Microtubule Cytoskeleton and Cell Patterning
10:00 Anna Akhmanova Microtubule Dynamics
11:00 Lukas Kapitein Motors and Transport
12:00 –12:30 Microscopy Facility Tour
13:00-14:30 Lunch and Self-study Jacobson et al Neuron 2006, 49:797
14:30 –15:15 Eugene Katrukha Pattern Formation
15:45 –17:00 Workshop on microtubules, transport and neuronal polarity Regulation of Microtubule Dynamics
Anna Akhmanova Cell Biology Faculty of Science Utrecht University The Netherlands Microtubules
mCherry-a-tubulin; MRC5 human lung fibroblast movie: Ilya Grigoriev Microtubules drive chromosome separation during mitosis
GFP-a-tubulin mCherry-histone
movie: Alexey Khodjakov (Wadsworth Center, Albany, USA) Cytoskeleton-active drugs Taxol – microtubule stabilizer
Taxus (yew) tree
Amanita Phalloides
Figure 16-23a Molecular Biology of the Cell (© Garland Science 2008) Cytoskeletal filaments are built from small subunits
Figure 16-7 Molecular Biology of the Cell (© Garland Science 2008) Cytoskeletal filaments built from multiple protofilaments are more stable
Figure 16-8 (part 2 of 2) Molecular Biology of the Cell (© Garland Science 2008)
Vg = kon C – koff
MICROTUBULES
Figure 16-11 Molecular Biology of the Cell (© Garland Science 2008) Microtubule dynamic instability
Dynamic microtubules visualized with Cy3-tubulin in 3T3 mouse fibroblast Dynamic instability in living cells
cell edge
25
m
m 20
15
10 distance, Ðàññòî ÿíì èå, êì 5 centrosome 0 0 10 20 30 40 time,Âðåì ÿ, min ì èí Microtubule Dynamics
Figure 16-16a Molecular Biology of the Cell (© Garland Science 2008) The role of GTP hydrolysis:
Not needed for assembly Required for depolymerization
GMPCPP Although the b-g linkage is normal, tubulin doesn’t hydrolyse it under standard conditions Microtubule polymerization kinetics
Does koff depend on the tubulin concentration?
Gardner et al, Cell 2011 Microtubule polymerization kinetics
Does koff depend on the tubulin concentration?
Gardner et al, Cell 2011 Microtubule outgrowth from a template requires tubulin concentration well above critical concentration
Wieczorek et al, NCB 2015 Microtubule-regulating factors
in vitro in vivo rate of growth <3-4 mm/min 10-25 mm/min rate of shortening 15-100 mm/min 12-40 mm/min
nucleation – g-tubulin ring complex polymerization – XMAP215/ch-TOG minus end anchoring/stabilization – ninein, CAMSAP severing – katanin, spastin depolymerization – stathmin, kinesin-13 (MCAK) stabilization – MAPs (tau, MAP2, MAP4) Microtubule Dynamics: what happens at the ends?
GTP
Polymerization GDP Depolymerization GTP cap Catastrophe
Rescue
Shrinking MT
Growing MT
Meta-stable intermediate state Plus end-tracking proteins (+TIPs) label the growing microtubule ends
GFP-EB3 and mCherry-a-tubulin in a MRC5 human lung fibroblast 0.5s/frame Plus end-tracking proteins (+TIPs) label the growing microtubule ends
EB3-GFP in a CAR goldfish fibroblast In vitro reconstitution of microtubule dynamics
GMPCPP-stabilised microtubule seed Other containing biotinylated +TIP(s) tubulin EB protein
Tubulin
Streptavidin Biotin PLL-PEG- Biotin
TIRF microscopy EB family members autonomously track growing microtubule ends
GFP-EB3 tubulin
kymograph
time
60 s 60
10 mm
distance +TIPs exchange rapidly at the plus ends of microtubules Catastrophes
Catastrophe induction
Loss of GTP cap
Slow growth, obstacles (barriers)
Catastrophe inducing factors Catastrophe is likely to be a multistep process
Catastrophes do not follow first-order kinetics
Odde et al., Biophys J 1995 Catastrophe is likely to be a multistep process
Catastrophes do not follow Catastrophes occur after several first-order kinetics intermediate steps
Odde et al., Biophys J 1995 Catastrophe induction: Kinesin-13
MCAK (XKCM1)=KIF2C KIF2A, KIF2B
neck Kinesin Dimerization motor
Gardner et al, Cell 2011 Kinesin-13 abolishes“microtubule aging”
Kinesin-13
Gardner et al, Cell 2011 Catastrophe induction by microtubule-destabilizing agents in cells
Control 5 nM Vinblastine
Growth rate 25.0
20.0
15.0
µm/min 10.0
5.0
0.0 0 3 5 7.5 Vinblastine (nM)
Catastrophe 25.0 frequency
20.0
1 -
15.0
min
10.0 10 10 s
5.0 2 µm 0.0 0 3 5 7.5 0 3 5 7.5 15 Vinblastine (nM)
Vinblastine (nM) Mohan et al., PNAS 2013 EB proteins sensitize microtubules to the action of microtubule-destabilizing agents
Microtubules grown in vitro Microtubules grown in vitro Concentration of the without EB3 in the presence of EB3 drug (nM) which stalls microtubule growth MTAs -EB3 + EB3 Colchicine 600 170 Combreta- 500 200 statin A4 Podo- 600 200
phyllotoxin 60 s 60 2 μm 2 mm Dolastatin 15 500 170 Control 200 400 500 Control 100 150 170 Vinblastine 2000 900 Dolastatin (nM) Dolastatin (nM) Vincristine 3000 2000 Vinorelbine 3000 1500
Mohan et al., PNAS 2013 Microtubule-targeting agents do not change the number of catastrophe- inducing steps but increase the rate of their occurrence
Number of intermediate Number of intermediate steps - EB3 steps + EB3 6.0 6.0
4.0 4.0
2.0 2.0
0.0
0.0
nM
150 100 150 100 150 100 100 200 750 100 150
400 300 200 300 150 200 200 400 750 900 100 150
200 100 100
100 400 500
1500 2000 1000 1500 1000
1000
150 Control Col CA4 Podo Dola VB VC VR Control Col CA4 Podo Dola VB VC VR
Rate of occurrence of intermediate Rate of occurrence of intermediate steps - EB3 steps + EB3
8.0 8.0
1
1
- -
6.0 6.0
min min
4.0 4.0
2.0 2.0
0.0
0.0
nM
100 150 100 150 100 150 100 200 750 100 150
750 150 200 300 400 150 200 200 300 400 900 100 150
100 200 100
100 400 500
1500 2000 1000 1500 1000
1000 Control Col CA4 Podo Dola VB VC VR Control Col CA4 Podo Dola VB VC VR
Mohan et al., PNAS 2013 Microtubule catastrophes depend on microtubule “age”
Some microtubule regulators can abolish age- dependence of catastrophes
Microtubule-targeting agents behave as if they accelerate aging process