Topic 11 Introduction to the Cytoskeleton and Actin

Transcription

1 BIOL 201 Cell Biology and Metabolism C. Aikins Notes II: Topic (Cell Cycle) Winter 2011 Topic 11 Introduction to the Cytoskeleton and Actin Overview Cells come in many shapes Ex. muscle cells are long, cylindrical, multi-nucleated cells Epithelial cells come in different shapes and sizes Simple columnar, simple squamous, transitional, stratified squamous (thin, flattened cells) With the same genetic information, different cells are able to adopt significantly different shapes Cell has a large degree of internal organization Apical domains often different from the basolateral domains Apical domain interacts with the lumen of an organ Basolateral domain communicates with the extracellular matrix Cells can import and export cargo Cytoplasmic dynein (bring towards centre), Kinesin family member (move away) Cells can move In contrast, bacteria and amoeba swim Cell constructs its own polymerized cytoskeleton to allow for organization and motility Made up of microfilaments (actin filaments), microtubules and intermediate filaments Lots of diseases associated with intermediate filaments, but there is significantly less known about them (tough to research) Cells can control the shape of the cytoskeleton 1

2 Roles of the Cytoskeleton Provide shape, structure and stability If a nerve wants to make a new connection with a neighbouring neuron, will extend itself towards the neuron by extending its cytoskeleton Initiated by extension of the actin filaments, followed by the microtubules Intracellular transport Provides a highway for organisms and substances within the cell Contractility and motility Allows muscle cells to contract (cells that were long and skinny become short and fat) Spatial Organization Polymers of the Cytoskeleton Cells produce small proteins that have the ability to self associate, polymerizing into long rods Microtubules poles resistant to compression Important for intracellular transport and in cell division Actin Filaments wires high tensile strength but flexible Two proto-filaments of actin coil around in each in a helix, forming the actin filament Intermediate filaments ropes elastic and flexible When considering an analogy to scaffolds, the microtubules are the poles Very stiff and resistant to compression (will buckle eventually) Actin filaments are the wires Have a high tensile strength, yet flexible as the same time Intermediate filaments are the ropes Both elastic and flexible (monomers can slide) Actin Actin is an ATPase Has an ATP-binding cleft in its core Binding of ATP is critical for the polymerization of actin Actin is the leading force for movement, with ATP driving it The helix of an actin filament has a periodicity is 36nm The filament is polar such that it has two distinct polar ends (+) end or barbed end and a (-) end Movement and polymerization occur at specific ends 2

3 Actin is one of the most common proteins in your body, especially motile ones Actin Localization: Microvilli cell protrusions made from actin filaments all oriented in the same direction Paralleled, bundled array of actin Line epithelial cells Cell cortex a layer of cross-linked actin filaments that sits immediately below the plasma membrane in all cells actin cage that underlines the plasma membrane Area of active contemporary research While secreting, must pass through this cell cortex (such as during exocytosis) Stress fibres actin cables within a cell (parallel bundles) Filopodia actin spikes stretching from the cell to make contact with a neighbour Parallel bundles Lamellipodium/leading edge at the leading edge of the cell underlying cell motility Cross-linked network called a dendritic network of actin filaments Contractile ring formed during cytokenesis Before movement, a cell is stuck to a surface by focal adhesion (stuck via rigid contact) Extension lamellipodium (actin-rich cellular protrusion) shoots out in direction of movement Cell orients itself using the different directions of the action fibres At the leading edge, actin filaments are growing to form a cross-linked network that pushes the membrane out New actin monomers must migrate to the cell membrane to be added to the extending actin filaments Adhesion lamellipodium forms a new adhesion with the surface Translocation actin stress fibres and cables pull the body of the cell forward De-adhesion and endocytic recycling old adhesion is released Draw moving cell! Mar 4 th lect 3

4 Actin Polymerization There are three phases of actin polymerization Actin in solution is called G-actin (globular actin) Nucleation 4 or 5 actin monomers stick together to form a nucleus In the presence of neighbours, actin does not want to stick together, therefore its slow Elongation once nucleus is formed, actin easily self assemble to form F-actin (Filamentous actin) fastest growing stage Steady state over time, actin is neither growing nor shrinking (no more net growth) Can attach a fluorescent molecule to actin such that it gets brighter as its becomes filamentous, showing how actin does not keep growing forever K ON on rate constant for actin Concentration dependent; the more actin monomers you have in solution, the faster the filament will elongate (+) end has a rate of C 12µm -1 s -1, where it is C 1.3µm -1 s -1 at the (-) end K OFF off rate constant for actin Not dependent on concentration, therefore will be the same no matter what the concentration of actin in solution Off-rate at the (+) end is 1.4s -1 and 0.8s -1 at the (-) end On- and off- rates are, within themselves, both different at the (+) and (-) ends Can find the net growth rate = (K on )([]) (K off ) There is a certain concentration where the on-rate and off-rate are equivalent: critical conc C + C critical concentration at the (+) end (0.60µM) C - C critical concentration at the (-) end (0.12µM) All of the actin you started with has associated to the (+) except for the last 0.12µM Actin treadmilling because the critical concentrations are not the same at each end, will treadmill between them There are concentrations at which polymerization is still occurring at the (+) end, but at the same time depolymerisation is favoured at the (-) end On-rate is greater than the off-rate at the given concentration at the (+) end, whereas the off-rate is greater than the on-rate at the given concentration at the (-) end 4

5 Subunits coming off the (-) end are being reattached to the (+) end, thus leading to intrinsic translocation can stay a constant length in critical concentration Mechanism by which a lamellipodium extends itself Different rates has to do with which surfaces are exposed and binding, such that there is a higher infinity between the (- )/bottom end of the actin monomer and the (+)/top end of the actin filament Actin is an ATPase Actin polymerizes in the ATP form as ATP-G-actin When it binds to the filament, the ATP is hydrolyzed and the phosphate is released, leaving ADP-actin The actin monomer that is released during depolymerisation is ADP-G-actin Controlling Actin: Actin Regulators Overview Grow the filament Formins Cut the filament ADF/Cofilin Cap the filament Capping protein Make branched networks Arp2/3 complex Cross-link the filaments eg α-actinin Cross-linkers proteins that cross link the filaments Actin filaments cannot cross-link themselves Can cross-link actin filaments to themselves, to cell membrane, etc. There are many different types of proteins, each of which can space out the filaments differently Fimbrin single enzyme has 2 actin-binding domains on either side, enabling the protein to cross-link by itself Present in microvilli, filopodia, local adhesions α-actinin protein has only 1 actin-binding domains, but dimerizes with itself Forms a dimers through α-helixes coiling into a coiled-coil Found in stress fibres, filopodia, muscle Z-line Filaments not as close together as fimbrin Spectrin tetrameter that cross-links actin in the cell cortex Present right below the membrane important for the formation of the cell cortex α-spectrin and β-spectrin dimerize, which dimerizes to form an actin cross-linking unit Each β-spectrin has an actin-binding domain, thus the molecule has an actin-binding sight at each end Creates a sort of hexagonal array of short actin Filamin found at the leading edge, in stress fibres and in filopodia Dystrophin links membrane proteins to actin cortex in muscle Arp2/3 Complex forms a branched actin network Actin branching nucleator Listeria monocytogenes Harnesses actin cytoskeleton building to infect the host 5

