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Microtubules and molecular motors


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Microtubules and molecular motors

  1. 1.  Serve as cytoskeleton to maintain cell shape Involved in changes in cell shape, & serve as a "temporary scaffolding" for other organelles. They function as diffusion channels for water and metabolites and even macromolecules, thus aiding intracellular transport. In mitosis, microtubules form the mitotic spindle along which chromosomes move. Some cellular proteins act to disassemble microtubules, either by severing microtubules or by increasing the rate of tubulin depolymerization from microtubule ends. Other proteins (called microtubule-associated proteins or MAPs) bind to microtubules and increase their stability. Such interactions allow the cell to stabilize microtubules in particular locations and provide an important mechanism for determining cell shape and polarity.
  2. 2. MICROTUBULES Microtubules, like actin microfilaments, exhibit both structural and functional polarity. Dimeric αβ–tubulin subunits interact end-to-end to form protofilaments, which associate laterally into microtubules. The wall is composed of 13 protofilaments of the protein tubulin, and later bind to microtubule- associated proteins (MAPs).
  3. 3. A stabilizing MAP consists of a basic microtubule-binding domain and an acidicprojection domain. The projection domain can bind to membranes, intermediate filaments, or other microtubules, and its length controls how far apartmicrotubules are spaced. The microtubule-binding domain contains repeats of a conserved, positively charged 4-residue amino acid sequence that binds the negatively charged C-terminal part of tubulin, thereby stabilizing the polymer.
  4. 4. The structure of a microtubule and its subunit. (A) The subunit of each protofilament is a tubulin heterodimer, formed from a very tightly linked pair of α- and β-tubulin monomers. The GTP molecule in the β-tubulin monomer is less tightly bound and has an important role in filament dynamics. Both nucleotides are shown in red. (B) One tubulin subunit (α-β heterodimer) and one protofilament consist of many adjacent subunitswith the same orientation. (C) The microtubule is a stiff hollow tube formed from 13 protofilaments aligned in parallel. (D) A short segment of a microtubule viewed in an EM. (E) EM of a cross section of a microtubule.
  5. 5.  Unlike microfilaments, micro- tubules are non-contractile polarized structures with a (-) end anchored to the centro- some, and a free (+) end at which tubulin monomers are added or removed. They exhibit : (1) treadmilling, the addition of subunits at one end and their loss at the other end, and (2) dynamic instability, the oscillation between growth and shrinkage. Balance depends on whether the exchangeable GTP bound to β-tubulin is present on the (+) end or whether it has been hydrolyzed to GDP.
  6. 6. Stages in assembly of microtubules.Free tubulin dimers form short, unstableprotofilaments (1), which then form more stable curved sheets (2). A sheet wraps around into a microtubule with 13 protofilaments. The microtubule then grows by the addition of subunits (3).The free tubulin dimers have GTP (red dot) bound to theexchangeable nucleotide- binding site on the β- tubulin monomer. After If the rate of polymerization is faster than the incorporation of a rate of GTP hydrolysis, then a cap of GTP- dimeric subunit into a bound subunits is generated at the (+) end, microtubule, the GTP on although the bulk of β-tubulin in a microtubule the β-tubulin is will contain GDP. The rate of polymerization is hydrolyzed to GDP. twice as fast at the (+) end as at the (-) end.
  7. 7. Dynamic instability model of microtubule growth and shrinkage. Only microtubules whose (+) ends are associated with GTP-tubulin (those with a GTP cap) are stable and can serve as primers for the polymerization of tubulin. Microtubules with GDP-tubulin (blue) at the (+) end, or those with a GDP cap are rapidly depolymerized and may disappear within 1 min. Becausethe GDP cap is unstable, the microtubule end peels apart to release tubulin subunits.
  8. 8. Microtubules (blue) organized aroundthe MTOC and spindle poles (1) establish an internal polarity to movements and structures in the interphase cell (left) and the mitotic cell (right). Assembly anddisassembly (2) causemicrotubules to probethe cell cytoplasm and are harnessed at mitosis to move chromosomes. Long-distance movement of vesicles are powered by kinesin and dynein motors. Both motors are critical in the assembly of the spindle and the separation of chromosomes in mitosis.
  9. 9. MOLECULAR MOTORS MOTOR PROTEINS- specialized motility structures in eukaryotic cells consisting of highly ordered arrays of motor proteins that move on stabilized filament tracks. They use the energy of ATP hydrolysis to move along microtubules or actin filaments. They mediate the sliding of filaments relative to one another and the transport of membrane-enclosed organelles along filament tracks.
  10. 10. Motorproteins pullcomponentsof the cyto- skeleton past each other. Motor molecules also carry vesicles or organelles to various destinations along “monorails’ provided by the cytoskeleton.
