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Lecture 10

Biology 2290F/G Lecture 10: Microtubules Pt. 2

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Western University
Biology 2290F/G
Ray Zabulionis

Section 2: Microtubules Pt. 2 Microtubules: Tracks for Transport - Vesicle transport (bi-directional) - Motor proteins require energy (ATP) - Microtubules are tracks for transport o Come from the centrosome towards the cortex o + end always facing outside - If we look inside the cell, we see dots on the microtubules o Dots move in two directions o Dots represent little organelles (vesicles etc.) - Things are moved along microtubules via motor proteins - Use squid axons to visualize this - We squished out all the innards of a squid axon to experiment - Found that we need ATP for motor proteins to work Axonal Transport - Squid axons are a model system - Labelled proteins travel at different speeds in cells (not diffusion) - We can also inject radioactive amino acids into squid axons - Take a large squid axon, inject amino acids that will be incorporated into proteins o Produces radioactive proteins in the squid axon, which will be made into all kinds of different things - Some structures made with radioactive proteins will be transported into the axon (anterograde transport) - Proteins start migrating away - Wait a specific amount of time after injection into cell body, then divide up the axon from the cell body into distinct fragments o Cut out the axon 1cm, 2cm, 3cm, 4cm etc. from your injection site, isolate the proteins in those four segments of the nerve relative to the injection site - Things in section 4 are the things that have travelled the furthest from you injection site - After 10 minutes (example), you have the 4 segments, you can run on SDS-PAGE, separating proteins according to size - Wait 20 minutes in second experiment, do the same thing - Repeat experiment with Time 3 and 4 - This shows us that there are groups of proteins that are travelling together - proteins move as groups, not individually - Also tells us that this is NOT diffusion! Things are moving at a set rate o Look at the blue bands - they're moving at 1cm/10 minutes, so they're going at a defined rate in a specific direction. This is not consistent with diffusion - We can then isolate these proteins by going into the SDS gel, cutting the bands out, purifying and identifying the proteins Kinesin, MT’s plus (+) end directed motor protein - Many types (14 known classes, 45 genes in humans) - 2 heavy chains – head, flexible neck and stalk - 2 light chains (variable) - heavy chain heads have ATPase activity and MT binding ability - light chains recognize cargo - MOST (+) directed - Kinesin is the (+) end directed motor protein involved in anterograde transport - Many different types of kinesin (only 4 will be covered here) - In general, they have 4 subunits: 2 heavy chains, 2 light chains - Different kinesins fold together in different ways - this produces different banding patterns - They move at different rates towards the (+) end - Two identical heavy chains are homophilic, which contain a head domain, a linker domain and a stalk o Head domain is the microtubule binding domain and the ATP activity domain o Head domains bind and hydrolyze ATP, using the energy to change the shape of the linker , moving the neck and the stalk - This allows it to move towards the (+) end - The stalk domain of the heavy chains binds to two light chains o Variable light chains = you can have different types of light chains o Light chains are responsible for binding cargo - Depending on combination of light chains, you can recognize different types of cargo - BUT there is one kinesin motor protein that is (-) directed o For the purposes of this course, assume that kinesin is a (+) end directed motor protein Structures and Functions of Selected Kinesin Members - Head domains are always ATPases - For cargo movement, cargo needs the appropriate receptor that is recognized by the specific light chain - Need to memorize these 4 kinesins!! - Kinesin 1 is conventional: 2 identical heavy chains, 2 light chains o Involved in anterograde organelle transport - Kinesin 2 is heterotrimeric: 2 heavy chains, binds to different sort of variable light chains found at the end of the stalk o Can bind to different receptors on the organelle - Kinesin 1 and 2 are the main anterograde motors involved in moving things to the (+) end of the microtubule - Kinesin 5 forms a bipolar structure: 2 heavy chains come together with 2 other heavy chains of kinesin 5 o 2 heavy chains on one side, 2 heavy chains on the other side, NO LIGHT CHAINS o Doesn't bind any cargo! o Called bipolar because both ends are the same (if you flip upside down it's still the same) o Heads of those kinesin molecules bind to microtubules, still will move to the (+) end of the microtubule o Important in microtubule sliding, which is essential in mitosis o If you have two antiparallel microtubules, kinesin 5 can bind to both microtubules, with both ends being (+) end directed motor proteins o Both ends want to walk to the + ends of their respective microtubules o What you get is relative sliding : imagine the kinesin 5 sitting still. One microtubule moves one way, the other one moves the other way because the heads are trying to get to the + end o The top microtubule will slide to the left, bottom one will slide to the right - Kinesin 13 is involved in microtubule dynamics: not really a motor protein, doesn't carry any cargo o Has microtubule binding domain; has ability to bind ends of microtubules and use ATP to pull dimers off o Depolymerizes the ends of a microtubule, can do both + and - end (recall: most of the time the - end is trapped inside the MTOC so not accessible, so usually the + end gets depolymerized) o Just has two little head domains Movement – usually anterograde - ATP hydrolysis causes conformational changes in kinesin - ATP is hydrolysed as each head moves 16nm - Kinesin 1 regulated as it is inactive when folded (no ATPase activity), which is released upon receptor binding 1. Leading head binds ATP 2. Binding of ATP induces conformational change causing the linker to swing