6 Polymerization of an actin comet tail drives listeria (bacterial pathogen) that propel around the cell and out into the extracellular fluid and into other cells Tricks out cells into making branched actin networks that look just like the leading edge in a motile cell Has taken over the proteins to built comet tails to help propel itself out Arp 2/3 branches the network with capping proteins capping the actin filaments and cofilin cutting actin filaments at the back end Taking any one of these elements out prevents growth Arp2/3 binds to the side of a previous actin filament Activated by WASp complex the two subunits, Arp2 and Arp3, undergo a conformational change Brings them close together, mimicking the base of an actin filament (form a nucleus for actin polymerization) Instead of waiting for nucleation, Arp2/3 quickens the process by nucleating itself New filament can extend off the side of the actin filament Branching occurs at a 70 ± 2/3 angle 70 angle is the optimum angle for the production of outward force and for polymerization If it is too direct, there is no room for another actin monomer to attach at base of filament at membrane Evolution has designed the Arp2/3 complex to bind and produce branched networks at the optimal angle for the extension of the branched network There are two problems with the polymerized branched networks: 1) How do you keep enough actin at the leading edge to maintain motility? Local concentration at the leading edge quickly falls, therefore after rapid extension of the lamellipodium the concentration will drop down towards the critical concentration, decreasing the rate of polymerization Cells somehow have a way of maintaining a large pool of actin at the leading edge move at a constant speed, must have a way of recycling actin (Profilin) 2) How do you prevent futile polymerization onto filaments that aren t actually touching the membrane? Need to ensure that the actin is added to the last actin filament, as there are many open (+) ends that are not pushing the membrane that you don t want to polymerize Need a capping protein Capping protein caps the protein to prevent any more growth Prevents futile polymerization of filaments that are not touching the membrane CapZ has just the right on-rate constants for capping filaments that are no longer touching the plasma membrane at the (+) end Takes about 1-2s for CapZ (slow) to find its way on to the actin filament Gives filaments a brief window to elongate Arp2/3 has already branched If capping protein were infinitely fast, the system would not work because as soon as it starts it would be shut down Would prevent the filaments that are going to push the membrane from actually pushing the membrane Tropomodulin caps filaments at the (-) end Profilin Actin recycling accelerated by Profilin ATP-exchange accelerating enzyme that recycles actin and prevents (-) polymerization 6

7 Before ADP-G-actin can reattach to the actin filaments, ADP must be replaced with ATP By itself, this nucleotide is not fast enough to keep up with polymerization Encourages the release of ADP from the ATP-binding site in actin Binds directly to the (+) end of ADP-G-actin, preventing actin from attaching to the (-) end Blocks (-) end polymerization As soon as profilin-actin is to bind to the actin filament, profilin falls off, allowing the (-) end to bind to the (+) end (-) end depolymerization is not fast enough, so what speeds it up? ADF/Cofilin ADF/Cofilin proteins that cut the filaments ADF Actin Depolymerisation Factor Torques actin open, creating many breaks Can generate new (+) ends by chopping up a long polymer, or disassemble proteins Need to accelerate the depolymerisation of actin off of the (-) to free it up and ship it over to the leading edge By itself, cofilin would not depolymerise fast enough Cofilin binds specifically to old filaments (ADP-actin), untwisting the helix Makes the helix more fragile and susceptible to breaking Cofilin starts severing filaments at the back end, with the monomers dissociating Profilin accelerates the nucleotide exchange Pool of actin can be used at the leading edge Thymosin-β 4 inhibitor of elongation, preventing actin from growing in each which direction Sequesters actin Holds actin in reserve, releasing it when actin is to be grown in own direction If not present, the pool of actin will build up and grow in all directions Overall (at the leading edge), missing actin disassembly at the bottom: Lysteria uses all of these to propel itself Uses Act A to tell actin to grow Formins proteins which control the polymerization of actin (actin polymerases) Sit at the fast-growing (+) end, controlling the rate at which actin monomers are being added Can increase or decrease the rate of polymerization (overall growth) You cannot stick a new monomer on the end of actin molecule without the formin opening can sort of cap it The rate at which the formin goes to the open state, can be used to control the rate of actin polymerization Two domains: 7

8 Have FH2 (Formin Homology Domain 2), 2 half-rings that can dimerize around the (+) of actin filaments, forming a loose clamp, which rocks its way up and down as new actin monomers are added FH1 domain of the formin reaches out, like arms, and grab new profiling actin monomers, feeding the FH2 dimerized clamp (2 arms) FH1 domain speeds up actin polymerization by 4x (already a very fast reaction) by gathering and readying actin nearby Clamp can slide along to elongate the filament Clamp modulates the on-rate kinetics (acts as catalyst), as it increases greatly with it They are not ATPases or GTPases Can continue on for around > 10 thousand of actin monomers, STAYS ON! Cells are always using formins to control the growth of actin filaments Controlling Actin Regulators Rho Family GTPases How do cells make different networks in different places? How do cells decide which network to put where? The amoeba, dictyostelium, is a motile cell that is attracted to camp Will move towards camp if it is sensed There is a chemical in the environment sticks to its membrane, allowing it to sense the camp and polymerize its leading edge in that direction Leading edge is a branched Arp2/3 network Sensing of a chemical in the environment and subsequent movement towards it is called chemotaxis Platelets at the sight of a wound emit platelet derived growth factor, a chemo-attractant signal that attracts platelets into the wound Cells will move via the extension, adhesion, translocation, de-adhesion mechanism Key discovery in the 1990s elucidated the Rho family GTPases by Dr. John Hall Rho Family GTPases the master regulators of actin cytoskeleton Transduce signals from the environment and regulate the actin regulators (tell working molecules what to do) Rho, Rac and Cdc42 Took fibroblasts (skin cells) and injected them with dominant active form of Rho, dominant active Rac and dominant active Cdc42, and got difference responses each time (dominant = always active form) Rho stress fibres were polymerized Rac extended lamellipodia in all directions Cdc42 cell shot out a lot of filopodia GTPase signalling works via a nucleotide exchange mechanism Rho can bind either GTP or GDP GDP-bound inactive form, protein is turned off don t make stress fibres absence of any signalling, Rho is in the GDP-bound state It is bound by GDI (Guanosine Diphosphate Inhibitor) GTP-bound active form, protein is turned on Turned on when an extracellular signal binds to a receptor in the plasma membrane receptor activates GEF, replacing GDP for GTP in Rho, activating Rho GTP-bound Rho will message/modify effector proteins to form stress fibres 8

