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Lecture 10

Biology 2382B Lecture 10: Microtubules

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Biology 2382B
Robert Cumming

Lecture 10 – Microtubules Microtubules: tracks for transport - Vesicular transport (bi-directional) - Function of microtubules: used for tracks of transport o Microtubules come from the centrosome in the middle of the cell and go towards the outside with + end going towards the outside - If want go to anterograde (towards the cell surface): go to + of the microtubule - Image live cells: see microtubules inside cells and structures on the microtubules o Things moving along both directions in the microtubules using motor proteins o Organelles can be identified and transported along microtubules (secretory vesicles, lysosomes  anything cell needs to transport) - MT move using motor proteins that require energy (ATP) - Analysis of MT using squid axons o Take squid axon and squish out the microtubules of the axon o In-vitro assay: and use it to investigate what is happening what is happening o Add or subtract things to see what you need in order to get movement of organelles ▪ ATP is required - Different types of axon prep but depending on how you prepare the microtubules and how you image, can see things moving along and in separate directions o Things moving in different directions o Things moving at different speeds – small = fast, big = slow o Different motors with different capabilities Axonal transport - Inject radioactive amino acids into cell body of a squid axon o Proteins that are made in the axon are going to be radioactive o Radioactive amino acids are transported along the axon o Anterograde transport: move away from the cell body o Transport proteins from the location you injected the radioactive amino acids - Proteins are not actually colourful, they are radioactive - Do an experiment and repeat it at different time points - Labelled proteins travel at different speeds in cells (not diffusion) - Wait a certain amount of time - Divide up the axon from the cell body into distinct fragments (1cm, 2cm, 3cm, 4cm from injection site) o Isolate proteins in those 4 segments of the nerve relative to the injection site o Segment 1 is closest and 4 is the furthest - Different segments contain radioactive proteins if they travelled that far - After 10 minutes, have 4 nerve segments and run them on a gel (SDS page) and separate them according to size o Know which proteins are in which segment o May or may not have proteins in some segments depending on how far it travelled travelled - Time 1 (10 minutes): run proteins, separate by size and the gel is radioactive so expose it to x-ray film and see bands that represents where the proteins are o Banding pattern is black  THERE IS NO COLOUR TO THE PROTEINS OR RADIOACTIVITY! o Where there are proteins, there are distinct bands o Isolated the proteins and see what is there after 10 minutes o No proteins in segment 3 and 4 because no protein travelled that far - Time 2 (20 minutes): take segments from another squid axon and repeat experiment o Inject more radioactive amino acids but wait 20 minutes o Isolate the same nerve fragments the proteins o Want the same proteins here – just moved o 3 large groups of proteins that were in segment 2 10 minutes ago are now in segment 4 (visualize the bands) ▪ Groups of 3 proteins travelling together o Protein bands that were in segment 1 are now in segment 2 o Some proteins move faster than others - Time 4 (40 minutes): take more radioactive amino acids, inject into third squid and isolate the 4 nerve segments o Proteins that were in segment 4 are gone  no proteins of that size anymore because they travelled beyond 4cm o Proteins in segment 2 are in segment 3 ▪ Travel at a distinct speed (1cm/10 minute) - There are groups of proteins that travel together (some proteins more individually too) - PROTEINS TRAVEL AS A COMPLEX AT DISTINCT SPEED - DIFFERENT COMPLEXES TRAVELLING AT DIFFERENT SPEEDS - This is not diffusion or random o Things are consistent and move at a set rate o Diffusion – could move forward or back - Proteins travel in one direction - Proteins moving as a complex towards the + end (anterograde – away from the cell body) - To know what the proteins are: SDS gel, isolate and purify proteins and sequence amino acids o Know what proteins are travelling together at a certain rate o This is how motor proteins were discovered Kinesin, MT’s plus (+) end directed motor protein - Kinesin: family of motor protein o + end directed motor involved in microtubules i anterograde transport - Different proteins and various combinations come together for transport - Different types of kinesins travel at different speeds Most kinesins have 4 subunits - 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 - 2 heavy and 2 light chains that come together o Different kinesins have different ability to come together = different banding pattern of different numbers of proteins (sometimes 2 or 4 moving together) - 14 different types that come together to make different types of kinesins o Move at different rates towards the + end - Prototypical kinesin: type 1 o Simplest form of kinesin  most abundant o Has 2 heavy chains and 2 light chains o 4 proteins make this motor molecule - 2 heavy chains for kinesin 1 are homophylic (identical) o Contains a head domain, a linker domain and a stalk - Head domain: microtubule binding domain and the ATP-activity domain o 2 head domains of the 2 heavy chains o Binds microtubules o Bind ATP, hydrolyze it (ATPase activity) o Use energy from ATP hydrolysis to change the shape of the linker (neck domain) o Change in shape allows it to move to the + end - Linker domain: allows the bending of the molecule (neck) - Stalk domain: binds to 2 variable light chains o On the heavy chains, attached to the linker domain - Light chains are variable = can have different types of light chains there o Responsible for binding to the cargo o Depending on the combination of the light chains, can recognize different types of cargo (ER vesicle, lysosome, etc) o Different cargo molecules are recognized by different combinations of light chains - Cargo must have the correct receptor for the light chains to recognize - Different combinations of light chains recognize different types of cargo to take them to the + end - Kinesin, + end directing motor protein: o 2 heavy chains with head domains that bind to the microtubules with ATPase activity o Heavy chains are towards the + end o At the other end of the heavy chain (end of stalks) are light chains that are variable and recognize specific receptors on a vesicle ▪ Depending on what the vesicle is, need the appropriate light chains to recognize it and take it towards the + end - Kinesis is a + end directed motor  assume it always goes towards + end (anterograde) Structures and functions of selected kinesin members - 14 types of kinesin - Anterograde transport motors: 2 main ones involved in organelle transport: kinesin 1 and kinesin 2 o Use a form of light chains to bind to the cargo to take it to the + end - Kinesin 1: most abundant o Prototypical with 2 heavy and 2 variable light chains (variable) o Can bind to a variety of organelles o Involved in anterograde organelle transport o Depending on light chain binds to different types of cargo - Kinesin 2: heterotrimeric o 2 heavy chains that are not the same and binds to a different types of light chain that are found at the end of the stalk that eventually recognizes its receptor o Involved in anterograde transport o 3 separate proteins (2 heavy chains and a different variable light chain) o Depending on the third protein (light chain), it can bind to different receptors on the organelle - Different organelles are recognized by different types of kinesins (1 and 2) that know to take it to the + end of the microtubule - Kinesin 5: o Forms a bipolar structure ▪ 2 heavy chains come together with 2 other heavy chains in an orientation where there are 2 heads on one side and 2 heads on the other side o No light chains  does not bind to cargo o Bipolar: both ends are the same  2 heavy chains on both sides ▪ Both poles are the same o Heads of kinesin molecules still bind to microtubules and still move to the + end o Involved in microtubule sliding: important during mitosis - Have 2 microtubules that are antiparallel (one going +  -, other going -  +) - Both heads bind to antiparallel MTs o Kinesin can bind to the 2 different microtubules ▪ Head domains on both sides ▪ Has ATPase activity and move towards the + end o Both ends are + end directed motors and both ends will go the the + end of the respected microtubule o Get relative sliding - movement is relative o Kinesin sits still and one microtubule moves one way and the other moves the other way o Heads are trying to get to the + end - Arrows = pointing towards the head = movement of head domain towards + end = microtubule moves the other way o Heads trying to get to + end o Top MT is going to the left, bottom MT is going to the right: 2 MT slide past