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Section 2

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Biology 2382B
Sashko Damjanovski

SECTION 2 Microtubules: Tracks for Transport • Vesicle transport (bi-directional) • Motor proteins require energy (ATP) • Using squid axons is one of the best models for tracking movement o Squids have large nerves (up to 1 mm in diameter) o Squid axons are easy to remove, easy to extrude – can get the cytoplasm out of this squid nerve and observe things moving around • Things of different sizes can move in different directions, in different speeds o Not random – things move on specific “railways” at specific rates Axonal Transport • Squid axons are a model system. • Labeled proteins travel at different speeds in cells (not diffusion) • Radioactive amino acids are injected into a nerve body such that the proteins that the nerve body makes will be radioactive • After about an hour, the cell has made proteins; because it is an axon, some of the proteins get transported along that axon • Axon is divided into four regions representing different distances from where the amino acids were injected • Proteins are isolated from the four segments, run protein gel o Because the proteins are radioactive, can be exposed to film, as an example o Bands represent proteins (bands are not usually colored) • Different binding patterns can be observed at one time point • Agroup of proteins may work together and may move at a constant rate 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) o At the end of the stalk (at the tail) • Heavy chain heads haveATPase activity and MT binding ability o UseATP to “walk” to the (+) end • Light chains recognize/bind cargo o Different types of cargo are bound by different types of light chains since light chains are variable • Most (+) directed, with some exceptions Structures of Selected Kinesin Members For this course, you are only required to remember 3 out of the 14 types of kinesins, which include: Kinesin 1, Kinesin 5, and Kinesin 13 • Kinesin 1 – organelle transport o Main type/conventional – a lot of what moves to the (+) end of a MT is because of kinesin 1 o Made of two heavy chains and two light chains o Important parts of heavy chains have head domains – head domains bind to MTs and useATPase activity to move to the (+) end o Light chains at the end of the stalk on tail domain can bind to different kinds of cargos and take the cargos to the (+) end • Kinesin 5 – microtubule sliding o Bipolar o Made up of two sets of two heavy chains – one kinesin 5 molecule has two heavy chains, another kinesin 5 molecule has two heavy chains o Two kinesin heavy chains on one side, two on the other – bound to each other through tail, tails are bound to each other such that it is bipolar o Can bind to microtubules o There are no light chains – these kinesin molecules bind to each other through stalks such that heads are on both ends (both ends are the same, both ends have a head – bipolar) o When you put this kinesin molecule in between antiparallel microtubules, the heads will bind to the microtubules and try to both walk to the (+) ends o This results in microtubule sliding (important during anaphase and mitosis) o Kinesin stays in place while each head tries to go in opposite directions, the microtubules slide past each other • Kinesin 13 – microtubule disassembly o Isn’t quite a motor protein – can act as a motor protein by moving along MTs, but it is not carrying cargo or anything o Basically just two head domains o Rips off dimers from the ends of microtubules o Can act on the (+) end or the (-) end, but in most cases, (-) end is capped in MTOC (so it can’t polymerize and depolymerize) o Most activity of disassembly is at the (+) end • Anterograde movement – towards (+) end, needsATP hydrolysis • ATP hydrolysis causes conformational changes in kinesin • With eachATP hydrolyzed, the head moves 16 nm o The head moves over the previous head that is bound to a subunit o Recall: each dimer is 8 nm and the head moves 16 nm o Sort of like “walking” o Ahead is bound on one monomer (dimer = 8 nm), another head is bound 8 nm away o But, each head travels 16 nm Dynein, MT (-) End Directed Motor Protein • Involved in retrograde transport • “Heavy chain” heads haveATPase activity and “stalk”. Linker attaches head to stem which in turn interacts with dynactin hetero complex (recognizes and binds to cargo) • ATP hydrolysis causes linker shape changes that drives movement • Dynactin both links dynein to cargo and regulates movement • Heavy chains have head and stalk region; when it folds up, head and stalk region have a strange shape o Head forms a 6-subunit structure and stalk sticks out of it o This head and stalk structure is basically considered the head structure – it is what hasATPase activity and it is what will bind to the microtubule • Just like kinesin, there are heavy chains, head structures binding to microtubules and they useATP o EXCEPT dynein goes to the (-) end instead • Just like kinesin, there is also a tail-like stem structure coming away from the head o But with dynein, the linker region between head and stem causes the bending during movement • Head and stalk is bound to MT, linker leads into stem inside stem is where you can bind the cargo • Unlike kinesin, other proteins come into play here • Stem binds to dynactin (a complex of proteins) o Dynactin is a complex of proteins where one end binds to dynein at the stem and the other end binds to cargo • Instead of having variable light chains bound to the tail (like in kinesin), this has variable proteins in dynactin complex (which is bound to stem) • Heads are moving the cargo, but dynactin is also bound to the microtubule and it slides along o Dynactin has microtubule-binding site, but doesn’t use that to actively move – it simply slides along the MT o Heads are close to the microtubule; is basically bound to microtubule o Dynactin can bind to cargo and to microtubule (acts like a cart) o Motor at the front moves towards (-) end and drags a cart behind it, to which the cargo is bound o Dynactin can have different proteins to bind to different cargos • The microtubules in a cell tend to be oriented in the