9 GTPase Activating Proteins (GAP) turn the signal off GAP will (accelerate rate that Rho will) hydrolyze GTP in the GTP-bound Rho to GDP, inactivating Rho Research in the actin cytoskeleton field has been geared towards determining the downstream and upstream proteins of Rho Which proteins are the receptors and GEFs upstream and which proteins are the effectors and GAPs downstream Three main pathways 1) Cdc42-GTP WASp Arp2/3 Actin polymerization Filopodia formation WASp has a Rho-binding domain (RBD) RBD generic name for a domain that binds to a Rho family GTPase Also has an Arp2/3 binding domain and an actin monomer binding domain WASp is folded up on itself such that these domains are hidden on the inside closed conformation RBD will directly bind to Cdc42-GTP (Cdc42 must be active!), causing the molecule to open up Arp2/3 activating domains activate the Arp2/3 complex, allowing them to start the nucleation of a new actin filament (WASp delivers its 1 st actin monomer from actin binding domain) Direct binding increases speed and keeps it localized (kinase/posphorylation cascade wouldn t work as well) 2) Rho-GTP Formin actin polymerization Stress fibre formation and contraction Formin exists in a closed conformation in cytoplasm, hiding the FH1 and FH2 domains The RBD of the binds directly to Rho-GTP, opening up the complex FH1 and FH2 can then accelerate actin filament polymerization 3) Rac-GTP WAVE Arp2/3 Actin polymerization Lamellipodia formation Don t need to memorize Par6, Myosin LC phosphatase or Rho kinase etc. Know that signalling pathways are distinct, which is why we get distinct responses but there is cross regulation between the pathways (eg Cdc42 activates Rac) Direct interaction between the intermediate proteins and the actin monomers allows for rapid actin polymerization For a motile cell to function: Cdc42 activation at the front Rac activation at the front (Arp2/3 activation) Polymerizes filopodia and lamellipodia at the front In the dictyostelium, Cdc42 resides only at the front near the chemotactic signal Rac activation Rho activation at the back (myosin II activation) 9

10 Leads to formation of stress fibres and subsequent contraction of myosin II filaments in both stress fibres and cell cortex Contraction important to pick up the back of the cell Rho activation inhibits Rac Scratch Assay experiment showed the importance of Rho family GTPase skin cells placed in a petri dish and the skin was scratched with a knife, skin cells migrate into the wound, healing it Converted all of the Rho family GTPases to their dominant negative form where they are permanently in GDP form Without any one of the master regulators, the cells were not able to move and cover the wound Thus, all three are independently required for successful closure of the wound Some proteins within the cell are affected by the chemotactic gradient, whereas others are not Receptors and G-proteins are always present all around the cell Some proteins undergo rapid polarization to one side of the cell These gradients are often very subtle, so the cell must be sensitive to very small changes in the signal Uses negative and positive feedback loops to amplify chemotactic signals to regulate the distribution of proteins throughout the cell Stress Fibres and Contraction During a contraction, the actin filaments of a bundle (contractile bundles) slide relative to one another, overlapping to form a thicker, shorter bundle This is how the back foot is pulled up requires force to pull focal adhesion, involving integrins, off the surface Myosin molecular motor protein that is capable of converting chemical energy into mechanical work/ force is an ATPase, has a nucleotide binding site to bind ATP Couples hydrolysis of ATP with a large scale conformational change such that contraction occurs Called a power stroke, translocating actin filament Also has an actin-binding site obviously There are three critical classes of myosin: I 10-14nm in size that are used in membrane association and endocytosis II dimeric molecules, 5-10nm in size, that form bundles Contraction of stress fibres and muscles Bundle of myosin produces a large motor that can pull on the actin filaments in which they are embedded V 36nm in size critical to organelle transport Two footed, actually walks along actin filaments 10

11 Topic 12 Contraction and Muscle Structure of Muscle Fibres Muscles bundle of muscle fibres bundle of multinucleated muscle cells bundles of myofibril units of sarcomeres filament bundles Muscle cells are very large, long tubular Can have up to 100 nuclei made up of bundles of myofibrils Myofibril one of the basic units of contraction when the muscle wants to contract made up of sarcomere units Sarcomere Basic unit of contraction (these are the part of the muscle that actually contracts) Has parallel arrays of actin and myosin filaments, bordered by Z disks Filaments interdigitate and slide relative to one another to contract sliding filament mechanism Sir Andrew Huxley (and Hugh Huxley) really amazing British scientist, half brother of Aldous Huxley Two types of filaments Thick filaments made up of myosin II filaments head domain force generating domain (ATPase) has ATP-binding pocket, binds ATP, curls has intertwined tail domains Neck domains connect the head and tail domains Anchored by M line Thin filaments actin filaments anchored at (+) end by Z disks Numbers: Thick filament with have ~100 myosin motor protein Each sarcomere has 100s of thick filaments 1000s of sarcomeres along each myofibril (10,000,000 myosin motor proteins!) 10mil if not billion motor proteins contracting when you contract a muscle! There are many features of the sarcomere: A-band length of the thick filament (dark band) I-band distance between the end of one thick filament and the beginning of another (light band) Length of thin filaments, across the Z-disk, between adjacent myosin heads Z-disk attachment site for the actin filaments Move relative to one another Distance from one Z-disk to the other is the length of one sarcomere How Myosin performs Work 11