each other - Kinesin 5: found between overlapping microtubules o No real cargo is moving microtubules o Causing sliding and undoing overlap - Kinesin 13: involved in microtubule dynamics - depolymerization o Not a motor protein, does not move anywhere o Does not carry cargo o Head domains - has a microtubule binding domain and ATPase activity o Binds to the end of the microtubule and using ATP, pulls dimers off o Depolymerizes microtubule ends  can do the + and the – end ▪ In most cases, - end is trapped in MTOC and is not accessible o Head domains that use ATP to rip apart the ends of microtubules (remove dimers) - Head domains are always ATPases - For cargo movement, cargo needs appropriate “receptor that is recognized by specific light chain” - Transport is anterograde and involves hydrolysis Movement – usually anterograde - Moving towards + end - ATP hydrolysis causes conformational change in kinesin - Each head moves 16nm/ATP hydrolysis - Head moves from beta dimer to the beta dimer ahead of where the other head is binding - Heads move past each other - If it is not moving, the two heads are 8 nm apart - Movement of one head with each ATP hydrolysis is 16nm and spacing between heads is 8 nm - If there is no cargo, don’t want kinesin molecule moving towards the + end o Don’t want it capable of movement if it is not carrying anything - Kinesin 1 regulated as it is inactive when folded (no ATPase activity), which is released upon receptor binding - Kinesin 1 is inactive when it is not bound to cargo/organelle/receptor o In absence of cargo, it is folded in the inactive form (no ATPase activity, no movement) - Cargo has a specific kinesin receptor that is recognized by the light chains o Receptor on cargo is recognized by light chains - When there is cargo that is recognized by the light chains, it opens the kinesin molecule, making it active o It can now bind to the microtubule and move to the + end - Only binds to microtubule and moves to the + end if it is bound to cargo - In absence of cargo, kinesin is inactive, folded up and it cannot move Cytoplasmic dynein, MT minus end directed motor protein - Involved in retrograde transport (- end) and is achieved by dynein - Move things from + end to – end - Dyenin: o Heavy chain head domain binds to microtubules and has ATPase activity o Linker domain that will bend (like kinesin) o Heavy chain has a stem/stalk that will eventually bind with the cargo - Makeup is the same: head domain, bend-neck linker region and stalk/stem domain that helps bind to cargo - Head domain: binds to MT and ATP hydrolysis causes shape change to drive movement - Like kinesin, have 2 heavy domains (2 head chains) that work together o 2 head domains that walk towards the – end o Head domain has an extra piece sticking out of it (stalk) – binds to microtubule - Heavy chains have head domains  head domains bind to microtubules using ATP to move to the – end o ATP hydrolysis bends linker region = change in shape o Stem region binds to cargo - Dyenin does not have any light chains o Heavy chains hydrolyze ATP o With ATP hydrolysis, linker region/neck bends and head domain moves - The way that dynein binds to cargo is different than kinesin - “Heavy chain” heads have ATPase activity and stalk (stack is part of head) o Linker and stem in turn interact with dynactin (hetero) complex to recognize and bind cargo o Complex can contain different types of proteins - ATP hydrolysis shape changes that drives movement The Dynactin Complex links dynein to cargo - Heavy chains and their stems bind to the dynactin heterocomplex - The dynactin (“hetero” because it can contain varying components) complex links dynein to cargo and regulates movement. - Dynactin complex binds to the cargo o Complex is made up of different types of protein o Depending on the protein that make up the complex, it can recognize different receptors on different cargo - Instead of using different light chains, its using different components in dynactin complex to represent different types of cargo to take it to the – end - Dynein can release cargo more readily than kinesin - All dynactin complexes have a protein called dynamitin - Its association with dynein regulated in art by dynamitin (inappropriate levels of dynamitin and dynactin and dynein “explode” apart) - Need dynamitin to link dynein to the dynactin heterocomplex o Too much or too little = explode = no transport - Dynamitin: o If misregulate it, it explodes and do not get transport o If don’t have right amount; the dynein will not attach = no transport o Regulates the transport ▪ Too much or too little, complex falls apart - P150glued binds to microtubules, but is not a motor protein o Heavy chain pulls things along and P150 just rides along and binds to the microtubule but does not do any of the work o Part of the dynactin complex o Aids in stability - Have 2 dynein heavy chains and the heads bind microtubules and hydrolyze ATP o 2 motor proteins move to the – end and have P150 glue that is pulled around o Binds to and rides along the microtubule but does not do any work o Extra stability to bind to the microtubule Kinesin and Dynein - Kinesin and dynein cooperate in the anterograde and retrograde transport of cargo - The motor proteins themselves can be cargo for other motor proteins - Kinesin and dynein work together (one to + and one to -) o These proteins need to be taken to those ends - Kinesin takes dynein to the + end o Any process that is anterograde - Dynein takes kinesin to the – end o Any process that is retrograde - Organelles can have both motor proteins on them but only one of them is being used o Organelle going to the + end: using kinesin to get there but it has dynein on it to get it back to the – end - Many organelles have both motor proteins bound to the them o Move both – end and + end - For this work, need motor proteins in the right area o If want anterograde, need kinesin to start somewhere close to the centrosome ▪ Has to start at the – end if want it to move it to the + end o If want retrograde, need dynein to be somewhere on the other side ▪ Need it at the + end if want to move it to the – end - Cargo for motor proteins can be OTHER motor proteins o Kinesin take dynamin to + end so dynein can take it the - end (at the periphery of the cell) - Need right motor protein in the right position to facilitate anterograde or retrograde transport - Anterograde (-  +) process = kinesin - Retrograde (+  -) process = dynein - Microtubules must be in the right state = motor proteins must be regulated o Modified to facilitate transport - Post-translational modifications of tubulin affect both MT stability and transport o Modify alpha subunit of dimers in MTs by acetylation to stabilize - Acetylation of a lysine residue of the α tubulin both stabilized the MT and promotes kinesin- 1 movement - Acetylating dimers will: o Stabilize the microtubule o Promotes kinesin 1 movement and facilitates transport of kinesin 1 - Speed of transport depends on kinesins and how they move different speeds o Modifying microtubules = speed things up = acetylate alpha Cilia and flagella - Cilia and flagella: two versions of the same thing o Cilia – short (2-10 μm)  many of these/cell ▪ Sweep material across tissue  bends and eating on cell surfaces to move stuff o Flagella – long (10-2000 μm) few of these/cell ▪ Propels cell  sperm cells moving when flagella moves - Both BEND and move things (cell or the things around the cell) to cause an effect Axoneme: the underlying structure of cilia and flagella - Axonemal structure: core structure of cilia and flagella and is responsible for causing bending and propellng o MT form this structure - Cilia and flagella move and movement is based on microtubules within them forming structure called axoneme - Over 250 proteins making an axoneme o Don’t know what most of them do o Many of them involved in regulating the bending - Axoneme made up of ring of doublet microtubules o A tubule (13 protofilaments), B tubule (10) o Stable – doesn’t polymerize/depolymerize - Axonome has 9 doublets and 2 singlets in the middle o 9+2 configuration of microtubules is the most typical (others exist) - Ring of doublet microtubules is most important functional part o Everything else is involved in stabilizing it and facilitating the movement of the microtubules relative to each other - Movement depends on axonemal dynein o Different than cytoplasmic - Stem of axonemal dynein is permanently linked to the A tubule - Head of the dynein is sticking away from the A tubule and reaching towards the B tubule of the next doublet - Axonemal dynein is on the doublets and permanently attached to the structure o Head domain can bind to B tubule and walk along the B tubule o Each doublet has a motor protein reaching towards the next doublet o If it does, get relative sliding = how you get bending - 250 proteins are there to hold things in position and to control how things move - Nexin: o Holds doublets together - All doublets are bound together by nexin o Nexin forms a ladder and is not found in all levels o Found in