Microtubule Cytoskeleton and Cell Patterning

Transcription

1 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

2 Regulation of Microtubule Dynamics Anna Akhmanova Cell Biology Faculty of Science Utrecht University The Netherlands

3 Microtubules mcherry-a-tubulin; MRC5 human lung fibroblast movie: Ilya Grigoriev

4 Microtubules drive chromosome separation during mitosis GFP-a-tubulin mcherry-histone movie: Alexey Khodjakov (Wadsworth Center, Albany, USA)

5 Cytoskeleton-active drugs Taxol microtubule stabilizer Taxus (yew) tree Amanita Phalloides Figure 16-23a Molecular Biology of the Cell ( Garland Science 2008)

6 Cytoskeletal filaments are built from small subunits Figure 16-7 Molecular Biology of the Cell ( Garland Science 2008)

7 Cytoskeletal filaments built from multiple protofilaments are more stable Figure 16-8 (part 2 of 2) Molecular Biology of the Cell ( Garland Science 2008)

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9 Vg = kon C koff

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12 Figure Molecular Biology of the Cell ( Garland Science 2008) MICROTUBULES

13 Microtubule dynamic instability Dynamic microtubules visualized with Cy3-tubulin in 3T3 mouse fibroblast

14 distance, mm Dynamic instability in living cells 25 cell edge Ðàññòî ÿí èå, ì êì time, Âðåì ÿ, min ì èí centrosome

15 Microtubule Dynamics Figure 16-16a Molecular Biology of the Cell ( Garland Science 2008)

16 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

17 Microtubule polymerization kinetics Does k off depend on the tubulin concentration? Gardner et al, Cell 2011

18 Microtubule polymerization kinetics Does k off depend on the tubulin concentration? Gardner et al, Cell 2011

19 Microtubule outgrowth from a template requires tubulin concentration well above critical concentration Wieczorek et al, NCB 2015

20 Microtubule-regulating factors rate of growth rate of shortening in vitro <3-4 mm/min mm/min in vivo mm/min 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)

21 Microtubule Dynamics: what happens at the ends? GTP Polymerization GTP cap GDP Catastrophe Rescue Depolymerization Shrinking MT Growing MT Meta-stable intermediate state

22 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

23 Plus end-tracking proteins (+TIPs) label the growing microtubule ends EB3-GFP in a CAR goldfish fibroblast

24 In vitro reconstitution of microtubule dynamics GMPCPP-stabilised microtubule seed containing biotinylated tubulin Other +TIP(s) EB protein Tubulin Streptavidin Biotin PLL-PEG- Biotin TIRF microscopy

25 EB family members autonomously track growing microtubule ends time 60 s GFP-EB3 tubulin kymograph 10 mm distance

26 +TIPs exchange rapidly at the plus ends of microtubules

27 Catastrophes

28 Catastrophe induction Loss of GTP cap Slow growth, obstacles (barriers) Catastrophe inducing factors

29 Catastrophe is likely to be a multistep process Catastrophes do not follow first-order kinetics Odde et al., Biophys J 1995

30 Catastrophe is likely to be a multistep process Catastrophes do not follow first-order kinetics Catastrophes occur after several intermediate steps Odde et al., Biophys J 1995

31 Catastrophe induction: Kinesin-13 MCAK (XKCM1)=KIF2C KIF2A, KIF2B neck Kinesin motor Dimerization Gardner et al, Cell 2011

32 Kinesin-13 abolishes microtubule aging Kinesin-13 Gardner et al, Cell 2011

33 10 s Catastrophe induction by microtubule-destabilizing agents in cells µm/min Control 5 nm Vinblastine 25.0 Growth rate Vinblastine (nm) Catastrophe frequency min µm Vinblastine (nm) Vinblastine (nm) Mohan et al., PNAS 2

34 60 s EB proteins sensitize microtubules to the action of microtubule-destabilizing agents Microtubules grown in vitro without EB3 Microtubules grown in vitro in the presence of EB3 MTAs Concentration of the drug (nm) which stalls microtubule growth -EB3 + EB3 Colchicine Combretastatin A Podophyllotoxin mm 2 μm Dolastatin Control Dolastatin (nm) Control Dolastatin (nm) Vinblastine Vincristine Vinorelbine Mohan et al., PNAS 2

35 Control Control Control Control Microtubule-targeting agents do not change the number of catastropheinducing steps but increase the rate of their occurrence 6.0 Number of intermediate steps - EB3 6.0 Number of intermediate steps + EB nm Col CA4 Podo Dola VB VC VR Col CA4 Podo Dola VB VC VR 8.0 Rate of occurrence of intermediate steps - EB3 8.0 Rate of occurrence of intermediate steps + EB3 min min nm Col CA4 Podo Dola VB VC VR Col CA4 Podo Dola VB VC VR

36 Microtubule catastrophes depend on microtubule age Some microtubule regulators can abolish agedependence of catastrophes Microtubule-targeting agents behave as if they accelerate aging process