12 The contraction is very fast Takes only 50ms for a Type-II fiber to contract Latent period 5-10ms period between the signal from the brain reaching the muscle cell and the muscle cell beginning to contract Contraction phase 40-45ms phase from fully elongated to fully contracted Type-I slower, but more force Crossbridge Cycle 1 Unattached state ATP-bound in nucleotide binding pocket and myosin head is in not attached to the actin filaments 2 Cocked state ATP is hydrolyzed to ADP + P i Myosin head, now bound to ADP + P i rotates into the cocked state Relatively reversible, myosin oscillates between cocked and unattached states, but mostly in cocked state The energy from the hydrolysis is stored as elastic energy as strain energy 3 Binding Myosin head binds to the actin filament after signalling from the brain 4 Power stroke bond to ADP + P i is broken, P i is released Elastic energy straightens/relaxes myosin release of strain energy (and release of P i ) is coupled to the movement of the actin filament 5 - Release ADP released ATP bound, releasing head from actin (affinity for the actin filament is decreased in ATP-bound state) Crossbridge cycle occurs 10 million times during a single muscle contraction There is sufficient crystal information of actin and myosin Cossbridge cycle actually happens, based on atomic level detail Actin and myosin have both been sequenced When an animal dies, rigor mortis results no new ATP to bind to myosin head to detach it from the filament stuck in bound state All of the thick filaments are stuck to the thin filaments and can no longer slide along each other The Sliding Filament Model Myosin Crowbar The myosin head actually works as a lever arm! Longer lever arm equals faster speed, saw by genetically modifying the myosin to increase lever arm length In evolution the amount of extra force you get by lengthening the lever arm had to be somehow balanced against the difficulty associated with constructing the crystalline lattice of thick and thin filaments into the sarcomere Actin length controls Once the precise distance between the Z-disks has been attained, must stop actin polymerization which wants to continue Actin filaments are capped by tropomodulin at the (-) end and CapZ at the (+) end The (-) end overlaps the thick filaments 12

13 The (+) end attaches to the Z-disk Very precise length, ± one actin monomer in all cells! An active area of research. How do they do this? Candidate proteins: titin and nebulin. Could act as rulers Muscle contraction initiation Kinases would be much too slow Motor neurons Reach out and touch a group of muscle cells from the brain, control muscle contractions by controlling calcium levels Ca 2+ is stored within the sarcoplasmic reticulum SR has tubular structure, interdigitates throughout myfibrils When the motor neuron triggers the muscle cell, the SR flushes out all of the Ca 2+ into the sarcomere Muscle contraction is very sensitive to Ca 2+ Over a very shallow concentration range, the rate of ATP hydrolysis skyrockets This sensitivity is brought about by troponin Troponin (TN) Ca 2+ binding protein, causes conformation change when bound Also bound to tropomyosin Tropomyosin (TM) a long protein that coils around actin filament, blocking myosin binding Troponin can pull tropomyosin away from actin, revealing the binding site OVERALL PROCESS Brain sends signal, motor neurons depolarize the membrane of muscle fibre (by touching it) Calcium channels in SR open, flooding the muscle with Ca 2- Troponin binds Ca, pulling Tropomyosin away from actin, revealing myosin biding site Myosin binds to the actin.. crossbridge cycle Once the contraction is completed, the sarcoplasmic reticulum will withdraw the Ca 2+ Ca 2+ is no longer bound to the troponin, assuming its original conformational shape Tropomyosin slides back over to cover the myosin II binding sites 13

14 Topic 13 Microtubules Overview and Early Observations Recall cytoskeletal Polymers Microfilaments (actin) Microtubules (αβ-tubulin dimer) Intermediate filaments (various) Another polymer system that makes up the cytoskeleton Substantially larger Form a hollow tube that serves about 25nm in diameter Keith Porter, in 1963, was the first to discover microtubules in plant cells using an electron microscope Discovered that there are 13 protofilaments forming a hollow tube Found in all eukaryotes Why Microtubules? provide stiffness & structure Persistence length the length of the filament required to bend to a 90 angle Although they are only 25nm in diameter, the length of microtubule that it would take to naturally fall into a 90 angle is 1mm ~10 million times its diameter! VERY STIFF! Provide a mechanism for long-range transport For larger cells in our body, we cannot simply wait for Brownian motion to govern the diffusion of substances across a cell Use microtubules as the internal highway for directed transport of organelles and sub-cellular structures Microtubules are found everywhere; all eukaryotic cells have microtubules exist in radial array pattern, with (-) ends at the centrosome, adjacent to the nucleus large proportion are growing and shrinking, very dynamic Mitosis Fundamentally requires the microtubule cytoskeleton as it forms the mitotic spindles Right before mitosis, the cytoskeleton is completely broken down It is rebuilt such that the cytoskeleton ensures that each daughter cell receives the required organelles bipolar structure, mitotic spindle This fact can be used to fight cancer, by controlling microtubules Neurons Axons and dendrities are chock-full of microtubules Each are essentially long parallel bundles of microtubules All of the products in neurons are produced in the cell body, thus they utilize the sophisticated microtubule system to ship them to the depths of the axon (to your toes!) Cilia hair-like protrusions that stick out of the cell into the extracellular space bundles of microtubules, can beat back and forth Originate from the basal body below the level of the membrane 14

15 Flagella Flagella of sperm is a very long microtubule-containing structure Microtubules exists as doublets There is an A tubule with a B tubule stuck on the side (not complete ring) Have a 9+2 rosetta arrangement 9 doublets exist around a central arrangement that has two singlet microtubules Microtubule Structure Dynamic Instability Tubulin the building block of microtubules Dimeric, polar molecule made of two subunits (never come apart) α-tubulin exposed surface at the (-) end (-) end is the end attaching to the centrosome Binds GTP during folding, but cannot use it β-tubulin exposed surface at the (+) end (+) end is the fast growing surface, extending into the cytoplasm Has GTP binding/hydrolysing doman Becomes the functional building block Has to ability to associate head-to-tail One α-β sticks to another α-β in a linear way to form a protofilament 13 of the protofilaments come together to form a tube Protofilaments come together such that a seam is formed because the α-tubulins are forced to lie adjacent to a β-tubulin (generally its α-α and β-β) Undergoes similar polymerization as the actin filaments Have on-rate and off-rate constants Rate constants are important because the rate of polymerization is a function of the concentration, whereas the rate of dissociation is a function of time There will likely be an exam question where you have to determine, using the rate constants and the given concentration, whether the chain will polymerize or not Critical concentration concentration above which the polymer will grow, at which the polymer will remain the same length, and below which the polymer dissociates The on-rate will be greater than the off-rate For microtubules, C - C > C + C The on-rate and off-rate constants are not very reliable as they are very difficult to measure Due to the way microtubules use its source of energy, GTP Tubulin is a GTPase GTP resides in the pocket of the β-tubulin In solution, tubulin is the GTP-bound state GTP-bound tubulin for a very stable polymer, whereas GDP-bound tubulin is an unstable polymer There is a transition zone where the tube changes from completely GTP-bound to completely GDP-bound Added as GTP-bound, then hydrolyses, but now the GDP-bound tubulin is stuck! Gives rise to the GTP-cap theory, as there is a cap of GTP-tubulins at the (+) end of the microtubule that keeps it stable and growing 15

