1. Modelling Microtubule Dynamics at Super-Resolution
Summer Project Plan
Nils Gustafsson
Supervised By:
Dr Lewis Griffin
Dr Thomas Surrey

2. Summary Microtubules In Vitro Microtubule Experiments
Microtubules and the Cytoskeleton
Dynamic Instability of Microtubules
In Vitro Microtubule Experiments
Analysis of In Vitro Experiments
Validation of In Vitro Experiments
Ground Truth Simulations
Simulating Experimental Data
Modelling Microtubule Dynamics

3. Microtubules and the Cytoskeleton
Multiple Cellular Functions
Mechanical Stability
Scaffold Structures
Force Generation
Cargo Transport
Cell Migration
Cell Differentiation
Cell Division
Drug Targets
Vinca Alkaloids
Taxanes
Microtubules (green) DNA (blue)
EB1 (yellow)
Fig. (top right) taken from Molecular Cell Biology 4th ed, Lodish
Fig. (bottom right) taken from Torsten Wittmann homepage, UCSF

4. Dynamic Instability
Microtubule (+)end tracking proteins (green) reveal rapid growth and shrinkage episodes in live cells.
Fine control of microtubule dynamics by microtubule associated proteins (MAPs).
Fig. (bottom left) taken from Molecular Cell Biology 4th ed, Lodish

5. Dynamic Instability
Fig. taken from C. Conde & A. Caceres, Nature reviews Neuroscience, 2009

6. In Vitro Microtubule Experiments
Stabilised GMPCPP seeds are bound to a cover slip
Fluorescently tagged tubulin subunits are introduced via micro-fluidics
Microtubules are nucleated at the seeds
Imaged by TIRF microscopy as they grow
Fig. (bottom left) taken from C Duellberg’s PhD Thesis
Fig. (bottom right) taken from The Dixit Lab research webpage, Washington University

7. Analysis of In Vitro Experiments
Custom analysis software tracks end positions
Using convolved model fitting
Sub-pixel precision alignment of frames allows averaged intensity profiles to be produced
Multiple channels can be analysed including MAP structures

8. Validation of In Vitro Experiment Analysis
Simple simulations have previously been used to determine resolution of taper lengths
We would like to be able to determine accuracy of tracking of dynamic characteristics of microtubule growth – such as growth fluctuations.
Fig. (bottom left) modified from Maurer, Cade, Bohner et. al. Current Biology, 2014

9. Simulating Experimental Data
Monte-Carlo Simulation of the 1D model defines a state sequence used to reconstruct microscope images
Considerations: states per frame, noise, movement, labelling densities, magnification……
Fig. (center left) modified from Gardner et. al. Cell, 2011

10. Modelling of Microtubule Dynamics
Accurate quantification of experiment leads to improved models
Models should include:
Growth velocities and fluctuations
Interaction with MAPs
Catastrophe/rescue frequencies
Fig. (left) modified from Gardner et. al. Cell, 2011
Fig. (right) modified from Maurer, Cade, Bohner et. al. Current Biology, 2014

11. Acknowledgements Microtubule Cytoskeleton Lab, Cancer Research UK
Dr Thomas Surrey  Laboratory Head
Dr Nicholas Cade   Principal Scientific Officer
Dr Iris Lueke   Senior Scientific Officer
Ms Claire Thomas   Senior Scientific Officer
Dr Sebastian Maurer Previous Group Member
Dr Christian Duellberg   Scientific Officer
Dr Jayant Asthana   Research Fellow
Dr Todd Fallesen   Research Fellow
Dr Franck Fourniol   Research Fellow
Dr Johanna Roostalu   Research Fellow
Dr Einat Schnur    Research Fellow
Dr Hella Baumann   Graduate Student
Mr Jonathon Hannabuss   Graduate Student
Ms Rupam Jha   Graduate Student
Mr Gergo Bohner   Diploma Student
CoMPLEX, UCL
Dr Lewis Griffin
Ms Stephanie Reynolds