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Lecture 9

Biology 2290F/G Lecture 9: The Cytoskeleton - Microtubules Pt. 1

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Western University
Biology 2290F/G
Ray Zabulionis

Section 1: The Cytoskeleton – Microtubules Pt. 1 The Cytoskeleton - Intricate network of protein filaments that extend throughout the cytoplasm - Microfilaments 7-9nm, microtubules 25nm, intermediate filaments 10nm - These are the different proteins that make up the cytoskeleton: microfilaments, microtubules, intermediate filaments - Named according to their size: smallest is microfilaments (actin), intermediate filaments, largest ones are microtubules (tubulin) - Memorize everything on this slide!! How is movement achieved? - Cellular trafficking of organelles - Cell migration (networks vs. ropes vs. rods) - Don't really need to memorize this! - Movement achieved by cell migration, movement within the cell Cytoskeleton roles - Organelle/protein trafficking, cilia/flagella, mitosis/cytokinesis, muscle contraction, cell adhesion, cell migration, extravasation Microtubules 1: Structure - Polymer of alpha and beta tubulin - Monomers 55kDa each - Alpha-beta dimer is the basic “subunit” - Have polarity - Hollow tubes, 25nm diameter, up to 100s of micrometres long - Memorize this slide - Largest component of the cytoskeleton in terms of diameter (25nm) - Made up of proteins called tubulin o These proteins can polymerize (monomers form dimers, forming microtubules) - Can be hundreds of micrometres long (way longer/bigger than a cell) o How is this possible? Microtubules make up cilia and flagella, which stick out of the cell - Different isoforms of tubulin; main component of microtubules are alpha and beta tubulin o Both about 55kDa each (very similar to each other) - When cell translates alpha and beta monomer, they instantly form a dimer, which is the basic building block - If cell is building microtubules it's using dimers - Cell almost never depolymerizes a dimer into a monomer, unless you're using super super strong denaturing agents o Dimer is pretty solid, very hard to pull apart o Alpha beta dimer forms end-to-end structures, forming a long chain called a protofilament (yellow thing in the picture) - Polarity: one side is different from the other side o In the dimer, one side is alpha, the other side is beta o Alpha beta dimer always polymerizes in the same direction: alpha-beta-alpha-beta - Make protofilaments by connecting dimers, then take 13 filaments and connect them into a hollow tube called a microtubule, which has polarity o Hollow tube is 25nm in diameter - One end of the microtubule has alpha subunits, other end has beta subunits - Alpha end called the (-) end, beta end called the (+) end, this is due to how polymerization works and how the cell grows, NOT DUE TO CHARGE - When protofilaments come together to form microtubule, a seam is formed o The ends of the protofilaments don't line up properly, and this is the seam - we don't know why this happens, but it just does Dimeric Tubulin Subunit - Dimer very stable - Alpha binds permanently to GTP, beta can hydrolyze GTP so can be bound to GTP or GDP - As the polymer (protofilament) grows, beta’s GTP is hydrolyzed - Dimer is very stable, has the ability to bind GTP o Both alpha and beta subunits can bind GTP - Beta subunit can also hydrolyze GTP into GDP (alpha can't do this) - Hydrolysis takes place as polymerization of microtubules occurs Arrangement of MT Protofilaments - Singlets (most common) – 13 protofilaments forming a single tube of 25nm diameter - Doublets and triplets (stable) can also be formed o Made of 13 protofilament tube with one or two 10 protofilament tubes attached - Protofilaments form microtubules - Most microtubules in the cytoplasm are in singlet structure o Formed by 13 protofilaments, can be hundreds of micrometres in length - Singlet microtubules are dynamic: always growing and shrinking - Can also have doublet and triplet microtubules o They all start off with singlet microtubule o Doublet microtubule has an "A" tubule with 13 protofilaments, and then you add a second "B" tubule that only has 10 protofilaments - double microtubule has total of 23 protofilaments o Doublet microtubules are stable - once you make them, they do not polymerize/depolymerize o Found often in cilia and flagella - Triplet microtubules have A tubule with 13 protofilaments, B tubule with 10 protofilaments, and C tubule with 10 protofilaments - total of 33 protofilaments o This is very stable, do not polymerize/depolymerize o Found in basal bodies and centrioles - Singlets aredynamic ; doublets and triplets astable Microtubules organize the interior of the cell - 2 types: cytoplasmic and axonemal (cilia etc.) - Singlets are cytoplasmic microtubules - These can grow and shrink all the time - Can alter themselves - During mitosis, cytoplasmic microtubules make up the spindle, separating chromosomes - Inside the axon, you also have microtubules o These microtubules are cytoplasmic microtubules o There are also axonemal microtubules, found in cilia and flagella, which are doublets o Don't confuse axonemal with axon (neuron)!!! o Axonemal microtubules are not cytoplasmic!! - Axon of a nerve cell is cytoplasmic - When you stain for cytoplasmic microtubules, you can almost always see that there seems to be one central area where the microtubules are growing from Microtubule Organization - Microtubule assembly is a dynamic process - Assembly centred around a “centre” MTOC - Microtubules in the cytoplasm are organized - Microtubule organizing centres (MTOC) - Centrosome is the main MTOC in most non-mitotic cells o During mitosis, the spindle poles are MTOCs (different from centrosomes) - In cilia and flagella, basal bodies are MTOCs - A lot of other MTOCs that we don't really understand - When we look at MTOCs, we should notice that the (-) end of the microtubule is ALWAYS in the MTOC o (+) is facing out, so that polymerization happens at the (+) end o Alpha is stuck in the MTOC, beta is sticking out, growing away from the MTOC o In basal body, cilia and flagella, the (+) end is still facing away o In axon, the (+) end is still facing away from the MTOC - When you're transporting something, you're going towards the (+) end - MTOC is (-) end - Exception: dendrites (nerve cells) have microtubules organized in random fashion, some with (+) end going towards cell body, some going out of cell body o This complicates cell transport - MTOC is capping the (-) end, meaning that this end is stuck in the centre, can't grow or shrink - only the (+) that grows/shrinks Microtubules Organization – Centrosome - The centrosome is the major (but not only) microtubule-organizing center (MTOC) in animal cells - Centrosomes contain centrioles - Centrioles not found in plants (still have many MTOCs) - Pericentriolar matrix (gamma-tubulin, augmin complex etc.) - Main MTOC in animals is the centrosome o Not the only one, but almost all animal cells have centrosome - Centrioles are largely what make up centrosomes o Barrel shaped structures, made of triplet microtubules o 2 of them are 90 degrees perpendicular to each other, located within the centrosome - Many other proteins found in centrosomes - Triplet centrioles shown in red, yellow microtubules are growing out of the MTOC, being polymerized o Note: they're not growing out of the centrioles!! - Centrioles probably responsible for duplicating microtubules o Not the main functional unit in terms of polymerizing stuff from the centrosome - All around the centrioles are hundreds of proteins, which are responsible for polymerizing the cytoplasmic microtubules (singlet) that grow from the centrosome - These proteins called the pericentriolar matrix (material around the centrioles o One protein called gamma-tubulin, another called augmin - Note: plants don't have centrioles!! (therefore they don't have centrosomes) o Plants still have many MTOCs, but they're just not centrosomes Centriole Details - Mother and daughter centrioles differ o Significance of this poorly understood - Centrioles surrounded by distinct pericentriolar material - Centrioles are two barrel-shaped structures made of triplet microtubules - One ring of triplet microtubules positioned 90 degrees to the other ring o Don't have to remember their exact position according to the picture! - The centriole structure is stable: don't grow/shrink, but they can replicate during mitosis o How they replicate is poorly understand o During replication, one centriole is different from the other: mother and daughter are different - In picture, the blueberry things are the mother centrioles o Though structures differ, function may not differ - we don't really know - Centrosome is important for polymerizing cytoplasmic microtubules o This polymerizing is carried out by pericentriolar material o One of the main proteins involved is gamma-tubulin Gamma-Tubulin Nucleates Polymerization - Gamma-tubulin ring complex (many proteins – augmin) provides nucleating sites for microtubules - Recall: alpha and beta tubulin make up the microtubulin dimer - Y-tubulin only found at the (-) end - Part of the y-tubulin ring complex (don't understand how this works) o Augmin is almost always present in this complex - This complex works to begin polymerization of a singlet microtubule - The alpha part of the dimer inside the MTO
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