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Western University
Biology 2382B

Section 1 Microtubules (pg. 757-791) 18.1 Microtubule Structure and Organization - mitotic spindle - flagella/cilia - components of axons o Made of 13 longitudinal repeating units called protofilaments o Brain microtubules were purified to find a protein called tubulin, which is a Microtubule Associated Protein (MAP) Microtubule Walls are Polarized Structures Built from αβ- Tubulin - Purified soluble tubulin is a dimer made of two closely related subunits; α and β tubulin of 55000 Daltons each (there is also γ-tubulin, which is used in regulatory function) - Each subunit of the tubulin dimer can bind one molecule of GTP - GTP trapped in the α-tubulin is never hydrolyzed - GTP in the β-tubulin is exchangeable with the free GTP, and can be hydrolyzed - MT consist of 13 laterally associated protofilaments forming a tube whose external diameter is 25nm - Each protofilaments is made up of alternating αβ-tubulin dimers, with each subunit type repeating each 8nm - Each protofilaments has an alpha at one end and a beta at the other end, giving the protofilaments an intrinsic polarity - MT have an overall polarity, one end is (+) and one is (-) - The (+) (fast) end is favoured by polymerization with exposed β subunits - The (-) (slow) end has exposed α subunits - Most MT in a cell consist of a simple tube, a singlet MT, made of 13 protofilaments (rare cases where a singlet contains fewer or more protofilaments) - Doublet & Triplet MT are found in specialized structures such as flagella and cilia (doublet) and centrioles and basal bodies (triplet) - Each doublet/triplet contains a 13- protofilament MT (A tubule) and one/two additional tubules (B and C) consisting of 10 protofilaments each Microtubules Are Assembled from MTOC’s to Generate Diverse Organizations - all MT are nucleated from structures known as Microtubule-organizing centres; MTOCs - In most cases the (-) end of the MT stays anchored in the MTOC - MTOC in interphase is the centrosome and is generally near the nucleus, producing a radial array of MT with the (+) ends toward the cell edges - This provides tracks for MT-based motor proteins to organize and transport membrane bound compartments, such as those comprising the secretory and endocytic pathways - MITOSIS: cells completely reorganize their MT to form a bipolar spindle, assembled from two MTOCs called the spindle poles - Neurons have long processes called axons, in which organelles are passed in both directions along MT. The MT in the axons are not continuous but have been released from the MTOC, but all have the same polarity - The MT in dendrites have mixed polarity (Reason unknown) - Flagella and cilia MT are from an MTOC called a basal body - Centrosomes in animal cells consist of a pair of orthogonally arranged cylindrical centrioles surrounded by pericentriolar material - Centrioles which are about 0.5μm long and 0.2μm in diameter, are highly organized and stable structures that consist of nine sets of triplet MT and are closely related in structure to basal bodies - It’s not the centrioles themselves that nucleate the cytoplasmic MT array, but factors in the pericentriolar material > - γ-tubulin ring complex – this protein complex is located in the pericentriolar material and consists of many copies of γ-tubulin, it is believed γ – TURC acts like a split washer template to bind αβ tubulin dimers - Centrisomes also anchor and regulate the (-) end of MT - Basal bodies have a similar MTOC at the base of cilia/flagella, the A&B tubules of their (mtoc) triplet MT provide a template for the assembly of the MT that make of the core structure of the cilia and flagella 18.2 Microtubule Dynamics - MT lifespan during mitosis: 1 minute - MT non-mitotic lifespan: 5-10 minutes - Microtubule lifespan is longer in axons and much longer in flagella and cilia Microtubules Are Dynamic Structures Due to Kinetic Differences at Their Ends - MAPs catalyze MT (de)polymerization - Disassembly of MT occurs at 4ºC and reassemble into MT again at 37ºC - A slow nucleation phase, followed by a rapid elongation phase and then a steady state phase in which assembly is balanced by disassembly - For assembly to occur, the αβ tubulin concentration needs to be above the critical concentration (Cc); when αβ is above the Cc, then polymerization occurs – and when it’s below Cc, the MT depolymerise - The higher the concentration above the Cc, the faster they MT polymerizes - When the concentration is below a certain point, there is tread-milling Dynamic Instability - MT undergo dynamic instability, meaning that they undergo periods of growth followed by periods of shrinkage - A growing MT has a blunt end, whereas depolymerising end has a frayed end - Growing MT with blunt ends terminate in GTP β tubulin, whereas shrinking ones with frayed ends terminate in GDP β tubulin. - Therefore, if the GTP in the terminal β tubulin of a MT become hydrolyzed, a formerly blunt-end growing MT will curl and fray - Growing MT are capped by GTP-β tubulin Drugs Affecting Tubulin - Colchicine causes depolymerisation of MT (binds to tubulin diamers so that they can’t polymerize) – joint pain of acute gout, relieves inflammation caused in gout by reducing the MT dynamics of WBC, rendering them unable to migrate efficiently to the site of inflammation - Taxol provides stability to growing MT, taxol