16 Dynamic instability The behaviour of MT to switch from happy growth with cap to rapid disassembly (and sometimes back) If the GTP-cap is lost because hydrolysis and phosphate-release catches up, the protofilaments undergo a change in structure Will curl outwards like a banana peel, rapidly disintegrating When GTP-hydrolysis occurs and phosphate is released, the GDP-bound dimer becomes kinked and wants bend backwards No longer happy in the protofilament because it does not want to be straight If the wave of GTP-hydrolysis catches up to the polymerization of tubulin, the whole microtubule falls apart, an event known as catastrophe Mitchenson and Kirshner discovered the theory by breaking the microtubules in the middle, and observing that the microtubule will disintegrate Catastrophe rate is related to the [tubulin], and thus the rate of polymerization, more tubulin will make a larger cap length increases the threshold Microtubule lattice stores energy in the form of strain Become a storage depot of elastic energy Because there are so many GDP-bound β-tubulins in the lattice trying to bend out and leave, the lattice is strained When the elastic energy is released, you can capture it and use it to pull substances around in cells (in mitosis, depolymerisation is coupled to movement of chromosomes) There are rare events in which the depolymerising microtubule is exposed to GTP-bound β-tubulin, adding on to the shrinking end and capturing it This is called a rescue Microtubule-Associated Proteins MAPs control the shape of the microtubule cytoskeleton Microtubules will self-assemble if let alone, however the cell has proteins to control the shape of the microtubules, the time it takes for them to be built, etc. These modify parameters of dynamic instability Depolymerases, Polymerases, Plus-TIP proteins, Severing Enzymes, Nucleation Factors Centrosome birthplace of microtubules nucleation centre found this out by putting cells in a cold environment (destroys MT), and then return to room temp, MT grew out of controsome Microtubules are arranged in a radial array where their (-) ends attached to the centrosomes, and the (+) ends are sticking out in to the cytoplasm The centrosome is made up of centrioles Centriole small tube-shaped organells Made of MT triplets (9) that form a radial arrangement, with blades out Exist in a pair, as there is a mother and daughter, arranged 90 to each other Made out of tubulin with a lot of accessory proteins One of the first things to duplicate when the cell wants to divide Centrioles are surrounded by a pericentriolar material 16

17 Pericentriolar material loosely defined matrix that contains all of the nucleation factors to produce a new microtubule and extend it into the cytoplasm Not terribly well defined Contains γ-tubulin ring complexes, γ-turc (primary nucleation factor for new MT) Nucleation Factors Proteins that give rise to new microtubules Don t specifically increase the length of the microtubules, just the number Can control the size (number of protofilaments) of the new microtubule γ-turc (γ-tubulin Ring Complex) nucleation protein made from α, β, and γ tubulin form a template ring (pre-formed bottom end) from which a MT can elongate with 13 protofilaments γ-tubulin is similar to α and β tubulin, but only goes into ring complex γ-tubulin organized by accessory proteins (Gamma tubulin Ring interacting proteins GRIPs) which help γ- tubulin polymerize into a 13pf ring such that it can provide a base template It is difficult to form a tubulin nucleus because be tubulin monomers to need to come together to form the nucleus (even tougher than forming the actin nucleus) γ-turc will mimic the tubulin nucleus, accelerating the process for nucleation Doublecortin (DCX) Located on X chromosome Measures MT thickness DCX only binds to 13pf MT (specific lateral cuvature) Mutations cause disease (double cortex syndrome) Males (hemizygous recessive) Type 1 Lissencephaly - very severly affected. Complete loss of the folds of their cortex Females (heterozygous) simplified cerebral cortex, but also have extraneous layer of grey matter under the simplified cortex, that forms their double cortex Usually single aa mutations! Cause of Disease: Failure in Neuronal Migration Severing Enzymes Proteins cut microtubules in the middle (by binding to the sides) Gives rise to a greater number of microtubules and microtubule ends There are plenty of enzymes to capture the (-) end and can prevent the GDP-bound tubulin from depolymerisation Katanin Form heximeric rings on the side of the MT Think they attach to the C-terminal tail of tubulin to pull it out, destabilizing and thus cutting the MT C-terminus sticks out, like a little end, into solution Mechanism is not completely set-in-stone, but we have a good idea Plus-TIP proteins +TIP tracking proteins, end binding proteins have allowed us to track MT ends Can identify the very distal tip ((+) end) of the microtubule, bind to it, and travel with it 17

18 Fluorescent tagging of EB1 (End-binding Protein 1) has allowed researchers to tag the end of microtubules and observe different characteristics of microtubule growth short peptide sequence (SXIP) on +TIPS, acts as a MT + tip localization signal EB1 Can specifically recognize plus ends, and other +TIPs hitch a ride has two CH domains (caponin homology) that only bind to (+) ends two EBH domain (end binding homology ) has +TIP binding site, where these can join (>50) Domains may form some attachment with GTP-bound tubulin, or may be some other structural features As far as we know, EB1 in no way inhibits microtubule polymerization If anything, it encourages it by bringing along other proteins Fission yeast (which only grow outwards and collide with polar ends = fission) End binding proteins are involved in the delivery of growth factors and other things that stimulate this polarized growth If you change where MT collide with ends, eg sides, will start sprouting protrusions (MT delivering cargo to wrong location) Hopping on to the end of a MT, different way to travel than long-range transport Depolymerases Also known as catastrophe factors Rapidly disintegrate the polymer by triggering catastrophes There are two categories: Kinesin-13 physically tear the microtubule lattice apart using the energy from ATP-hydrolysis induce curled protofilament state, the normal intermediate conformation of MT shrinkage Stathmin acts indirectly by sequestering free tubulin, making it unable to repolymerize By taking away all the free tubulin in the solution, will slow the rate of growth such that it cannot keep up with GTP-hydrolysis triggers a catastrophy MCAK (Prof. Brouhard s experiment, mitotic centromere associated kinesin) Attached microtubules to a glass slide Microtubules were modified to be stable and fluorescently tagged (both sides had GTP-caps, therefore both were (+) ends and could be depolymerised) Discovered the microtubules were being actively depolymerized from both sides by Kinesin-13 The ends of the control microtubules were blunt, whereas, when added, the depolymerase MCAK forced the microtubule into the kinked, banana formation Polymerases Control the growth of microtubules (often accelerate) XMAP215 polymerase protein that binds and extends tubulin has many tubulin binding domains When graphing the [tubulin] on the x-axis and the rate on the y-axis, the slope of the graph represents K ON When adding XMAP215, increases the K ON by a factor of 5 18

