1. 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 Laboratory course: Model organism C. elegans Week 4: 1.What is trafficking? 2.How is cargo transported? 3.Motor-cargo specificities 4.Studying trafficking using kymograph analysis

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

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

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

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

7. 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 Motor Cargo-binding 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/kif1a kif1a/kif1a (Yonekawa, JCB, 1998)

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

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

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

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

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

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

15. 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 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 => 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. Isolated primary C. elegans neurons Current research example