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Lecture 9

Biology 2382B Lecture 9: The Cytoskeleton

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Western University
Biology 2382B
Robert Cumming

Lecture 9 – The Cytoskeleton Cytoskeleton – general characteristics/sizes/distributions The cytoskeleton - Photo is proteins that make up cytoskeleton - Cytoskeleton: intricate network of protein filaments that extend throughout the cytoplasm of most cells - 3 types of cytoskeletal proteins: o Microfilaments (7-9 nm) o Intermediate filaments (10 nm) o Microtubules (25 nm) - Largely made according to their size o Smallest in terms of their diameter: actin o Intermediate ones: intermediate filaments o Largest: microtubules  made of tubule QUESTION: how is “movement” achieved? - Cellular trafficking of organelles - Cell migration (networks, vs ropes vs rods): cell moving involving the cytoskeleton - Things moving in a cell: anterograde and retrograde transport - They move because of the cytoskeleton - Some cytoskeletal components can form a network (like a mesh) o Network of protein - Some components can act like a rod or a stick and are hard so you can poke things with them o Move things by poking - Some components are like ropes  can pull with ropes Cytoskeleton roles - Organelle/protein trafficking o Anterograde and retrograde transport - Cilia/flagella - Mitosis/cytokinesis - Muscle contractions - Cell adhesion - Cell migration - Extravasation Biological molecules are 3-dimensional and take up space! - Molecules exist in 3D space and take up space - Have a particular size and shape - When talking about something happening in a cell, need to think about the 3D space - Cell is not empty – everything is full  there is no empty space - Example: RBC is in blood stream and blood stream has water and protein in it - Microtubules and intermediates, actin and myosin take up space - Nothing occurs in empty space Microtubules 1: structure - Largest component of the cytoskeleton in terms of diameter o 25 nm diameter o Microtubules can be up to 100s of m long - Made up of protein tubulin o Proteins can polymerize o Small monomers form dimers and the dimers polymerize and form long structures called microtubules - Microtubules can be longer/bigger than a cell o They make up structures such as cilia and flagella o Flagella: back side of a single cell organism/sperm - There are different isoforms of tubulin o Main components of microtubules are alpha and beta microtubulin - Polymer of  and b tubulin o Both monomers 55kDa each o Similar to each other - When a cell translates the alpha and beta monomer, they almost instantly form a dimer o Alpha beta dimer is the basic building block o If a cell is building microtubules, its using dimers o If it is depolymerizing microtubules, it depolymerizes it to dimers ▪ Almost never depolarize the dimer into monomers - If isolating microtubules from a cell, unless you’re using a very very strong denaturant, you see dimers not monomers - In the cell, the dimer is the building block o b dimer is basic “subunit” - Protofilament: end to end alpha beta chain o Alpha beta dimers can polymerize and form end-to-end structures o Can bind to other dimers and get a chain - Have polarity: one side is different than the other side (nothing to do with charge) - Dimer: one side is alpha and one side is beta o Always polymerizes in the same orientation o When make a protofilament, one end is always alpha and one end is always beta ▪ It does not alternate alpha beta - Can take 13 proto filaments and put them together into a hollow tube structure = microtubule - Hollow tube structure is 25 nm in diameter and all the protofilament that make up the microtubule has polarity so microtubule has polarity o One end is alpha subunit and the other side is beta subunits - Dimer, protofilament and microtubule has polarity – difference between the two ends - Alpha end (-), beta end (+)  the way polymerization works (nothing to do with charge – has to do with growth) - Growth prefers to occur at the + end - Almost all have cells have microtubules – many proteins and tubulin in our cells - Alpha beta dimer is 8nm in size - As things move along the microtubule, can predict how things are moving because we know the sizes of things o Predict how motor proteins are binding - If things are moving at 4nm increments, its going from every monomer to monomer - If things are going at every 8nm, it is going from alpha to alpha or beta to beta - When protofilaments come together to form a microtubule, there is a structure called a seam - Seam is when things don’t line up properly - Dimer is stable Dimeric tubulin subunit - Dimer VERY stable - Both alpha and beta subunit can bind GTP - Difference between alpha and beta: o Beta can hydrolyze GTP  GDP ▪ Beta can be bound to GTP or GDP depending on if it was hydrolyzed o Alpha is always bound to GTP - Hydrolysis takes place as you get polymerization of the microtubule -  binds permanently to GTP,  can hydrolyze GTP so can be bound to GTP or GDP - As the polymer (protofilament) grows, ’s GTP is hydrolyzed - Hydrolysis is involved in forming a protofilament Arrangement of MT protofilaments - Protofilaments come together to form microtubules - Most