19 After binding to the end of tubulin, can bind and add on tubulin monomers again and again Act in a similar context to formins during actin polymerase XMAP215 acts like a catalyst, lowering the activation energy between reactants and products (accelerate both forward or backwards depending on concentrations) In a solution with no substrate (tubulin monomers), will actually depolymerize Kymograph time/space plots (puts a movie into an image) Time on the x-axis and length on the y-axis If a small amount of tubulin added, no growth, equilibrium reached XMAP215 acts by stabilizing a weakly-attached tubulin dimer Normally there is a transition state where, by itself, the dimer will weakly associate with the lattice and then dissociate Does not increase on-rate, but decreases off-rate There is always a polymerase at the end of a polymer controlling the rate of addition There are many similarities between the regulators of actin polymerization and microtubule polymerization Polymerase Formin vs. XMAP215 Both have domains which bind do monomer, and one to polymer, catalyze coming together between the two things Depolymerase N/A vs. MCAK Actin does have something that actually pulls of actin monomers +TIP proteins Capping protein vs. EB1, etc. both can distinguish side and end, but capping protein shuts down growth Severing Cofilin vs. Katanin Nucleation Arp2/3 vs. γ-turc Both fucntion by providing a template for the new filament, which looks like the base of a tube Long-Range Transport Cells too large to allow random diffusion and Brownian motion to accomplish transport for them Bacteria don t have to worry about this Especially necessary in nerve cells, some are as long as 1m in our bodies Transport is controlled, in response extracellular cues We can see this with melanocytes, pigment producing cell (think melanin) Have Melanosomes melanin-containing vesicles Respond to [camp] at large amounts, disperse melanosomes radially small amounts, aggregate melanosomes at centromere (and thus nucleus) There are a variety of enzymes that have the ability to produce force and transport cargo Discovery came in the mid-1980s by studying the nerve cell of a squid Squid has a giant axon that can be analyzed Observed vesicles moving up and down the length of the axon Used electron micrographs to observe the organelles attached to the microtubule via attachment proteins Were able to purify these proteins out of the squid to determine the proteins of long-range transport Design problems for long-range transport: 19

20 Movement How is transport accomplished; as in, how does kinesin put one foot in front of the other? Directionality How is some cargo moved in and some moved out? Specificity How does the cell know what cargo to select? Kinesins Proteins that move cargo directed towards the (+) end = OUT Discovered in 1985 out of the squid s giant axon Dimer, has two head domains, a coiled-coil stalk and a cargo-binding tail (light chains) Head domains are the feet World s smallest bi-ped Along a stationary microtubule, will pick up one head domain and move it forward Binds to the vesicle through a kinesin receptor that binds to it s light chains Carries the vesicle along in a hand-over-hand (or foot-over-foot) mechanism When the catalytic core was crystallized, turns out that it had a similar catalytic core to that in myosin, both using the energy from ATP-hydrolysis to drive movement There are absolutely no similarities in the DNA Convergent evolution arrived at the same solution; the catalytic core, which couples ATP hydrolysis causes a large scale conformational change in the molecule Each kinesin protein can take ~ steps (per second!) before they fall off, a distance of about 1µm Falls off because the mechanism of walking is difficult for a small protein For example, how does the protein know that one of the head domains has stepped down such that the back head domain can now lift up Multiple kinesins walking an organelle, so it does not fall off Mechano-chemical Cycle Each one of the feet has an ATP-binding pocket At the start, in solution both heads are ADP-bound In the ADP-bound state, the up-state, it does not like to bind to the microtubule 1 MT Binding Kinesin collides with MT, the front foot releases ADP, and is now tightly bound to tubulin 2 Swing Forward head binds ATP in the nucleotide-binding pocket causes a conformational change in the neck linker which swings the back foot forward **Kinesin must be careful not to hydrolyse ATP and thus detatch back foot, before front foot attaches 3 - Step New forward head releases ADP (both feet down!) Trailing head hydrolyzes ATP and releases P i Cycle now repeats New forward head accepts ATP, swinging the back foot, which is ADP-bound, forward through the conformational change of the neck-linker Research is being done to figure out the exact timing of the ATP-binding/ADP-hydrolyzing If ATP in the back foot hydrolyzes too early, it will be ADP-bound and fall off of the microtubule Figure that ATP hydrolysis is inhibited until the front foot feels some sort of strain (by taking a step) 20

21 With both feet on the ground, the protein is stretched out, introducing the strain and need to hydrolyze ATP in the back foot Statistics of the rate constants of ATP hydrolysis will determine when the ATP is hydrolyzed too early But, after binding, back foot must release before front foot binds ATP and tries to swing People argue about the intrinsic order or disorder of the coiled stalk It has a high rotational freedom, as it can wind and unwind a little during the steps Has a long stalk to provide space between the cargo and the microtubule and to provide it with more rotational freedom Binds in between the α-tubulin and the β-tubulin Each time a head swings forward, it is going from -8nm to +8nm relative to the bound foot The foot moves a distance of 16nm, but the centre of mass of the kinesin moves only 8nm Dynein protein that moves cargo towards the (-) end, towards centromere A lot larger and a lot more complicated than kinesin Don t know nearly as much about it, hard to purify or make Has two ring-shaped, ATPase domains Each is made of 6-7 subunits, each an ATPase site ATP is not hydrolyzed directly at the binding site of the microtubule Has a separate microtubule binding domain The two domains are attached by a long, intertwined stock When the molecule hydrolyzes ATP, the ring domain spins, flinging the cargo relative to the ring Now in the post-stroke state (originally was in the pre-stroke state) Don t know much about the translocation step If the dynein is stationary, the microtubule is moved; whereas if the microtubule is stationary, dynein is moved Dynein is often dimerized to prevent it from falling off of the microtubule One of the stocks is always attached to the microtubule Flagella Rosetta arrangment of Doublet microtubules, 9 making an outer ring, and a central pair D MT: A tubule with B tubule (half moon) stuck to it Outer-arm and Inner-arm dynein spread like spokes between them The pulling of dynein against neighbouring MT that drives the beating Activation of dynein bends MT doublets, The motor walks towards (-) ends, but constrained by nexin links (linking the tubules) Coordinates pulling in an oscillating action Sort of like a tug of war, where winning team pulls and loosing team gets pulled off After they execute their power stroke, and fall off and the other side pulls Cargo-binding adapter/accessory proteins Select specific cargos Adapter proteins attach different cargos to different motors Cells control the adapter proteins Different motors like to go to different parts of the cell Could control by phosporylation 21

