Transcript Lecture

Laboratory course: Model organism C. elegans
Week 4:
1.
2.
3.
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, kinesins and dynein
v20-02-vesicle_transport.mov
+
-
-+
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
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.
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
Cargo-binding
Motor
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
(Yonekawa, JCB, 1998)
wt/kif1a
kif1a/kif1a
Dynein alone cannot attach to vesicles or mitochondria: it needs
another “helper” named dynactin
DYNACTIN
DYNEIN
MT
binding
HC contains
AAA domains
Dynein moves cargo backwards
Dynactin is an adaptor to connect dynein to the vesicle and the microtubule
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Vesicle
Joseph Roland 2002
The motor toolbox for intracellular transport
• Motor domains = blue
• Cargo binding domains = purple
Dendritic vesicles
Axonal vesicles
Backward transport
Vale, 2003, Cell
Synaptic vesicles move bidirectional: coordinated activity of antagonistic motors?
Taken from: Cell Biology, Pollard & Earnshaw
… or tug-of-war between antagonistic motors?
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 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”.
t
x
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
All Particles
unc104(ok217) cells
Velo. w/o pauses (µm/s)
Total run length (µm)
Change direction per 100 s
Change direction per 10 µm
Pausing per 100 s
Pausing per 10 µm
Pausing duration (s)
Persis. of mov. at uni. velo.(s)
# Events
# Anterograde movements
# Retrograde movements
# Unidentified movements
# Axons
# Dendrites
# Commissures
# Unidentified
# Particles
# Movies
Pause Calibration Ave. (s)
Velocity due to particle size
Counts:
7
Large STDEV+/- Medium
Aver.
0,39
0,13
L versus M
L vers. S
T-Tests
0,19
0,43
5,47
4,12
2,06
2,19
1,44
16,45
9,00
490
25
30
7
25
0
0
0
62
25
0,062
21
STDEV+/- Antero
STDEV+/0,19
0,32
0,13
3,56
5,19
3,00
2,53
4,37
2,37
1,26
2,34
1,18
1,16
1,98
0,73
1,03
1,36
0,87
7,16
19,37
9,78
3,37
7,85
2,56
245
Events neither antero nor retro:
45 % antero
57
55 % retro
43
Retro
STDEV+/0,47
0,17
5,94
3,84
4,09
2,79
1,94
1,37
2,54
1,61
1,68
1,31
14,68
7,90
9,62
3,69
186
59
% antero events
% retro events
32
STDEV+/- Small
STDEV+/0,30
0,14
0,48
0,22
M vers. S
0,18
0,001
Velos of particles with no CD and one event only (linear and directed movements)
Aver.
0,62 STDEV+/0,26 Aver.
0,34 STDEV+/T-Test
0,0017
0,12
In living worms
In primary
C. elegans
neurons
Current research example
Isolated primary C. elegans neurons