forward and dock into the head. This motion swings the former trailing head to become the leading head 3. New leading head finds a binding site on the microtubule 16nm ahead of its previous site 4. Leading head releases ADP and coordinately the trailing head hydrolyzes ATP to ADP + P. P isireliased and the linker becomes undocked - Moving to the + end uses ATP hydrolysis - Ignore the mechanism shown on the right! - MEMORIZE: each head moves 16nm per ATP hydrolysis!! - When not moving, the two heads are 8nm apart, but when it walks, they are 16nm apart - Don't have to know how ATP hydrolysis causes this movement, but the point is that ATP hydrolysis causes the head to move 16nm - If there's no cargo, you don't want the kinesin moving to the + end - When kinesins aren't bound to their receptor, they're folded in their inactive form - Kinesin 1 cannot bind microtubules if it's not bound to cargo!! o Kinesin molecule is opened up when it's bound to the cargo's kinesin receptor Cytoplasmic dynein, MT minus end directed motor protein - Involved in retrograde transport - “heavy chain” heads have ATPase activity and stalk (stalk is part of head) - Linker and stem in turn interact with dynactin (hetero) complex to recognize and bind cargo - ATP hydrolysis shape changes that drives movement - Dynein has heavy chains again, similar structure to kinesin - We have a large head domain that binds microtubules and has ATPase activity - We have a linker domain that bends (like kinesin), we have a stem/stalk that eventually leads to binding of cargo - There's an extra "stalk" sticking out of the head domain (it's part of the head domain) o The whole head + stalk thing is what binds microtubules - The two head domains interact together and walk along the microtubule - ATP hydrolysis bends the linker region - Stem region binds the cargo eventually The dynactin complex links dynein to cargo - The dynactin (“hetero” because it can contain varying components) complex links dynein to cargo and regulates movement - Its association with dynein regulated in part by dynamitin (inappropriate levels of dynamitin and dynactin cause dynein to explode apart) - P150glued binds microtubules, but is not a motor - Dynein uses the dynactin complex to link to cargo - The complex can contain different types of proteins - The head domains of the dynein will move towards the (-) end - Heavy chains and their stems will bind the dynactin heterocomplex o The dynactin heterocomplex binds to the cargo o The complex is made of different proteins, which can bind different types of cargo o Instead of using different light chains, the dynactin complex is used to recognize different types of cargo - All dynactin complex all have dynamitin; essential to link dynein to the dynactin complex o Without dynamitin, transport cannot occur - P150glued can bind to the microtubules, but it's not a motor protein! o Think of it as a train car o The heavy chain is a locomotive, the p150glued is a cart behind the engine o It helps carry things, helps with stability (like a carriage) Kinesin and dynein cooperate in anterograde and retrograde transport of cargo – often the motor proteins themselves are cargo! - Post-translational modification of tubulin affect both microtubule stability, and transport - Acetylation of a lysine residue of the alpha tubulin both stabilized the MT and promotes kinesin-1 movement - Kinesin and dynein work together; one goes to the + end, one to the - end - One motor protein can be the cargo to the other motor protein!! o Kinesin can take dynein to the + end - As an organelle is going to the + end, it uses kinesin to take it there, but it can also have dynein to take it back to the - end o So both proteins are often found on the organelle, but only one is used at any one time - Any process that's anterograde is kinesin, anything retrograde is dynein - For this to work, we need the motor proteins to be in the right area - We need kinesin to be found somewhere near the centrosome, dynein needs to be found somewhere near the cortex - Microtubules have to be in the right state o Can be modified to facilitate transport through post-translational modification that can affect stability and transport o E.g. acetylation (adding acetyl group) of the alpha subunit of the dimer - when this happens, the microtubule is stabilized and it promotes kinesin 1 movement/migration - Many types of modifications - just have to memorize ACETYLATION o Take home: acetylation is good for migration Cilia and Flagella - Two versions of the same thing - Cilia 2-10 micrometres, flagella 10-2000 micrometres - Flagella: propel cells (few) - Cilia: sweep material across tissue (many) - Cilia usually short and numerous - Flagella usually long, less numerous - When they bend, they cause things to move (can be cells or tissues) - Flagella causes cell movement - Cilia help to sweep material across tissue Axoneme: the underlying structure of cilia and flagella - Over 250 proteins - 9+2 array of microtubules typical (others exist) - outer doublets consist of A and B tubules - Axonemal dynein - Cilia and flagella can move as a result to axonemes - Axonemal structure is the core structure of cilia and flagella; responsible for their ability to bend - Over 250 proteins involved in regulation of axonemes (don't know all their functions) - There's a ring of doublet microtubules (recall: they're stable) - This example has 9 doublets; also has 2 singlets in the middle o Known as a 9+2 configuration of an axoneme; this is the most common structure o Other organisms can have 8+2, 8+1, 10+1, etc. - Important: there's a ring of doublet microtubules that allows function o Everything else stabilizes movement - Movement depends on axonemal dynein o Different than cytoplasmic dynein!! o Axonemal dynein is permanently linked to the A tubule - Stem of axonemal dynein is bound to the A tubule; head of dynein sticks away from A tubule, reaching towards the B tubule of the next doublet - Head domain of A tubule can bind to B tubule of next double and bend - The configuration of how close the dimers are to each other helps control how things move - M
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