can form networks and bundles o Tubulin is a very linear structure, but actin is much more flexible Actin: Structure - Vertebrate isoforms – 4alpha (muscle), beta and gamma - G-actin (globular) polymerizes into F-actin (filamentous) microfilaments - Different forms of actin: alpha (found in muscles), beta (corticle actin), gamma o Different forms have different functions - When actin is translated, it forms G-actin (globular actin), which has 4 domains - It has a 4-leaf clover shape with an ATP-binding cleft o Has polarity due to the ATP cleft - When actin polymerizes, it does so in a distinct orientation where the ATP-binding cleft is always in the same orientation - All actin molecules oriented in the same directed - G-actin can polymerize into F-actin - Actin is quite thin; how do we know the + and - ends? Actin: Polarity - If you take an actin filament and add myosin S1, it will "decorate" the actin o Forms arrowheads that show you the actin's polarity o Arrowheads always point to the (-) end Polymerization of actin filaments occurs preferentially at the (+) end - We also stabilize the microfilament when we "decorate" with myosin S1 - We can now use it to look at polymerization rates - We need G-actin to be in the ATP state for polymerization to occur - Actin is preferentially polymerized at the (+) end o (-) polymerizes slower - As we add more and more monomers to a petri dish, mass increases, but at some point, we hit the critical concentration, after which we start polymerizing the microfilament o All new monomers we're adding are going towards the growing microfilament - Always a set amount of monomers that remain in solution to stabilize critical concentration - Either this graph or the tubulin graph will be on the midterm!! - After polymerization, there is hydrolysis to ADP - If above critical concentration in ATP form, you get polymerization - Idea of steady state only works if things are in ATP form o Doesn't happen in a cell; only works in vitro - Ideally in the graph, there should be a bit of a lag above critical concentration because nuclei need to be formed - Steady state only occurs when you get to critical concentration, and you add and subtract subunits o Disregard steady state for most situations Actin Assembly - “steady state” not functionally important - Above concentration, a nucleus is formed o We then get elongation at a much faster rate, polymerization occurs at (+) end - If you have a nucleus, elongation is much slower Actin Polymerization and Critical Concentration - (-) and (+) ends have different critical concentrations!! o Cc for (+) is lower than that for the (-) end - MEMORIZE: C needs c.12 micromolar of G-actin in ATP state to polymerize o C ceeds 0.6 micromolar of G-actin in ATP state to polymerize - What happens if we're between those two critical concentrations? (e.g. 0.4 micromolar)? o So we end up with polymerization at the (+) end, and depolymerization at the (-) end ▪ We get something called "treadmilling" that occurs, where both polymerization and depolymerization are occurring simultaneously - In this image, polymerization is occurring at (+) end, depolymerization at (-) end - Looks like blue monomers are moving to the (-) end, but imagine that it's all relative movement Regulation of Actin Polymerization - Cellular concentration of G-actin is 400 micromolar, but C ic 0.12 micromolar - All should be polymerized, then, right? - Thymosin sequesters actin and provides a reservoir - Profiling promotes actin polymerization by charging G-ADP into G-ATP actin - Cofilin enhances depolymerization - In general, concentration of G-actin in cell is 400 micromolar o Theoretically, all should be polymerized! - Thymosin (protein) sequesters actin and provides a reservoir o When it binds G-actin, the G-actin can't be used for polymerization - Profilin is a protein that promotes actin polymerization by charging G-ADP into G-ATP actin o Recall: ADP form cannot polymerize!! - Cofilin depolymerizes actin microfilaments - All these proteins affect polymerization Actin capping proteins – block assembly and disassembly - CapZ binds to the (+) end, while tropomodulin binds the (-) end, and both prevent assembly and disassembly - Actin-disrupting drugs: o Cytochalasin depolymerizes actin filaments o Phalloidin stabilizes actin filaments - Capping proteins: proteins that bind to the (-) or (+) end of the actin, blocking assembly and disassembly - Useful for determining critical concentration for either (-) or (+) end - CapZ is the (+) end, tropomodulin is the (-) end cap - Recall: phalloidin can be tagged with fluorescent molecule, and will then bind to actin filaments Assembly (and Branching) - Formins assemble unbranched filaments - Actin is found in very high levels in the cell, so it's very highly regulated - (+) end is almost never free to polymerize because it will do so too quickly - Formin acts as a nucleating agent (brings together several actin monomers, forming a little nucleus onto which other monomers can be