  11. 11.  All known motor proteins that move on actin filaments are members of the myosin superfamily. Motor proteins that move on microtubules are members of either the kinesin superfamily or the dynein family. The myosin and kinesin superfamilies are diverse, with about 40 genes encoding each type of protein in humans. The only structural element shared among all members of each superfamily is the motor "head" domain. These heads can be attached to a wide variety of "tails," which attach to different types of cargo and enable the various family members to perform different functions in the cell.
  12. 12. Functions of myosin tail domains.(a) Myosin I and myosin V are localized to cellular membranes by undetermined sites in their tail domains. As a result, these myosins are associated with intracellular membrane vesicles or the cytoplasmic face of the plasma membrane. (b) In contrast, the coiled-coil tail domains of myosin II molecules pack side by side, forming a thick filament from which the heads project. In a skeletal muscle, the thick filament is bipolar. Heads are at the ends of the thick filament and are separated by a bare zone, which consists of the side-by-side tails.
  13. 13. Cycle ofstructural changes used bymyosin towalk along an actin filament.
  14. 14. Summary of the coupling between ATP hydrolysis and conformational changes for myosin II. Myosin begins its cycle tightly bound to the actin filament, with no associated nucleotide, the so-called "rigor" state. ATP binding releases the head from the filament. ATP hydrolysis occurs while the myosin head is detached from the filament, causing the head to assume a cocked conformation, although both ADP and inorganicphosphate remain tightly bound to the head. When the head rebinds to thefilament, the release of phosphate, followed by the release of ADP, triggerthe power stroke that moves the filament relative to the motor protein. ATP binding releases the head to allow the cycle to begin again. In the myosin cycle, the head remains bound to the actin filament for only about 5% of the entire cycle time, allowing many myosins to work together to move a single actin filament.
  15. 15.  KINESINS move towards the (+) ends of tubules, while dyneins move towards the (–) ends. Kinesin is responsible for movement of vesicles and organelles in the cytoplasm, dynein regulates 2-way traffic and dynamin serves as a motor for sliding movements during microtubule elongation.
  16. 16. Kinesin and kinesin-related proteins. (A) Structures of four kinesinsuperfamily members. Conventional kinesin has the motor domain at the N-terminus of the heavy chain. The middle domain forms a long coiled coil, mediating dimerization. The C-terminal domain forms atail that attaches to cargo, such as a membrane-enclosed organelle.These kinesins generally travel toward the minus end instead of theplus end of a microtubule. (B) Freeze-etch EM of a kinesin molecule with the head domains on the left.
  17. 17. Summary of the coupling between ATP hydrolysis and conformational changes for kinesin. At the start of the cycle, one of the two kinesinheads, the front or leading head (dark green) is bound to the microtubule,with the rear or trailing head (light green) detached. Binding of ATP to the front head causes the rear head to be thrown forward, past the bindingsite of the attached head, to another binding site further toward the plusend of the microtubule. Release of ADP from the second head (now in thefront) and hydrolysis of ATP on the first head (now in the rear) brings the dimer back to the original state, but the two heads have switched their relative positions, and the motor protein has moved one step along themicrotubule protofilament. In this cycle, each head spends about 50% of its time attached to the microtubule and 50% of its time detached.
  18. 18. CENTROSOME Also called the centrosphere or cell center, which refers to a specialized zone of cytoplasm containing the centrioles and a variable number of small dense bodies called centriolar satellites. Considered to be a center of activities associated with cell division, usually adjacent to the nucleus. The Golgi apparatus often partially surrounds the centrosome on the side away from the nucleus. It consists of an amorphous matrix of protein containing the g-tubulin ring complexes that nucleate microtubule growth. They serve as basal bodies and sites of anchor for epithelial cilia. Plant and fungal cells have a structure equivalent to a centrosome, although they do not contain centrioles .
  19. 19.  The matrix of the centrosome is organized by a pair of centrioles. An electron micrograph of a thick section of a centrosome showing an end-on view of a centriole. The ring of modified microtubules of the centriole is visible, surrounded by the fibrous centrosome matrix.
  20. 20.  Centrioles are self-duplicating organelles that exhibit continuity from one cell generation to the next. They double in number immediately before cell division but they do not undergo transverse fission. Paired centrioles are called diplosome. The long axes of the two centrioles are usually perpendicular to each other. Centrioles become prominent in mitosis. In prophase they separate and a new procentriole develops adjacent to each. Microtubule organizing centers (MTOCs) become nucleation sites around each centriole to form the fibers of the aster and the mitotic spindle. MTOCs determine cell polarity including the organization of cell organelles, direction of membrane trafficking, and orientation of microtubules. Because microtubule assembly is nucleated from MTOCs, the (-) end of most microtubules is adjacent to the MTOC and the (+) end is distal.
  21. 21. A centrosome with attached microtubules. The minus end of each microtubule is embedded in thecentrosome, having grown from a Ý- tubulin ring complex, whereas the plus end ofeach microtubule is free in the cytoplasm.