microtubules in the cytoplasm are singlets - Singlets - most common – 13 protofilaments forming a single tube of 25nm diameter o Singlet microtubule is the working microtubule that can be found in most of the cytoplasm - When you stain for tubulin inside a cell, most of it is singlet, cytoplasm microtubule - Singlets are very dynamic and are always growing and shrinking (polymerizing and depolymerizing) - Doublets and triplets microtubules (stable) can also be formed o Made of 13 protofilament tube with one or two 10 protofilament tubes attached - Start off as single microtubule with 13 protofilaments - If you’re making a doublet, singlet is called the A tubule (A tubule with 13 protofilaments) o Doublet: add another tubule to it and it only has 10 protofilaments (B tubule) - Doublet microtubules: 23 protofilaments o A tubule with 13 – like a singlet o B tubule with 10 o 2 rings of protofilaments (13 and 10) - Doublet microtubule vs. singlet: o Double are stable – when you make them, they are stable and don’t polymerize or depolymerize ▪ They are found in structures called cilia and flagella - Triplets: A (13), B (10), C (10) o 33 protofilaments o Like double, the configuration is stable - don’t polymerize or depolymerize o Found in basal bodies and centrioles - SUMMARY: o Singlets: dynamic and always growing and shrinking and found in most of cytoplasm o Doublet: stable, found in cilia and flagella o Triplets: stable, found in basal bodies and centrioles - If stain the cell for alpha and beta tubulin, will see microtubules throughout the cytoplasm - Cytoplasm microtubules are singlets (most common in cell) and they can polymerize and depolymerize  make up most of the MTs in the cytoplasm o Constantly growing/shrinking o Can alter themselves - Have cytoplasmic microtubules during mitosis – during interphase they make up the mitotic microtubules o They are dynamic and they move and their movement is what separates the chromosomes - Microtubules organize the interior of the cell - 2 “types” – cytoplasmic and axonemal (cilia etc) - Axonemes: part of a structure of a neuron – MTs in neurons o Have an axon (bring signal towards cell body) and a dendrite (bring signal away from cell body) - Inside the axon, have microtubules and they are cytoplasmic microtubules o Cytoplasmic microtubules are singlets and found in cytoplasm o Axons of a nerve cell cytoplasm  microtubules in axon - Axonemal microtubules are in cilia and flagella and are doublets o Not cytoplasmic  not the same as axons Microtubule - When stain for microtubules, that there is one area that they are growing from o Interphase and during the mitotic apparatus o The poles of the mitotic apparatus: where the microtubules are growing - Microtubules in the cytoplasm are organized - Microtubule assembly is a dynamic process o Grow and shrink and because they are dynamic, they need to be organized - Cells have lots of areas called microtubule organizing centers (MTOC) o There are different types of MTOC - Assembly centered around a “centre”  MTOC - If it is a MTOC, it doesn’t say anything about its function other than microtubules grow from it Minus (-) ends of microtubules are associated with MTOC (note dendrites) Microtubule organization – centrosome - Centrosome: main MTOC in most non-mitotic cells - During mitosis when you form the mitotic apparatus, the spindle poles are MTOC but look different than the centrosome - In cilia and flagella, have basal bodies (MTOC) - T he minus ends are always in the MTOC - Centrosome: all the microtubules grow from the plus end away (why the + end is the + end) o Polymerization grows from the + end o Alpha is stuck in the MTOC and beta is the end that is growing (add alpha beta alpha beta) - + end is the end that grows away from MTOC in non-mitotic cells - In mitotic apparatus, have 2 MTOC but the + ends are always away from the center - In a basal body and axon, the + end is away from MTOC o If transporting things along the microtubule in an axon, going to the + end, away from the cell body  have direction o If start at the cell body and go to + end of microtubule, go away - In the case of dendrites (nerve cells) have microtubules organized in a random fashion o Some have + end going towards cell body and some going away o Transport is more difficult o Some face one way and some face the other way - If want to go to + end in most microtubules, know that is away from the center - Almost all MTOC are - in the cell body and + end is away from the center but not like a dendrite - MTOC is capping the - end (it is stuck there whether it’s a centrosome or basal body) o - end can’t grow or shrink o + end does the growing and the shrinking - MTOC is where microtubules grow and binds to and traps the - end and + end grows away from the cell body - If transporting anterograde, going to + end away from MTOC - 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) o Plants do not have centrosomes o Plants have MTOC but they are not called centrosomes because they do not have centrioles - When polymerization occurs, it grows from MTOC - - end (alpha) is MTOC and it is capped (other proteins bound to it so it can’t grow or shrink) - Centrioles: circular arrangement of triplet microtubules and 2 of them are 90 