added) o Formin always sits at (+) end, so can regulate how fast that end polymerizes/depolymerizes - Formin is regulated by RhoGTP o RhoGTP is required to activate formin - If formin inactive, it will not nucleate, might still bind to actin, but will end up capping it permanently - no depolymerization/polymerization - You have to be above critical concentration to polymerize - Nucleating factors (e.g. formin) regulate the speed of things - If formin is bound to the (+) end but is not active, then it basically just caps the + end o RhoGTP has to be in the GTP form in order to activate formin - Polymerization occurs at the + end! Activated Arp2/3 mediated filament branching - Arp2/3 is a protein that regulates actin branching o Requires nucleation promoting factor (NPF) such as WASp or WAVE - both of which have to be activated (by Cdc42 and Rac respectively) - Recall: + end is always the end that's growing - Arp2/3 is regulated by cdc42 or Rac, both of which must be in GTP form - Basically, you have a microfilament; if you want to form a branch form, you need activated Arp2/3 o We need Cdc42 to be in GTP form, which will activate WASp or WAVE - We need some sort of activation molecule; in this case, we have Cdc42 that activates WASp, and Rac that activates WAVE - Both Cdc42 and Rac must be in GTP form to allow branching - Regulation is required because actin polymerization is very powerful o Cell and its organelles can move through this, so the cell tries very hard to regulate polymerization o If we don't regulate things, a lot of unwanted actin growth can occur o The listeria parasite can use unwanted actin growth to propel itself from cell to cell o Listeria ActA acts as an NPF, polymerizing actin and pushing Listeria throughout the cell and into the next cell Arp2/3-dependent actin assembly during endocytosis, phagocytosis and actin dynamics - In endocytosis and phagocytosis, movement is relative - actin polymerizes (requires nucleating agent) o In endocytosis, we can attach the PM to the actin network, and as actin grows into the cytoplasm, we pull away part of the plasma membrane , forming a vesicle that we can use for endocytosis o So actin polymerization can facilitate endocytosis - In phagocytosis, it's the opposite o Actin polymerization pushes the plasma membrane out away from the cell surface o If the + ends of the actin push against the plasma membrane, pushing it outwards to form a phagocytic vesicle that will engulf something Actin-Binding Proteins and Cellular Structures - Bundles, networks and support - Three classes of actin-binding proteins: bundles, networks, and supports - If you have parallel actin microfilaments with short proteins in between them, you can form bundles o Fimbrin and alpha-actinin can both facilitate bundle formation o E.g. intestinal cells have microvilli that increase surface area - their actin is bundled up o Don't confuse microvilli with cilia! - We also have proteins that can take actin and form networks (because actin can branch) o E.g. spectrin and filamin can bind to actin and cause even more crosslinking and cross-bridging - complex network of actin o Meshwork is found in the cell cortex, supporting the plasma membrane - We have other proteins that link the actin cytoskeleton to the PM o Actin is found at the cortex, regulates cell shape, can push/pull on the PM o For it to be able to do that, ideally it should be connected to the PM o Proteins such as dystrophin link actin to the PM Red blood cells depend on actin binding proteins to support the cell membrane, as do microvilli and muscles - Red blood cells have a biconcave shape o This is because the underlying actin structure is that shape o Allows the RBC to be squished without damage o In order for it to have that shape, the actin cytoskeleton has to be linked tightly to the PM - If we look at the underside of a RBC, we see the actin cytoskeleton, and we see how it's linked to the PM - Structurally, we see that the actin is crosslinked into a network by spectrin o The actin is linked to the PM by Band 4.1 (grey square), which links actin to transmembrane proteins o Ankyrin is binding spectrin to the PM ▪ Anchors the actin cytoskeleton to the PM as well - In microvilli (which has actin bundles that extend the cell surface, held together by fimbrin), we also need the bundles to hold onto the PM o This is achieved by the protein Ezrin, which links the actin cytoskeleton to transmembrane proteins of the PM in microvilli o When Ezrin is phosphorylated, it binds to the PM - this is how it is regulated o During mitosis, we don't necessarily want the actin cytoskeleton to be attached, so through regulation, we can release actin from the PM through dephosphorylation - Dystrophin links actin cytoskeleton to a protein in the PM; plays important role in muscle cells o Dystrophin links the actin to the membrane protein that links to extracellular matrix stuff (i.e. tendon) o If dystrophin doesn't function, then the muscle will contract with too much