22 Destination specification There is new research investigating the post-translational modifications of microtubules This indicates where its going to take its cargo, road signs There are certain chemical properties of the microtubule that specify its destination For example, in nerve cells, some microtubules are specific to axons and others are specific to dendrite When the kinesin attaches to the microtubule, it knows where it is destined for Flagella Has the 9+2 array of microtubules Two types of microtubules stuck together A-microtubule 13 protofilament ring B-microtubule half ring stuck on the side of the A-microtubule In the centre of the 9 doublets is the central pair of regular, singlet microtubules Packed between the 9 doublets are dyneins and nexins Nexin mechanical cross-linkers that keep the ring stable Dyneins are cross-linking the microtubules, enabling them to produce force such that they push and pull on the microtubules on either side of the flagella Activation of dyneins on one side bends the microtubule doublets One side given the cross-section of the tail, on one half it would activate at the same time Dyneins walk towards the (-) end, but are constrained by nexin links Allows the sperm to whip their tail There is no mechanism to allow for only one side to be activated at once because the process would be too slow Instead, it is like a tug of war One side pulls harder than the other, causing the other side to give in and fall off, permitting the tail completely bends Once the tail is completely bent, all of the dyneins can no longer pull, thus the dyneins on the side that originally lost pulls back, whipping the tail back A stable oscillation and whipping of the tail results 22

23 Topic 14 Mitosis and Cell Division Review of Mitosis In humans, microtubules must divide the chromosomes such that each daughter cell gets all of the required chromosomes Problems with chromosome distribution often result in embryonic death or of the fetus (can sometimes spontaneously abort) Aneuploidy abnormal number of chromosomes in a cancer cell Interphase phase during which the cell is not undergoing mitosis Chromosomes are extended and filamentous (duplication and cohesion) Microtubules have already started to form Centrosome duplication Prophase chromosomes begin to condense Breakdown of Interphase MT display and its replacement by mitotic asters Mitotic aster separation Centrosomes begin to move to either side of the cells Prometaphase nuclear envelope breaks down, allowing the microtubules to interact with the chromosomes The two centrosomes send out the microtubules to capture the chromosomes Chromosomes captured, bi-oriented and brought to the spindle equator Metaphase Chromosomes aligned at the metaphase place (along the centre of the mitotic spindle) Mitotic spindle composed of microtubules Equator of the spindle centre of the mitotic spindle Metaphase plate congregate of chromosomes Mitosis will not occur until the complete genome is in the centre Anaphase chromosomes are separated, polarizing to either side of the cell Between anaphase and telophase, a constriction along the centre of the cell is formed Tightly controlled by actin filaments APC/C activated and cohesins degraded Anaphase A: Chromosome movement to poles Anaphase B: Spindle pole separation Telophase reconstruction of the new nucleus in each of the daughter cells Fully assemble contractile ring Cytokinesis Reformation of Interphase MT array Contractile ring forms cleavage furrow 23

24 The Mitotic Spindle Machinery Recall a mitotic chromosome contains two sister chromatids that are stuck together at the centre Because they are stuck together, they will be able to move together How do you pull these centrosomes apart? How do you build a mitotic spindle? 1) Push the centrosomes apart 2) Put the spindle in the right place (not always down the centre!) 3) Make the microtubules flux 4) Connect the kinetochores to the MT 5) Segregate the chromosomes Microtubule/Cancer drugs Taxol When a cancer patient takes taxol, cells undergoing mitosis will freeze and die. Their MT are frozen. Important for breast cancer patients especially But, MT are important for many things. There are a lot of neuronal side effects Monastrol Tartgets Kinesin-5 (eg-5), specifically mitosis (not just MT in general) Homo-tetramer (two kinesin-1 dimerize) Add Monastrol: centrosomes collapse on each other and you get a monopolar spindle spindle poles collapse Thus Kinesin-5 essential for bi-polarization 1 PUSH CENTROSOMES APART: Kinesin-5 Tetrameric kinesins that slide anti-parallel microtubules Coiled tail part of the pushing force spreading centrosomes, four motor domains (heads) Walks towards (+) end, pushing them apart towards (-) end 2 SPINDLE SET UP Asymetric cell division Not all cells divide down the middle asymmetric division Important in development, big and little ones have different developmental fates 24

25 Fate determining molecules Eg budding yeast, in neurnal and somatic cells in C Elegans Dynein positions the spindle Always have dynein coating the inner surface of the cell membrane at the cortex These try to walk to the (-) of the MT, so it pulls the MT in Responsible for the asymmetric divisions Spindle rocking C. Elegans spindle pulls osscilate (rock) helps asymmetric division One centrosome wiggles and changes from even to asymmetric Getting rid of dynein disrupts spindle positioning centrosomes drift around First loose oscillation and then displacement 3 MICROTUBULE FLUX Microtubules flux toward the poles Use fluorescence speckle microscopy track specks of fluorescent tubulin in space Near the centrosomes (spindle poles) Kinesin-13 (MT depolymerisers) Depolymerise MT at (-) end, sucking them in to the spindle pole Head domains hydrolyze ATP similar to motor kinesin, but they couple it into a force producing curling of protofilaments 4 ATTACH KINETOCHORES TO MICROTUBULES Kinetochores are big sleeves for microtubules Have an inner and outer layer very recently identified structure/proteins involved MT don t end at the kinetochore end on immediately, penetrate outer and inner kinetochore. Kinetochore wraps around MT like a sleeve Structure: Ndc80 complex (main component) extend off MT at a slight angle. Rings of them array themselves around the MT (so the end is surrounded by Ndc80 heads) In yeast, Dam1 ring complex actually forms a collar structure that encircles the tip of the MT Need to have a collar on the MT because it is MT depolymerisation that drives chromosome movements GDP tubulin molecules with strain energy, can couple depolymerisation (and thus release of strain energy) to anaphase chromosome movement Because of these sleeves the movement of chromosomes must be coupled to the growth and shrinkage of the MT Kinetochore structure: While this is the main force, also a lot of motor proteins at the kinetochore Dynein, Kinesin-7 (+) end directed that helps establish kinetochore MT attachments Kinesin-4 on the chromosome arms. chromokinesins : have a DNA binding motif on their tails. Part of how the chromosomes initially find the metaphase place Kinesins-13 MCAK, help depolymerize MT Overview: Cross linking of antiparallel MT that push centromeres apart (kinesin-5 also act during anaphase) Cytoplasmic dynein pull on centrosome critical for positioning mitotic spindle in space At the poles you have shrinkage induced by kinesin-13 causing MT flux 25

