Microtubule and Cell Patterning

10:00 Anna Akhmanova 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 , transport and neuronal polarity Regulation of Microtubule Dynamics

Anna Akhmanova Faculty of Science The Netherlands Microtubules

mCherry-a-tubulin; MRC5 human lung fibroblast movie: Ilya Grigoriev Microtubules drive chromosome separation during

GFP-a-tubulin mCherry-

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 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, -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