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
Laboratory course: Model organism C. elegans
What is trafficking?
How is cargo transported?
Studying trafficking using kymograph analysis
Oliver I. Wagner, PhD
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
the energy factories
of the cell
3D EM image of a mitochondrion
(computer-generated from series
of 2D EM images)
Hirokawa and Takemura, 2005, Nat Rev Neurosci.
Kinesin superfamily proteins (KIF)
The mechanisms of kinesin I movement on microtubules is well known
KIF1A knockout mice: defect in synaptic precursor transport and neuronal cell death
(Yonekawa, JCB, 1998)
another “helper” named dynactin
Dynein moves cargo backwards
Dynactin needs is an adaptor to connect dynein to the vesicle and the microtubule
Joseph Roland 2002
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
- movie length about 5 min.
- width of neuron about 150 nm
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.
The „paper“ is
A stable spot in the axon remains as a line on the “paper”.
A moving spot will leave an individual trace on the „paper“.
=> with time and distance we can calculate velocity, pausing, run length etc.
Translation of a particle movement from a movie-sequence into a kymograph
Example of data evaluation using the kymograph technique into a kymograph
In living worms into a kymograph
C. elegans neurons
Current research example into a kymograph
Isolated primary C. elegans neurons