between the doublets but not throughout the whole length of the cilia (only in certain spots) - Cilia and flagella arise from inside the cell and go past the cell surface - If take a cross section from the top of the flagella, see axonemal structure (doublets and singlet in the middle – singlets are stable unlike cytoplasmic microtubules) - In between doublets, occasionally will find nexin (holds things in place – forms a ladder structure) Axoneme and basal body - Axoneme continuous – attach to Basal Body in cell (MTOC) o Made of tripley MT o Basal body similar to centriole o Stable o 2 basal bodies 90° to each other o Centrioles do not directly polymerize MTs ▪ Paracentriolic material does o Basal bodies are directly responsible for polymerizing the axonemes - 9 basal body triplet MTs (A and B pass through transition zone, C does not) - Microtubules in the axoneme are continuous and come from an MTOC (basal body) - Cilia and flagella come from basal body (MTOC of cilia or flagella) - Basal bodies are made of triplet microtubules - Triplet microtubules grow towards the cell surface o Go through the transition zone where they lose the C tubule ▪ Go from triplet microtubules to doublet with A and B tubules - Number of triplets in basal body = number of doublets in the axoneme - If have axoneme with 9 doublets = basal body had 9 triplets - No relationship between singlet/lack of singlet in basal body and what ends up in the axoneme o Unknown where they came from and some have it and some don’t - Have 2 cilia and flagella, have 2 basal bodies that are made up of triplet microtubules that are 90 degrees to each other o Almost identical looking to centrioles - Centrioles and basal bodies are almost the same thing except centrioles do not polymerize microtubules directly o Pericentriolar material o Triplets grow towards cell surface, turn into doublets but those doublets are directly connected to the basal body triplets - BASAL BODIES: o Stable triplets kind of like centrioles and they directly polymerize microtubules into the axoneme - Doublets are in a ring and held together by nexin - Dimers have axonemal dynein in between (permanently attached to A tubule) and heads reach out to B tubule  different than cytoplasmic dynein - Axonemal structure is part of cilia and flagella  polymerized from MTOC (basal body) o Basal body is at the base of all cilia and flagella - 2 basal bodies together at 90 degrees: o Similar to centrioles  made up of triplet mictotubules and they are stable o Basal bodies directly polymerize the axoneme structure vs. centrioles do not directly polymerize singlet microtubules that arise from centrosomes - Nexin connects A and B tubule between the doublets o Found like a ladder structure (found in certain places) - A tubule stem: axonemal dynein permanently bound - Head: functional part of dynein reaching towards the next B tubule Axenome bending - Bending causes movement or move things across the cell surface - Have doublet microtubules and they are found in the axoneme (made of A and B tubules) - A tubule (13 protofilaments): permanently bound to stem of axonemal dynein - Occasionally there is nexin holding the doublets together - Bending generated by sliding of microtubules against each other - powered by axonemal dynein - "A" tubule of one doublet "walks" along neighbor “B”. o Heads bind to “B” – “A” is permanent cargo - Heads, have MT binding properties, will reach towards the B tubule of the adjacent doublet o Head domains bind to B tubule to the next doublet - Result= MTs slide past each other – but linked to basal and nexin - Bending: generated by axonemal dynein moving and causing MTs to slide - A tubule is permanently bound to the stem of the axonemtal dynein What happens if there is no nexin? - Take doublets and remove nexin but still have axonemal dynein on A tubule: - Dynein is - end directed motor and will move along the microtubule trying to get the - end and it requires ATP - 2 doublets together with axonemal dynein in between them = relative sliding o Dynein heads try to move to the - end  MT doublets slide past each other - Relative motion because the heads are trying to get to the - end - Microtubules are in the same orientation (+ on one side, - on other side) - Similar to kinesin 5 causing sliding in between singlets. Difference here is that the microtubules are in the same orientation (parallel). The microtubules were in opposite orientation (antiparallel) in the kinesin 5 example. - If get
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