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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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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


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


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)


Mitochondria are

the energy factories

of the cell

3D EM image of a mitochondrion

(computer-generated from series

of 2D EM images)


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)


Kinesin receptor control cargo attachments

Axon

Dendrites

Hirokawa and Takemura, 2005, Nat Rev Neurosci.


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


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)


Dynein alone cannot attach to vesicles or mitochondria: it needs

another “helper” named dynactin

DYNACTIN

DYNEIN

Dynein moves cargo backwards


Dynactin needs is an adaptor to connect dynein to the vesicle and the microtubule

+

-

Vesicle

Joseph Roland 2002


The motor toolbox for intra- needs

cellular transport

Dendritic vesicles

Axonal vesicles

Backward transport

  • Motor domains= blue

  • Cargo binding domains= purple

Vale, 2003, Cell


Synaptic vesicles move needsbidirectional: coordinated activity of antagonistic motors?

Taken from: Cell Biology, Pollard & Earnshaw



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


A Kymograph is the UNC-104::GFP particlestranslation 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.




In living worms into a kymograph

In primary

C. elegans neurons


Current research example into a kymograph

Isolated primary C. elegans neurons


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