stops cells from dividing by inhibiting mitosis, it has been used to treat come cancers (ie. Breast and ovary- these cells are very sensitive to the drub) Regulation of Microtubule Structure and Dynamics - MT are stabilized by side and end binding proteins called MAPs - There are many different proteins that stabilize MT, many of them show cell specificity. - Best studied are the tau family of proteins which includes; tau, MAP2 and MAP4 - These proteins have a modular design, with 18-residue positively charged sequences, repeated three to four times, that binds to the negatively charged tubulin surface and a domain that projects from the MT wall. - Tau proteins are believed to stabilize MT and also act as spacers between them - MAP2 is found only in dendrites, where it forms fibrous cross-bridges between MT and links MT to intermediate filaments, IF - Tau is present in both axons and dendrites - In many cases the activity of the MAPs is regulated by the reversible phosphorylation of their projection domain - Phosphorylated MAPs are unable to bind to MT; thus phosphorylation promotes depolymerisation - Microtubule-affinity-regulating kinase (MARK/Par-1) is a key modulator of tau proteins - Some MAPs are also phosphorylated by a cyclin-dependant kinase (CDK) that plays a major role in controlling the activities of proteins in the course of the cell cycle - Some MAPs associated with the (+) end of MT, and in some cases only the (+) ends of growing MT - This class of proteins are known as +TIPs, and they perform varied functions when present at the MT tip - Some selectively stabilize the (+) end against shrinkage or enhance the frequency of rescues (promote continuous growth) - Other +TIPs are associated with proteins used for attachment to an organelle or cell wall (migrating cell leading edge) - One specific +TIP is EB1, which is associated with the (+) end of the MT as well as the seam; location is known, function is not entirely know; some package and move etc etc Section 2 Microtubules are disassembled by End Binding and Severing Proteins - binding kinesin-13 to remove dimers from the (+) end which will cause depolymerisation, ATP usage is needed for this reaction - Stathmin binds tubulin dimers in the curve and may also cause GTP hydrolysis, removing the cap at the (+) which will induce shrinkage – it has been found that phosphorylation near the leading edge of motile cells inactivates Stathmin/Op18 - Katanin severs and releases anchored MTs and this also removes the cap at the (+) end which will allow for depolymerization to occur Microtubules: tracks for transport - Vesicle transport is bi-directional - Motor proteins doing the movement and they need ATP in order to move anything - Axonal transport: observed from a squid giant axon, uses ATP to move things up and down the MT Kinesin-1 Powers Anterograde Transport of Vesicles Down Axons Toward the (+) end of MT - 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 are (+) directed - The head binds to the MT and uses ATP to “walk” along the MT - The light chain recognizes cargo - - Kinesin 1&2, work by walking towards the (+) end, pulling a type of “cargo” (ie. Organelles) - Kinesin 5: works between MT, heads on both ends and walk in opposite direction causing a sliding - Kinesin 13: works in the disassembly of MT by removing dimers from the (+) OR (-) end Kinesin Movement: By starting with the leading head in a nucleotide-free state, under which conditions it is strongly bound to a β- subunit, with the trailing head in a weakly bound state containing ADP. The energy associated with binding ATP to the leading head induces a forward motion of the linker domain of that head that then physically docks into the core head domain. This movement results in the linker domain pointing forward and physically swinging the trailing head into a position where it becomes the leading head. The new leading head finds the next β subunit, which induces dissociation of the ADP and tight binding. Importantly this step also induces the now trailing head to hydrolyze ATP to ADP and release Pi and be converted into a weakly bound state. The leading head is now ready to bind to ATP and start the cycle over again. Dynein - Involved in retrograde transport - Heavy chain heads have ATPase activity - Dynein associated with a dynactin heterocomplex is what is used to recognize and bind to cargo - Works by moving towards the centre of the cell Cilia and Flagella - cilia 2-10μm - flagella 10-2000μm - Flagella are used to propel the cell forward - Cilia are used to sweep material across the tissue - Cilia and Flagella are both bending - Axoneme: the underlying structure of cilia and flagella - Over 250 proteins - 9(doublets)+2(singlets) array of MT typical (others exist) - Outer doublets consist of A and B tubules - All doublets are kept in place by Nexin and Radial Spoke Head - Doublets cause the bending because Dynein head binds to the tail and wants to walk to the (-) end - Axoneme continues and attaches to the Basal Body in the cell - Basal Body is similar to centrioles; 9 basal triplet MTs (A and B tubules pass through the transition zone whereas the C tubule does not. Ciliary Binding - Generated by sliding of MT against each other- powered by dynein “A” tubule of one doublet “walks” along neighbour “B” - Result is that MTs slide past each other but are linked to Nexin and Basal and therefore bending occurs - Movement is well regulated but we don’t know how - No nexin causes the A and B tubules to become parallel to each other Karyokinesis and Cytokinesis - cytokinesis divides cytoplasm during mitosis, by using actin and myosin filaments - Mitosis contains two kinesis - Centrosome Duplicates and facilitates novel mitotic MT dynamics - G0 MT half life of about 10 minutes - Half life drops to about 30 seconds during mitosis Mitotic Apparatus - after the centrosomes have duplicated and move to opposite ends of the cell, the ability of the MTOCs to nucleate MT is increased greatly as they accumulate more pericentriolar material. Because MT radiating from these two MTOCs now resemble stars, they are often called mitotic Asters - The three types of MT that make up the mitotic apparatus are; - Polar MT, which move towards the opposite ends of the pole with no chromosomal contact - Astral MT: extend from the spindle poles to the cell cortex. By interacting with the cortex, the MT perform the critical function of orienting the spindle with the axis of cell division - Kinetochore MT: link the spindle poles to the kinetochores on the chromosomes Centromere: attachment site for microtubules, kinetochore proteins mediate attachment of chromosomes to MTs - the (+) end is actually free, KC is grabbing MT away from the (+) end - the (+) end can (de)polymerize if needed in order to push or pull the kinetochore into place - the (+) end of the MT attaches to the outer kinetochore SPINDLE: Kinetochore and Polar Microtubules - have to capture all the chromosomes from both poles - need to be moved to the metaphase plate, centre between two poles - well regulated (not sure how) - Spindle finds out if the chromosomes are attached at both ends, then moves to the centre - Dynamic MT the push and pull the chromosome to the centre by polymerizing or depolymerizing the (+) end of the MT - In order for the kinetochore to move up the MT as it is being polymerized or depolymerised, there needs to be plus end and minus end motor proteins moving it; the motor proteins that are moving it are Kinesin or Dynein - Separation only occurs when everything is captured and everything is lined up Anaphase A: chromosome from centre to the pole, so the distance from the chromosome to the pole SHRINKS (driven by microtubule disassembly). Kinetochore MT is being depolymerised. Dynein is also attached to the microtubule and walking it to the end. There is also some very, very minor shrinkage at the minus end at the pole in order to help bring the chromosome to the pole. Anaphase B: Pushing the poles apart and elongating the cell. In the spindle, in addition to kinetochore MT there is Polar MT that are reaching from one pole to the other, they over lap each other and become parallel to each other. Kinesin 5 works in between the two polar MT, sliding them apart (pushing the poles apart). The importance of this part is so that all the chromosomes are far enough apart so that when cytokinesis occurs to pinch off the cell, no chromosomes are being lost. Chromosomal movements in Anaphase B is driven by motor proteins - At the same time that the poles are being pulled apart, they are also being pulled to the plasma membrane - Astral MT are participating in Anaphase B. At the end of the astral MT is Dynein [(-) end directed motor] stuck to the plasma membrane and astral MT, because it’s stuck to the MT and PM and is pulling towards the (-) which will in turn pull the MT towards the PM. But if the MT is being pulled towards the PM, then the (+) end of the MT is depolymerizing in order to prevent it from being shoved through the PM - Mandatory Check Points: Chromosomes MUST line up, MUST be separated, the poles MUST move apart Section 3 Actin Filaments (pg. 713-731) Microvilli: finger-like projections on epithelium that increase the area of the PM on the apical surface in order to increase absorption Cell Polarity: the ability of cells to generate functionally distinct regions – an additional and fundamental example of cell polarity is the ability of cells to divide: they must first select an axis for cell division and then set up the machinery to segregate their organelles along that axis - A cell’s shape and its functional polarity are provided by a 3d filamentous protein network called the cytoskeleton: it extends throughout the cell and is attached to the PM and internal organelles, providing a framework for cellular organization (not set in stone like the bone skeleton) - The cytoskeleton is composed of three major filament systems, all filament systems are composed of a polymer of assembled subunits – these undergo regulated (dis)assembly that give the cell it’s flexibility Microfilaments: polymers of the protein actin organized into functional bundles and networks by actin- binding proteins. - MF are important in the organization of the PM, including surface structures such as microvilli - MF can function on their own or serve as tracks for ATP-powered myosin motor proteins, which provide contractile function or ferry cargo along the MF Microtubules: the second type of filament to make up the cytoskeleton (organizational – mitotic apparatus – see sections 1&2) Intermediate Filaments: tissue-specific filamentous structures providing a number of different functions, including structural support to the nuclear membrane, structural integrity to cells in tissues, and structural and barrier functions