same direction. [All (+) ends are towards the outside of the cell, away from centromeres.] • With exocytosis, for example, kinesin is involved. With endocytosis, dynein is involved. • Many processes are going on and you can potentially figure out which motor protein is being used • Acellular process going towards (+) end may use kinesin; a cellular process going towards the (-) end may use dynein Cilia and Flagella • Two versions of the same thing, only differ in length • Cilia = 2-10 μm • Flagella = 10-2000 μm • Flagella: propel cells (few) • Cilia: sweep material across tissue (many) • Microtubules make up cilia and flagella • Underlying structure is called axoneme • Both bend, but because they are different in size, they can do various things when bending • Cilia bends and beats (moves things around in terms of liquid) and can cover cell surfaces o Usually cells have lots of cilia on them o For example, cilia on trachea constantly beat and bend to get dust out of our lungs o Are short, in general • Flagella are usually longer and less abundant in cells o For example, sperm cells have one flagella o Usually involved in propulsion Axoneme: the Underlying Structure of Cilia and Flagella • Over 250 proteins • 9+2 array of microtubules typical (others exist) • Outer doublets consist ofAand B tubules • Axonemal dynein • NOTE: Memorize structure • Axoneme: ring of doublets of microtubules and other proteins o 9 doublets + 2 singlets • There are other configurations (e.g. 9+1, 9+0, 10+1, etc.) o 9+2 is typical in our cells • Doublets have anAring and a B ring (RECALL: 13 protofilaments inAtubule, 10 protofilaments in B tubule) • Stable structure – doesn’t tend to polymerize and depolymerize o Also stable in terms of shape o Held in shape by a bunch of proteins (e.g., radial spokes, nexin) • Radial spoke heads (proteins) stick away from the doublets, holding them in place • Nexin (protein) lines the outer ring; connects the doublets together • Axonemal dynein (a special dynein) comes off of theAtubule o Dynein that is found in axonemes o Stem of axonemal dynein is attached to theAtubule o Atubule is the “cargo” o Head domain sticks away from theAtubule and reaches toward the B tubule of the next doublet o So, dynein is present between each of the doublets, where stem is attached to theAtubule and the head is reaching towards the B tubule o As the head tries to reach towards the B tubule, it will try to move towards the (-) end, which will cause bending Axoneme and Basal Body • Axoneme continuous – attach to Basal Body in cell • The basal body is similar to centriole. 9 basal body triplet MTs (Aand B pass through transition zone, C does not) • Axonemes in cilia and flagella come from MTOCs called basal bodies • Basal bodies are made up of triplet microtubules • RECALL: Triplet microtubules haveA(13 protofilaments), B (10 protofilaments), and C (10 protofilaments) rings • The triplet microtubules in basal bodies polymerize the axonemal microtubules o Polymerization occurs until you reach the cell surface o As microtubules get to the cell surface, they go through a transition zone and the C tubule is lost o Go from a triplet to a doublet microtubule as it enters the axoneme o 9 triplets in the basal body becomes 9 doublets in the axoneme (since the C tubule is lost) • Number of doublets in the axoneme = number of triplets in the basal body • Unlike the centriole, these microtubules play a direct role in polymerization o Microtubules in the axoneme come right from the same microtubules (Aand B microtubules) in the basal body o Growth occurs right from the microtubules o Different from centriole, centrioles don’t polymerize (have a percentriolar matrix where things polymerize from) • In the center are center singlets o Number of center singlets does not seem to be related to what goes on in the basal body o Here, there is one center singlet in the basal body, which is lost in the transition zone; at the end, there are two center singlets (9+2) • In cells that have two flagella, two basal bodies are visible that are 90° to each other o The two basal bodies (or barrels of triplet microtubules) look just like centrioles, act as MTOCs o Except here, they are involved in the direct polymerization • Basal bodies, like centrioles, are stable o Made of triplet microtubules o But only basal bodies are responsible for the direct polymerization of theAand B tubules (unlike centrioles) Ciliary Beating • Generated by sliding of microtubules against each other – powered by dynein • "A" tubule of one doublet "walks" along neighbor “B” • Result = MTs slide past each other – but linked to basal and nexin • Doublets are attached together by dynein, a (-) end-directed motor protein o Dynein will want to walk to (-) end o As it tries to walk to the (-) end, there is relative motion o As dynein moves to the left, it will move the MT to the right (relative sliding occurs) • If the doublets are not attached to each other (no motor between them), they will slide past each other • Inside the axoneme, however, they are not free o They are attached together (by nexin) and are held in place (by radial spokes and the basal body – basal body at the bottom won’t allow movement, as well) o Can’t slide; motor tries to move, bending occurs • Need things to be fixed in place o Need nexin and radial spokes to hold things in place so that as the motor protein is moving, there is relative bending o If they weren’t held in place, they would just slide past each other • This has to be very localized – bending has to occur on one side and one side only in order for bending to actually occur • If all the dynein heads bind to all of the doublets and all of them move at the same time, this can just blow itself apart o Bending will not occur and everything will just move relative to each other, and maybe twist itself apart • Movement/bending has to be localized in terms of the ring of doublets • Bending is also localized along that axoneme o Bending occurs as a wave that travels down the axoneme Intraflagellar Transport Moves Material Up and Down • Movement is not related to bending • May be related to stability and signalling
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