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INTRACELLULAR MOTILITY Lecture 6 January 23rd, 2006

INTRACELLULAR MOTILITY Lecture 6 January 23rd, 2006. Intracellular transport requires microtubules. Stain with an anti-gamma tubulin antibody (red). Gamma-tubulin at initiates synthesis at one end (-) (green). Microtubules assemble from organizing centers. Pericentriolar material.

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INTRACELLULAR MOTILITY Lecture 6 January 23rd, 2006

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  1. INTRACELLULAR MOTILITY Lecture 6 January 23rd, 2006

  2. Intracellular transport requires microtubules Stain with an anti-gamma tubulin antibody (red). Gamma-tubulin at initiates synthesis at one end (-) (green).

  3. Microtubules assemble from organizing centers Pericentriolar material. Each centrosome has 2 centrioles Despite the amorphous appearance, the pericentriolar material of an MTOC is an ordered lattice that contains many proteins necessary for the assembly of microtubules.

  4. The selective stabilization of microtubules can polarize a cell • A newly formed microtubule will persist only if both its ends are protected from depolarization. In cells, the minus ends of microtubules are generally protected by the organizing centers from which these filaments grow. The plus ends are initially free but can be stabilized by other proteins.

  5. How are molecules transported in the cell? Membrane-bound vesicles and proteins are transported many micrometers along very well defined routes and delivered to specific addresses Rapid transport (3mm/s) 250mm/day Slow transport 1mm/day Anterograde transport - towards synapse Retrograde transport - towards cell body

  6. Microtubules provide tracks for movement of vesicles along the axon

  7. Axonal transport does not require an intact cell Extruded axoplasm assays - Cytosol is squeezed from the axon with a roller onto a glass coverslip. Addition of ATP shows movement by videomicroscopy Vesicle movement in this system is about 1-2um/s similar to fast axonal transport.

  8. Experiments that unraveled the mechanism of axonal transport • Synaptic vesicles added to microtubules in the presence of ATP • No Binding and No Movement • -Addition of squid nerve axoplasm preparation (tubulin-free) • Binding and Movement observed • -Squid axoplasm + microtubules + AMPPNP • Binding but No Movement AMPPANP + Microtubules + Brain extract Binding and isolation of the microtubule fraction Release of bound protein with ATP Identification of kinesin

  9. Movement on Microtubules Requires Motor Proteins • Progression of organelles along axons requires microtubules and the motor proteins: kinesin and dynein. Also dependent on motor proteins: • Transport of vesicles for exocytosis/endocytosis or between the endoplasmic reticulum and Golgi • Extension of the endoplasmic reticulum • Integrity and reassembly of the Golgi apparatus • Beads coated with kinesin binds to microtubules and moves along by rotation. • Dynein promotes movement in the opposite direction.

  10. Movement occurs in individual protofilamentsand it is directional Antrograde transport Retrograde transport

  11. Kinesin Dimer of a heavy chain complexed to a light chain Mr= 380kD Three domains: Large globular head Binds microtubules and ATP 2) Stalk 3) Small globular head Binds to vesicles To date 12 different family Members have been identified

  12. Kinesins interact directly with microtubules (movie)

  13. dynein kinesin 10nm Light chains kinesin Heavy chains dynein microtubule 25nm Minus end Plus end Molecular structure of dyneins and kinesins Dyneins - composed of 2-3 heavy chains with a total Mr of 1,000kD - interact with microtubules indirectly (microtubule-binding proteins)

  14. Interaction between cargo and motor protein is indirect

  15. How to build directionality • and specificity? • Multiple motor proteins can • bind to a given cargo • -Each kinesin/dynein transports • a specific cargo

  16. Movement of pigmented granules in a cell

  17. Intracellular transport, positioning of organelles and growth of ER requires motor proteins and microtubules

  18. In addition to kinesin and dynein, myosin can also function as a motor protein on actin filaments

  19. MICROTUBULE DYNAMICS AND MOTOR PROTEINS ARE REQUIRED FOR MITOSIS DUPLICATION OF CENTRIOLES PRECEEDS MITOTIC SPINDLE EM; centrosome (colored red) has two centrioles. PC is pericentriolar material. MTOC; Ab against centrosomal protein

  20. Microtubules are responsible for separation of chromatids during mitosis • Dynamic instability of microtubules increases during mitosis - lifetime interphase - 10min - lifetime mitosis - 30sec KRPs are essential for separation and migration of centromeres Dynein contributes to the centrosome movement and spindle orientation

  21. chromatids chromatids • Anchorage of microtubules occurs in the kinetochore through specialized proteins. kinetochore kinetochore microtubule Centromere kinetochore Middle layer CDE I CBF2 CDE II Chromatin fibrils CDE III Outer layer Inner layer CBF3

  22. Anaphase Chromosome Kinetochore microtubules containing fluorescent tubulin Early Anaphase Expose to laser light Pole (MTOC) nonfluorescent but functional region of microtubules kinetochore microtubules depolymerization at (+) ends and kinetochores move toward poles Target region for laser light 0 min 5 min 10 min 15 min Anaphase Free fluorescent tubulin no depolymerization region of depolymerization 2mm

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