in skin, hair and nails (keratin – Epithelial) Microfilaments and Actin Structures - Normally has a cortical function, “cortical” meaning on the outside or just under the PM - Exocytosis: golgi – PM (need to pass from a MT to a AF) - Forming networks and bundles Structure - built from monomers - different isoforms 4 alpha, beta and gama actin - G-actin (globular) polymerizes into F- actin (filamentous) microfilaments - Cleft in the structure of G-actin is where the ATP binds - The ATP cleft gives the actin polarity (+/-) ends – and therefore G-actin polymerizes with polarity - If you take purified actin and add S1myosin, you can “decorate” the actin, a “barbed/arrowhead” structure where the point is always pointing to the (-) end - In the initial nucleation phase, ATP-G-actin monomers slowly form stable complexes of actin. These nuclei are rapidly elongated in the second phase by the addition of subunits to both ends of the filament. In the third phase, the ends of actin filaments are in a steady state with monomeric G-actin - G-actin is polymerized faster at the (+) end of the filament - The rate of addition of ATP-G actin is much faster at the (+) end than at the (-) end, whereas the dissociation of G-actin is similar. This difference results in a lower critical concentration at the (+) end. At steady state, ATP-actin is added preferentially at the (+) end, giving rise to a short region of the filament containing ATP- actin and regions containing ADP-Pi – actin and ADP-actin toward the (-) end. Regulation of Actin Treadmilling by Profilin and Cofilin - Cellular levels of G-actin are ~400μm but the Cc for polymerization is 12μm! Therefore there needs to be some regulation in how much is polymerized - Thymosin: a small protein that binds to ATP- G actin in such a way that it inhibits addition of the actin subunit to either end of the filament. Can be very plentiful (human platelets) - Profilin: a small protein that binds G-actin on the side opposite to the nucleotide-binding cleft. When profiling binds ADP-actin, it opens the cleft and greatly enhances the loss of ADP, which is replaced by the more abundant cellular ATP, yielding a profiling ATP actin complex. This complex binds back to the (+) side - Cofilin: small binding protein that binds specifically to F- actin in which the subunits contain ADP, which are the older subunits in the filament towards the (-) end. Cofilin binds by bridging two actin monomers and inducing a small change in the twist of the filament. The twist destabilizes the filament, breaking it into short pieces. By breaking the filament, cofilin generates many more free (-) ends and therefore greatly enhances the disassembly of the (-) end. The released ADP-actin subunits are recharged by profiling. Actin Capping Proteins - CapZ: binds to the (+) end and prevents growth or dissociation. Made of two closely related subunits and binds with a very high affinity - Tropomodulin: binds to the (-) end, inhibiting growth or dissociation. - Actin Disrupting Drugs: Cytochalasin (depolymerises actin filaments) and Phalloidin (stabilizes actin filaments) - Assembly and Branching - There are two classes’ of actin nucleating proteins; formin and the Arp2/3 complex. - Formin leads to the assembly of long actin filaments - Arp2/3 leads to branched networks Formins - Found in all eukaryotic cells, diverse with seven different classes. Although diverse, they all have two adjacent domains in common, the FH1 & FH2 (the formin homology domain) - Two FH2 domains for a dimer (the doughnut thing) and provide a basic nucleating function of formins - FH2 complex binds to two actin subunits, holding them so that the (+) end is toward the FH2 – “rocks” back and forth between the alternating subunits that are being added - The FH1 is adjacent to FH2 and is rich in proline residues that are sites for the binding of several profiling molecules (ADP -> ATP actin). The FH1 complex serves as a “landing zone” in order to increase the local concentration of ATP actin that are used by the FH2 complex. *Rapid Assembly of the (+) end f-actin. - Formins bind in such a way to prevent capping of the (+) end – therefore no termination of assembly - Formin activity is regulated on the PM by RhoGTP. Rho is on the PM, and when it is activated (and ONLY when it is activated) by GTP it binds to a Rho-Binding-Protein (RBP) which then activates FH1 and FH2. - Responsible for stress fibres and in the contractile ring of mitosis Arp2/3 Complexes - A protein machine consisting of seven subunits, two of which are actin-related proteins (Arp) - Alone it is a poor nucleator when added into an actin assembly assay - To be active, Arp2/3 needs to bind to both a regulatory protein (i.e. WASp) a preformed actin filament – with both of these things, Arp2/3 is a potent nucleator - When it binds to the side of F-actin in the presence of an activator, it changes conformation so that two actin-related proteins, Arp2 & Arp3, resemble the (+) end of an actin filament; thus providing a template for the assembly of a new filament - The new (+) grows as long as there is ATP G-actin available or until it is capped - The angle between the old and new filament is 70º - which is the angle observed in branched filaments at the leading edge of motile cells, which is believed to be formed by the action of activated Arp2/3 - The activati
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