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王歐力 助理教授 Oliver I. Wagner, PhD Assistant Professor National Tsing Hua University

Laboratory course: Model organism C. elegans. Week 4: What is trafficking? How is cargo transported? Motor-cargo specificities Studying trafficking using kymograph analysis. 王歐力 助理教授 Oliver I. Wagner, PhD Assistant Professor National Tsing Hua University

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王歐力 助理教授 Oliver I. Wagner, PhD Assistant Professor National Tsing Hua University

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  1. Laboratory course: Model organism C. elegans Week 4: What is trafficking? How is cargo transported? Motor-cargo specificities Studying trafficking using kymograph analysis 王歐力助理教授 Oliver I. Wagner, PhD Assistant Professor National Tsing Hua University Institute of Molecular & Cellular Biology College of Life Science

  2. Trafficking in C. elegans neurons Vesicles and mitochondria move along actin or microtubule tracks attached to molecular motors as myosins, kinesinsanddynein v20-02-vesicle_transport.mov

  3. Axonal transport of vesicles - + • Synaptic vesicles and mitochondria are transported via • kinesinsfrom the cell body of the neuron to the termini • (growth cone) • The molecular motor dyneintransports them back - + - Mitochondria synaptic vesicle + (growth cone)

  4. Mitochondria are the energy factories of the cell 3D EM image of a mitochondrion (computer-generated from series of 2D EM images)

  5. Model of kinesin-based vesicle transport • Kinesins bind via their motor domain to microtubules while the tail (cargo) domain is connected to the vesicle • The vesicle connection is mediated by kinesin receptor proteins (linker proteins)

  6. Kinesin receptor control cargo attachments Axon Dendrites Hirokawa and Takemura, 2005, Nat Rev Neurosci.

  7. Cargo-binding Motor Kinesin superfamily proteins (KIF) • KIF1A is a monomeric kinesin: in C. elegans it is called UNC-104 • It is the main synaptic vesicle transporter in neurons The mechanisms of kinesin I movement on microtubules is well known 16_7.mov

  8. KIF1A knockout mice: defect in synaptic precursor transport and neuronal cell death • Reduction in the density of synaptic vesicles in nerve terminals, accumulation of vesicles in the cell body • KIF1A plays a critical role in the development of neuropathies resulting from impaired axonal transport WT wt/kif1akif1a/kif1a (Yonekawa, JCB, 1998)

  9. Dynein alone cannot attach to vesicles or mitochondria: it needs another “helper” named dynactin DYNACTIN DYNEIN Dynein moves cargo backwards

  10. Dynactin is an adaptor to connect dynein to the vesicle and the microtubule + - Vesicle Joseph Roland 2002

  11. The motor toolbox for intra- cellular transport Dendritic vesicles Axonal vesicles Backward transport • Motor domains= blue • Cargo binding domains= purple Vale, 2003, Cell

  12. Synaptic vesicles move bidirectional: coordinated activity of antagonistic motors? Taken from: Cell Biology, Pollard & Earnshaw

  13. … or tug-of-war between antagonistic motors?

  14. Determination of motor activity by analyzing motility of UNC-104::GFP particles • bidirectional • velocity of 1 μm/s • fast axonal cargo transport - movie length about 5 min. - width of neuron about 150 nm Living worm

  15. A Kymograph is the translation of a moving spot, on a line in one direction, into a two dimensional projection area with time and distance. movie Kymograph The „paper“ is continuously moving. A stable spot in the axon remains as a line on the “paper”. A moving spot will leave an individual trace on the „paper“. t x => with time and distance we can calculate velocity, pausing, run length etc.

  16. Translation of a particle movement from a movie-sequence into a kymograph

  17. Example of data evaluation using the kymograph technique

  18. In living worms In primary C. elegans neurons

  19. Current research example Isolated primary C. elegans neurons

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