26 At the kinetochores also have kinesin-13 that allow kinetochore to couple the deoplymerization to the movement of the MT In plants and egg cells, there are no centrosomes Each centrosome contains two centrioles Centriole is made of triplet microtubules Microtubules nucleate off of γ-tubulin ring complexes of the centriole Grow towards the chromosomes between the centrosomes Topic 15 Cell Cycle Overview Cell cycle Refers to the passage of cells in their lives through mitosis into their life as a new cell, the replication of DNA, this mitosis again M G 0 / G 1 (2n in humans) S G 2 (4n) M Cell division There are two central requirements for cell division i) Increase your size can t just keep dividing ii) Duplicate your DNA How do they know they ve done this? Cells cannot measure (easily) Lengths (in um), Volumes (in um 3 ), sizes etc Current time Cells can measure concentration Low kinase would lead to low phosphorylated/not phosphorylated substrate ratio, while high kinase concentration would lead to a larger one Concentration comes from accumulation accumulation comes with time (set time to make 50 protein product etc) Cell Cycle Control Cyclin concentration Tim Hunt looked at sea urchins Acts as the timer Cyclins are made continuously, destroyed periodically Synthesis and degradation corresponded with the cell cycle Yeast Budding yeast (bread and beer), fission yeast used to study cell cycle control studies Exposed to mutagenic compounds easy to look at mutant types Mutant yeasts were used to identify a gene that controls entry to mitosis (Leland Hartwell) Cell division cycles mutants cdc genes Also used to identify a kinase that controls entry to mitosis (Paul Nurse) cdc2- mutants, just kept growing without division cdc-2 is cyclin dependent kinase 26

27 Cyclin-dependent kinase (CDK) Binds to cyclin to define the active molecule don t phosphrylate things unless bound to cyclin Thresholds of CDK activity activate DNA synthesis (S) and mitosis (M) A certain threshold is met = DNA duplication occurs. The next one triggers mitosis etc Core reason behind everything can take everything else out and cyclin/cdk will still drive the cell cycle Cyclin-cdk also called maturation factor Similar logic underlines circadian rhythms Sensing daytime vs nightime Clk (clock) and Cyc bind to an emoter transcribe period and timeless During the day they accumulate, until they can inhibit their own production, then their [] drops Cell cycle check points Things can go wrong: bad environment (can t increase size), DNA damage Wee1 kinase keeps Cyclin-CDK under control until cells have grown sufficiently Can shut down cyclin-cdk no matter how much you have you can t divide Can phosphorylate a tyrosine on cdk inactive Wee1 deficient = small cells Cdc 25 activates Cyclin-CDK to initiate cell division Cdc 25 deficient = elongated cells (without it could never turn on cdk) Removes the inhibitory phosphorylation, and allows cells to continue to mitosis Antagonistic regulation Wee1 competes with Cdc25 If you have an over expression of one, gives the phenotype of the other being deficient Complex cell cycle regulation Checkpoints DNA-damage checkpoint freezes cells in place in the presence of DNA damage Spindle-assembly checkpoint, spindle-position checkpoint Cyclin-CDK phosphorylates the DNA replication machinery Prereplication complex sits on the ORI, and gets phosphorylated duplicates DNA Cyclin-CDK phosphorylation breaks down the nuclear envelope Lamin filaments make up the nuclear envelope in hatch work structure Get phosphorylated laminal network breaks apart Metaphase Anaphase Now in mitosis 1) Segregate your chromosomes (perfectly! metaphase anaphase) have to destroy the bond between the sister chromatids (mitotic kleisin) 2) Degrade your Cyclins both involve protein degradation = controlled protein destruction! Protein degradation = control Proteins are degraded by the proteasome after poly-ubiquitination Proteins get tagged by ubiquitin lygase makes a long chain = destroy this! 27

28 Anaphase-Promoting-Complex (APC) M-phase Cyclins are poly-ubiquitinated by the Anaphase-Promoting-Complex Ubuquitin kinase (targets proteins for destruction) Gets us out of mitosis MUST be very well controlled APC on its own cannot ubiquitinate Gets its target-specificity and activation from binding factors Binding factors activate Different binding factors control what APC will tag and thus destroy! Cell can hold back Metaphase-Anaphase transition by inhibiting APC partners Cdc20 activates the APC (to poly-u securin) so as to initiate the M-A transition Cell can hold back Cdc20 After Anaphase APC binding parter = Cdh1, controlled by posphorylation Phosphorylated = inactive, does not interact with APC De-phosphorylated = active, APC+Cdh1 mitotic cyclins degraded Cdh1 activated only after chromosome almost entirely in daughter cells (or else you d have a huge mess) Anaphase must wait until all chromosomes are bi-oriented Bi-oriented its opposing kinetochores on either side are connected to MT that lead to opposite spindle poles waiting means preventing proteolysis of cohesion molecules cohesions the molecules that are holding the two chromosomes together shaped like rings. Heterotrimers Separase cuts cohesin molecules (Kelisin) Separase is kept in check by Securin Securin blocks the proteolytic site of separase Securin is ubiquitinated by Cdc20-APC, RELASEING securing! In the end it all comes down to keeping Cdc20 in control! Cdc 20 inhibition: Mad2 (Mitotic Arrest Deficient Proteins if mutated, can t stop mitosis) Cdc20 is inhibited by binding to Mad2 Mad2 exists in 2 conformations Open conformation: CANNOT inhibit Cdc20 Closed conformation: INHIBITS Cdc20 (captured Mad1, hugs Cdc20) Unaligned chromosomes catalyze the conversion of Mad2 from open closed (which binds to Cdc20) In the cytoplasm have lots of Open-Mad2, Mad1 sits on the unattached kinetochore Mad2 now binds to Mad1 Closed-Mad2 released and inhibits Cdc20 A little more complicated, the first two actually stay, and then the later Mad2s can come get changed and be released as Closed-Mad2. These free C-Mad2 can also catalyze the change from openclosed in the cytoplasm A single unattached chromosome must be powerful enough to stop the entire division process = VERY LOUD PROCESS 28

29 Bi-orientation releases Cdc20 Mad1-Mad2 tetramer is released from the kinetochore no longer acts as a catalyst In cytoplasm interacts with p31 instead of the Open-Mad2 that is normally getting cycled Now the p31-mad1/mad2 tetramer can break up the C-Mad2-Cdc20 molecules release Cdc20 How do you know it is bi-oriented? Chromosomes start out attached to the spindle in a variety of ways Force based: Bruce Nicklas micro-needle wizard If not pulled in both directions, no tension! Can induce mitosis by skewering a chromosome with a needle How would tension produce changes in Mad1/Mad2 binding? We don t know TENSION causes the kinetochore to STRETCH STRETCH leads to physical separation of components Outer plate moves away from the inner plate Somehow couples to signalling that leads to Mad1/Mad2 tetramer release Metaphase-Anaphase transition overview (Forward direction) Chromosomes become aligned (stretch based sensing mechanism Causes release of Mad1/2 tetramer (by properly aligned kinetochores) Mad1/2 tetramer interacts with p31 interacts with the Closed-Cdc20 Cdc20 (along with open Mad2 and p31) released Cdc20 interacts with APC complex Cdc20-APC poly-ubiquitinates securin Securin degraded released from separase Separase destroys cohesins separation of chromosomes in anaphase Cell proceeds to anaphase very quickly after the last chromosome is properly aligned 29