force in an attempt to move the extracellular matrix o Muscular dystrophy involves a trouble in linking the extracellular matrix to the actin cytoskeleton, so that muscles will damage themselves until none of them work anymore o Dystrophin is crucial in linking in muscle cells to extracellular matrix things such as tendons and bones Myosin: Actin’s Motor Protein - Myosin II (muscle) is most abundant o Heavy and light chains o Head is an ATPase o Neck binds light chain o Tail binds “cargo” - Actin also has to provide transport for myosin, which is actin's motor protein o Myosin is the only motor protein that moves toward the + end o Similar to kinesin in many ways - Myosin has two heavy chains, which have a head, neck and tail domain o Head domain is the actin binding site, binds to microfilament ▪ Also has ATPase activity that allows movement o Neck domain bends with ATP hydrolysis o Tail domain is what binds cargo - Myosin also has light chains, which regulate movement o Two types: essential and regulatory (don't worry about this too much), regulate step size and speed of movement - In muscle, type II myosin forms a dipolar structure , with many dimers coming together, and all their tail regions binding together on the inside o Forms a thick filament - If we cleave off the tail domain, we see that it binds cargo or other tail domains - We can also get S1 fragmentation where the fragment is only the head and neck domain o Used to study how myosin works, and the S1 myosin can bind to actin, pointing to the (-) end Myosin Classes - Myosin I is atypical: short monomer that has a very short tail o Can bind to PM, plays role in endocytosis o Is also a (+) end directed motor, which can move the PM o Not necessarily involved in most organelle trafficking - that job goes to myosin V - Myosin V is similar to kinesin: two heavy chains, head domains bind to actin o Tail ends bind to different types of cargo - has variability o Has lots of light chains - Myosin II involved in muscle contraction and other contractions o Forms thick filament; bipolar structure that finds itself in between actin filaments - Notice that there are different step sizes o Step size is dependent on the neck: the shorter the neck, the smaller the step size Sliding filament assay can be used to detect myosin-powered movement - Length of myosin II neck determines the rate of movement - Bind myosin molecules to a glass plate, and then we add fluorescent actin (phalloidin dyed) - Look at what happens to the actin as it's moved by the myosin - Look at what happens to #1 o By putting on myosin, we see that #1 actin filament moves at a certain speed o Looking at it carefully, we can measure step size and speed o Speed is dependent on neck length o The longer the neck, there's usually more light chains associated with it - that's why myosin V has largest step size - Don't have to memorize the specific step sizes Conformational Changes of Myosin Start in rigor state 1. Binds ATP, head released from actin 2. Hydrolysis of ATP to ADP + P, myisin head rotates into “cocked” state 3. Myosin head binds actin filament 4. “power stroke”: release of P and elastic energy straightens myosin; moves actin filament left 5. ADP released, ATP bound; head released from actin - Myosin uses ATP hydrolysis to move to the (+) end - This process is cyclic: no beginning and no end - "start" in a rigor state because the diagram starts in rigor state; the process doesn't actually start specifically in rigor state - Myosin thick filament has many head domains, some of which are bound to actin - In absence of ATP, myosin stays bound to actin - this is rigor state (myosin bound to actin) - In presence of ATP, ATP binds to head domain, which will let go of the actin o Done through conformation change in head domain, causing release of actin - ATP is then hydrolyzed, causing another conformational change so that the head domain moves towards the (+) end, binding somewhere down the (+) end o We now have ADP and phosphate - no change in relative positions of actin and myosin at this stage - thick filament has not moved - When we release the phosphate, another shape change occurs, and the head bends so that the actin moves relative to the thick filament o Myosin head now has ADP bound to it; when it releases ADP, you're back in rigor state waiting for ATP to bind again - In general, we get sliding of the actin filament Skeletal Muscle Sarcomere - Myosin II filaments are the A band (this doesn’t change size) - Z disks come closer together during contraction - We have two vertical protein bands called Z-disks - Z-disks made of multiple proteins - From the Z-disks emanate actin microfilaments, like fingers that project between the Z-disks o Actin does not touch the actin fingers from the next Z disk!! - In between actin fingers are actin thick filaments - We can see that all the actin is organized where the + end is at the Z disk, - end is in the middle of the sarco
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