Lecture 8: Microtubules

Microtubules Microtubule is a stiff hollow tube Microtubule is composed of 13 parallel protofilaments Microtubule is very difficult to bend Microtubule is hollow inside Microtubule is the straightest and the stiffest structural element in the cell The tubulin subunits all point in the same direction, which make a microtubule a polar filament Alpha-tubulin is exposed at one end and beta-tubulin at another Tubulin subunits assemble head to tail to create polar filaments Tubulin is a heterodimer composed of alpha-tubulin and beta-tubulin proteins Alpha-tubulin and beta-tubulin are bound by non-covalent bonds Each alpha- and beta-tubulin subunit has a site for GTP binding The GTP inside the alpha-tubulin is trapped and cannot be exchanged or hydrolyzed The beta-tubulin is a GTPase Straight conformation of the GTP-bound end makes it less prone to dissociation The protofilaments, which contain GTP, are straight; those with GDP are curved The curved ones disassociate easier, since they lose their lateral connections GTP-bound plus ends are more likely to be stable and add more subunits; GDP-bound end is more likely to be unstable and lose subunits Proteins that bind along the side of microtubules Proteins which bind along the sides of microtubules are called MAPs (microtubule-associated proteins). They can cover all the length of microtubules Specific MAPs, MAP2 and tau, can affect the distance between microtubules and other cell components. The free end of MAP2 can contact other MAP-coated microtubules and organize them Which filament is the hardest to bend? • A. Microtubule • B. Actin • C. Intermediate filament Dynamic instability If an end has GTP, it will most likely add subunits (rescue) If it has GDP, it will most likely shrink (catastrophe) If the GTP hydrolysis catches up with the addition of new subunits, an end can start shrinking If a new unit can be added, it can get stabilized and switch to growing Each end can change from growing to shrinking and back The phases of growing and shrinking involved addition or loss of clusters of subunits This phenomenon is called dynamic instability Dynamic instability is more pronounced for microtubules than for actin filaments Dynamic instability happens at certain concentration of tubulin Gamma-tubulin regulates the nucleation of microtubules Microtubules are nucleated at microtubule-organizing center (MTOC) Microtubules are nucleated at their minus end Plus end is going outward from MTOC A gamma-tubulin complex (gamma-TuRC) serves as a template to assemble new tubulin subunits into the filament ARPs and gamma-tubulin Both are very similar to the actin or microtubules subunits Very conserved Both serve to overcome the barrier for nucleation of the filaments Arose by gene duplication and divergence to create a protein with nucleation function Centrosome In most of the animal cells, the microtubule organizing center is well defined and called a centrosome Centrosome is located close to the nucleus Microtubules emanate from the centrosome in an astral conformation Microtubules are nucleated at centrosome at their minus ends; plus ends grow and shrink by dynamic instability Microtubule organizing center serves to ______ • A. Overcome a problem of inefficient nucleation of filaments • B. Promote actin polymerization for the cell movement • C. Promote the strength of the cellular layers Centrosome structure Centrosome is composed of: A fibrous centrosome matrix More than 50 copies of gamma-tubulin ring complex Other proteins, most unknown Two centrioles at a right angle relative to each other Most plant cells and fungal cells do not have centrioles High plant cells nucleate microtubules at the nuclear envelope Centrioles A centriole consists of a short cyclin der of modified microtubules and a large number of accessory proteins Alternating growing and shrinking of the plus end is called ______ • A. Dynamic instability • B. Treadmilling Microtubule motor proteins: dyneins and kinesins Kinesins Kinesins have similar structure to myosin II Most of them move to the plus end of microtubules Dyneins: Structural unrelated to kinesins or myosins Move to the minus end of microtubules Kinesins Motor domains are similar C domain of kinesin-1 is for carrying cargo Kinesin-3 carries membrane-enclosed organelles Kinesin-5 is for sliding microtubules in opposite direction Kinesin-13 is a catastrophe factor, lost motor activity Kinesin-14 moves to minus end instead of plus end Kinesin movement ATP hydrolysis is the rear domain The rear domain with ADP is released from a microtubule Exchanges of ADP to ATP in the front domain The step movement: rear domain moves to the front Which of the following does not move along microtubules? • A. Myosin • B. Kinesin • C. Dynein Dyneins Minus-end directed Have several chains with motor domains, which are larger than kinesin motor domain. Also a number of light chains Dyneins are very fast: about 14 um/sec, five times faster than kinesins Dynein movement Dynein molecule has a shape of a ring with a stalk. Stalk connects to a microtubule One of the subunits of the ring is an ATPase The formation of the ring is associated with the dynein movement The dynein cycle is similar to the kinesin cycle Attachment of the motor proteins to the cargo Dynein attaches to the membrane of an organelle through a protein complex In some cases dyneins can interact directly with their cargo Kinesins attach to the special transmembrane receptors located at the membrane of organelles Dyneins move Golgi complex A. To the centrosome B. To the plasma membrane Motor proteins mediate intracellular transport of membrane-enclosed organelles A major function of motor proteins is intracellular transport and positioning of membrane-enclosed organelles Kinesins are involved in transport of mitochondria, secretory vesicles precursors, etc. Myosins also have a significant role in transport along the actin filaments (endosomes, melanosomes, etc.) mRNA can also be transported along the filaments to the specific cellular sites where the corresponding protein will be needed. This has a role in development Motor protein activity can be regulated by phosphorylation Kinesins and dyneins mediate polarized transport along microtubule highways Kinesins move to the plus end Plus end is at the cell membrane Kinesins extend the network of ER tubules laos to the edge of the cell. If microtubules are polymerized with a drug, ER collapses to the center of the cell Golgi is located close to the centrosome where the minus ends are. Dyneins move Golgi apparatus to the centrosome. In the absence of microtubules Golgi is dispersed in the cytoplasm Intermediate filaments Intermediate filaments can be nuclear or cytoplasmic Cytoplasmic intermediate filaments: Not in all eukaryotic cells, and only in some metazoans, including vertebrates Many different types More prominent in cell, which are subject to mechanical stress Nuclear intermediate filaments: Only one kind: lamins Line the interior of the nuclear envelope In all eukaryotic cells The structure of intermediate filaments Parallel polypeptide chains form dimers Two pairs of dimers connect in antiparallel fashion to form tetramers The tetramers form filaments by lateral connections No GTP or ATP binding sites Non-polar, symmetrical ends Intermediate protofilaments are kept together by strong lateral contacts The lateral bonds in intermediate filaments are stronger than in microtubules; intermediate filaments are very hard to break The intermediate filament can tolerate stretching and bending, forming long rope-like structures Microtubules breaks easier when they are bent than intermediate filaments Intermediate filaments are organized into high order structure by cross linking Protein plectin links intermediate filaments to each other, to actin filaments, microtubules, and other cell membrane structures Septins Septins are additional set of filaments in the cell They are non-polar, form rings and cage-like structures Recruit actin-myosin ring in cell division Serve as barriers to diffusion when proteins need to be localized