degrees to each other  in a structure called a centrosome o 2 structures that are 90 degrees are in a barrel structure - Microtubules that are growing are not actually contacting centrioles (not growing from there) - Centrioles in centrosome: responsible for duplicating centrosome during mitosis for the daughter cells o Do not play a direct role in polymerizing the microtubules that grow from MTOC o Centrioles are not the functional unit with respect to polymerizing the cytoplasmic microtubules - Around the centrioles are proteins o Proteins are responsible for polymerizing the cytoplasmic microtubules that grow from the centrosome o Group of proteins: pericentriolar matrix ▪ Material around the centrioles ▪ Gamma tubulin and augmin help polymerize microtubules - The proteins in pericentriolar matrix around the centrioles are responsible in aiding polymerization of the microtubules in the cell - Centrioles define a centrosome (main MTOC in animal cells) but other MTOC exist Centriole details - 2 barrel shaped structures made of triplet microtubules that are 90 degrees o 1 ring of triplet microtubules that is 90 degrees to another triplet ring - Ring: triplet microtubules  structure is stable o 13, 10, 10 filaments - Centrioles do not change in size (do not grow or shrink) o They can replicate during mitosis - When they replicate, one centriole is different from the other one o Can tell mother from daughter - Mother and daughter centrioles differ - “Pericentriolar material” o Proteins around the centriole are actually doing the polymerizing ▪ Protein involved: gamma tubulin - Triplet microtubules found in a structure called a centrosome o Centrosome is important for polymerizing cytoplasmic microtubules γ-Tubulin Nucleates Polymerization - Found in pericentriolar matrix - Have alpha and beta tubulin that make the dimer and the large structures of most microtubules - Gamma tubulin: only found at - end and helps start the polymerization process o Part of a group or proteins that makes a complex (gamma tubulin ring complex) - γ -tubulin ring complex (γTuRC) (many proteins - augmin) provides nucleating sites for microtubules o Augmin is always present and helps with polymerization of MTs - Gamma tubulin and augmin work together to begin the polymerization of a singlet microtubule o Microtubule starts with alpha part of the dimer inside the MTOC where the beta part of the dimer is always the growing edge o Add alpha beta alpha end o Facilitate the polymerization of the + end growing away - Gamma tubulin ring complex is at - end, it caps the - end o Can’t grow or shrink because it has protein group attached to it o - end is stable - Growing and shrinking occurs at the + end - Look for gamma tubulin – use antibodies for alpha and beta tubulin: o Alpha and beta will be inside the microtubule o Gamma tubulin is at one end (- end) and the rest of microtubule will be alpha and beta ▪ Only found at one end Polarity of tubulin polymerization - Polymerization is growth o To make something, start off with something smaller - For polymerization to occur, it works better with a nucleus (nucleating agent) - Flagella do not have a nuclei - Different type of nuclei exist - Nuclei: center of something of which you’re going to grow something else - Nucleating agent (nuclei): o Gamma tubulin ring complex – acts like a nucleus where things can grow o Small group of already polymerized microtubules ▪ They can act as as nucleating agent from which things can grow - Nucleation site accelerates initial polymerization - Microtubules: - end and + end  can be represented by a flagella o Take a piece of a sperm or amoeba that has flagella o Isolate a piece of the flagella and it has microtubules o Flagella has polarity (has a + end and a – end) - Microtubule assembly and disassembly occur preferentially at the plus (+) end - Flagella has polarity and it is stable (cannot grow or shrink) o Made of doublet microtubules = it is stable o Flagellar nucleus is stable - Have nucleating agent of a particular size that will not grow or shrink (stable) - Have a piece of doublet microtubules (nucleating agent) and start adding material to initiate polymerization o Need alpha beta dimers to be in the GTP form o Dimers need to be above a certain concentration - If add a little, nothing happens  need to add until you get above critical concentration o When you get above a critical concentration, get polymerization - Get polymerization at both the (-) end and the plus end but it is much faster at the (+) end - Polymerization can occur at a (-) end but in a cell, there is never a free (-) end o MTOC always has a capping nucleus at the (-) end - In vitro and experimentally can get small amount of growth at (-) end but in reality, in vivo in a cell, the (-) end is always capped - Growth occurs primarily and faster at the (+) end  more active - If above the critical concentration, get growth and polymerization - If below critical concentration, get depolymerization - microtubules shrinks at both ends (shrinks faster at + end) - - end is capped and CANNOT grow or shrink (can occur but only experimentally – it is not an important functional aspect of microtubules in the cell) M
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