Chapter 17. Microtubules. Chapter 17. Microtubules. Chapter 17. Microtubules. Chapter 17. Microtubules. Chapter 17. Microtubules

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1 Chapter 15. Mechanism of Vesicle Formation A reminder: two things I said that we should to keep an eye on for each of the components of the cytoskeleton: The role of polymerization and depolymerization The role of accessory proteins. 1 2 Structure of microtubules. Microtubules are structurally polar. Microtubules display length changes because of polymerization and depolymerization. 3 Fig Drugs can be used to experimentally change the ratio of polymer to dimer. Colchicine binds to dimers and preventing them from polymerizing leads to disassembly of MTs. Taxol stabilizes the polymer leads to net assembly of MTs. Microtubules are functionally polar. In vitro evidence. Minus end Axoneme Plus end 5 6 1

2 Microtubules are functionally polar. In vitro evidence. Minus end Axoneme Plus end The two ends of the microtubule are different. Reason for the names (plus end, minus end) Dimerpolymer molecular binding constants are different for the plus and minus ends. 7 8 The two ends of the microtubule are different (cont). The binding constant for the plus end is higher than that for the minus end. Thus. 9 Both ends can grow if free tubulin dimer concentration is high enough. Both ends will shrink if the free tubulin concentration is low enough. The plus end can grow even at concentrations where the minus end will depolymerize. The two ends of the microtubule are different (cont). Cellular concentrations of free tubulin dimers are generally great enough to cause (slow) net polymerization at the plus end, but low enough to cause rapid depolymerization at the minus end. 10 Chapter 17. Microtubule polarity in cells (modified) The centrosome as a microtubular organizer, 11 Fig Fig

3 Microtubules are dynamic in vitro and in vivo In vitro (Fig 1712) Microtubules are dynamic in vitro and in vivo In vivo Technical challenge Evidence Question of the day Recall the drugs colchicine and taxol. Explain how each of these drugs supports the idea of dynamic instability of microtubules. Use a dividing cell as your experimental model. These dynamic changes involve the plus ends of the microtubules. Minus ends are typically capped by proteins that prevent subunit exchange Two potential problems with the same solution: Problem #1: How can the two ends be kinetically distinct without violating the second law of thermodynamics (or can we make a perpetual motion machine with microtubules?) Problem #2: How do some microtubules shrink while others grow while all MTs are suspended in the same cytosol? (= At the same concentration of tubulin dimers!) 17 One answer to these questions: Tubulin is a GTP binding protein. One of the two GTPs bound to the tubulin dimer is hydrolyzed after incorporation into the microtubule. Thus the minus end will be composed of the oldest dimers and those most likely to have hydrolyzed their GTP to GDP. Thus tubulin GDP has a different (and lower) binding constant from other dimers and therefore tends towards depolymerization. 18 3

4 How can we explain plus end mediated dynamic instability? The mechanism of dynamic instability the tubulin GTP cap Fig Other microtubular binding proteins interact with the plus ends. Some are preferentially associated with growing ends. Some are preferentially associated with shrinking ends. Some are preferentially associated with paused ends. Do these proteins respond to, or cause, microtubular transitions? How might such plus end capping proteins work? Capping proteins Terminology NOT the same as the tubulin GTP cap. The importance of minus end capping proteins (including the centrosome) and plus end capping proteins in stability and longevity. (Fig 1714)

5 Microtubules organize the interior of the cell. Microtubules can form structural networks. Microtubules can link to other organelles. Microtubules often polarize cells. Microtubules can interact directly or indirectly with other cytoskeletal components. Microtubule associated motor proteins can generate motility by walking along the microtubule. (Fig. 1717b) The two types of microtubule associated motor proteins. (Fig. 1717) Both kinesins and dyneins have MT binding sites and cargo binding sites. Kinesins usually move towards the plus ends of MTs. Dyneins always move towards the minus end of MTs. (Fig. 1717) Fig

6 Organelles can move along microtubules. An in vitro example: Organelles can move along microtubules. Another example: the nerve cell (Fig 1715) An in vivo example: pigment granules in fish melanophores. (Video) The position of the ER and Golgi appear to be determined by dynein and kinesin respectively. Distribution of these organelles 33 ER is generally spread out along MTs Golgi is generally near the centrosome. Fig Experimental evidence for the MT dependent positioning of the ER and Golgi. Depolymerize MTs with drugs. ER collapses, Golgi dissipates Wash out drug MTs grow, ER extends, Golgi reforms 35 6