  22. 22.  In EM, each centriole is found to be a hollow cylinder closed at one end and open at the other. The central cavity is occupied by small dense granules. In transverse section, its wall is composed of 9 evenly spaced triplet microtubules (9 x 3). Each triplet (A, B and C) is set at an angle of about 400o to its respective tangent. Subunit A is nearest to the centriole axis; short fibers connect it to subunit C of the adjacent triplet.
  23. 23. Orientation of cellular microtubules. (a) In interphase animal cells, the (-) ends of most microtubules areproximal to the MTOC. Similarly, the microtubules in flagella and ciliahave their (-) ends continuous with the basal body, which acts as the MTOC for these structures. (b) As cells enter mitosis, the microtubule network rearranges, forming a mitotic spindle. The (-) ends of all spindle microtubules point toward one of the twoMTOCs, or poles, as they are called in mitotic cells. (c) In nerve cells, the (-) ends of all axonal microtubules are oriented towardthe base of the axon, but dendriticmicrotubules have mixed polarities.
  24. 24.  The restructuring of the microtubule cytoskeleton is directed by duplication of the centrosome to form two separate MTOCs at opposite poles of the mitotic spindle. The centrioles and other components of the centrosome are duplicated in interphase cells, but they remain together on one side of the nucleus until the beginning of mitosis. The two centrosomes then separate and move to opposite sides of the nucleus, forming the two poles of the mitotic spindle. As the cell enters mitosis, the dynamics of microtubule assembly and disassembly also change dramatically. First, the rate of microtubule disassembly increases about tenfold, resulting in overall depolymerization and shrinkage of microtubules. At the same time, the number of microtubules emanating from the centrosome increases by five- to tenfold.
  25. 25.  In combination, these changes result in disassembly of the interphase microtubules and the outgrowth of large numbers of short microtubules from the centrosomes. As mitosis proceeds, the two chromatids of each chromosome are then pulled to opposite poles of the spindle. This chromosome movement is mediated by motor proteins associated with the spindle microtubules. In the final stage of mitosis, nuclear envelopes reform, the chromosomes decondense, and cytokinesis takes place. After cell division, each cell acquires 2 centrioles, one from the parent cell, and one which arose as a procentriole. If mitotic cells are exposed to drugs like colchicine (binds to monomeric tubulin and prevent polymerization), vinblastine and taxol (disrupt microtubule dynamics), microtubules disappear and mitosis is arrested because of inadequate formation of the mitotic spindle. These drugs are useful in the treatment of certain cancers.
  26. 26. Effect of the drug taxol on microtubule organization. (A) Molecular structure of taxol. Recently, organic chemists havesucceeded in synthesizing this complex molecule, which is widely used for cancer treatment. (B) Immunofluorescence micrograph showing the microtubule organization in a liver epithelial cell before the addition of taxol. (C) Microtubule organization in the same type of cell after taxol treatment. Note the thick circumferential bundles of microtubules aroundthe periphery of the cell. (D) A Pacific yew tree, the natural source of taxol.
  27. 27. CILIA & FLAGELLA Characteristic 9 x 2 arrangement of microtubules Tubulin forms doublets composed of subunit A, a complete microtubule with 13 protofilaments, joined to a C-shaped subunit B with only 10. Lateral arms composed of the MAP axonemal dynein project from subunit A to subunit B of the next. Major motor portion of the flagellum is called the axoneme.
  28. 28. Ciliary dynein is a large motor protein assembly composed of 9-12polypeptide chains (A) The heavy chains form the major portion of the globular head & stem domains, & many of the smaller chains areclustered around the base of the stem. The base of the molecule binds tightly to an A microtubule in an ATP-independent manner, while the large globular heads have an ATP-dependent binding site for a B microtubule. When the heads hydrolyze their bound ATP, they move toward the (-) end of the B microtubule, thereby producing a sliding force between the adjacent microtubule doublets in a cilium or flagellum. (B) Freeze-etch EM of a cilium showing the dynein arms projecting at regular intervals from the doublet microtubules
  29. 29. The bending of an axoneme. (A) When axonemes are exposed to the proteolytic enzyme trypsin, thelinkages holding neighboring doublet microtubules together are broken.Addition of ATP allows the motor action of the dynein heads to slide one pair of doublet microtubules against the other pair. (B) In an intact axoneme (such as in a sperm), sliding of the doublet microtubules is prevented by flexible protein links. The motor action therefore causes a bending motion, creating waves or beating motions
  30. 30. The contrasting motions of flagellaand cilia. (A) The wavelike motion ofthe flagellum of a sperm cell. Waves of constant amplitude movecontinuously from the base to the tip of the flagellum. (B) The beat of a cilium, which resembles the breast stroke in swimming.