Thomas Surrey: Projects

Microtubule cytoskeleton architecture and dynamics

The laboratory is interested in understanding how the microtubule cytoskeleton determines the dynamic structure of intracellular space.

Different specific intracellular arrangements of the microtubule cytoskeleton are required for distinct cell functions. During cell division the cytoskeleton undergoes dramatic rearrangements; these are crucial for the correct distribution of the genetic material to the two newly forming daughter cells - a process which presents a formidable mechanical task.

How the molecules of the cytoskeleton perform all these actions correctly is a fascinating question. To answer this, we need to understand the dynamic interplay between a large number of cellular components; these include the cell membrane, biological polymers, and a variety of mechanically active enzymes which generate forces. The microtubule cytoskeleton is therefore at the interface between biochemistry and mechanics, and mediates the transition from molecular organisation to cellular function.

Figure

Reorganisation of the microtubule (green) cytoskeleton inside a cell during cell division. DNA in blue. Scale bar 20 µm (from Olichon and Surrey, J. Biol. Chem. 2007).

From single molecules to collective behaviour: systems biochemistry

To understand how the cytoskeleton produces dynamic order, a major challenge is to derive cellular behaviour from molecular information. Here, the real question is how the various components of the cytoskeleton act together as a system to produce coherent behaviour.

The basic building blocks for the different architectures of the microtubule cytoskeleton are always the same: tubulin assembling into dynamic polymers, associated proteins regulating microtubule assembly and dynamics, molecular motors moving cargo along microtubules, and proteins attaching microtubules directly, yet dynamically, to other cellular structures such as the cell cortex or chromosomes during cell division.

The combination of a distinct set of such activities, a particular 'protein interaction network', leads to a specific microtubule architecture with defined dynamic properties and functionality. However, the question remains: under which conditions does the network generate which intracellular architecture?

Although it is becoming increasingly clear which molecular players are involved in cytoskeleton organisation, the rules or physical principles determining how they work together still remain a mystery. In general, it is a major challenge to predict the internal organisation of the cell and its function, solely from the knowledge of the composition of a cell and of the properties of the relevant components. The microtubule cytoskeleton provides an excellent opportunity to address this question.

Modern fluorescence microscopy techniques have allowed major advances to be made in this field; we use these techniques to visualize the movements of individual molecules of the cytoskeleton, and we are developing novel biochemical reconstitution approaches to observe how minimal subsystems of the cytoskeleton self-organise. These arrangements can be directly compared to what the same molecules do inside cells. Our goal is to be able to understand and to predict how the entire cytoskeleton functions dynamically, based on the biochemical and biophysical properties of cytoskeletal molecules.

Local regulation of microtubule dynamics

The control of the polymerisation/depolymerisation properties of microtubules is crucial for proper cytoskeleton functioning. Dynamic microtubule properties are regulated in cells globally (e.g. responding to the cell cycle state) and also locally in response to local interactions.

It has become clear over the last 10 years that there are a multitude of proteins interacting selectively with microtubule ends to affect their dynamic properties. Several of these proteins interact with each other, thus forming a dynamic protein interaction network; this determines the fate of the microtubule and its interaction with other cellular structures. How this protein network functions as a system is not understood. 

Using an in vitro reconstitution approach combined with total internal reflection (TIRF) microscopy, we have recently elucidated the molecular mechanism by which the key member (EB1) of this local protein interaction network localises specifically at microtubule ends, and how it recruits some of the other proteins of this network (Bieling et al., Nature, 2007; Bieling et al., J. Cell Biol., 2008)

We have shown that a specific conformation of the tubulin subunits exists over an extended region at growing microtubule ends to which EB1 binds. Interestingly this is a conformation related to the GTP cap (Maurer et al., PNAS, 2011), and EB1 binds close to the binding site of the hydrolysable GTP (Maurer, Fourniol et al., Cell, 2012). Using microfluidics, we demonstrated that longer caps make microtubules more stable (Duellberg et al., eLife, 2016) and cap size fluctuations can be understood quantitatively based on the reaction network of cap formation (Rickman, PNAS, 2017). Presently, we investigate how these fluctuations and the nanoscale structure of microtubule ends relate to the fundamental property of dynamic microtubule instability.

Microtubule dynamics

(A) Cryo-electron microscopy reconstruction of the microtubule binding domain of EB1 on a microtubule (Maurer, Fourniol et al., Cell 2012). (B) TIRF microscopy image sequence and (C) time plots of the microtubule end position (red) and the corresponding EB1-GFP intensity (green) in a tubulin washout experiment (Duellberg et al., eLife, 2016). Scale bar is 3 µm. (D) Scatter plot of delay times between washout and catastrophe versus EB1-GFP intensities at the time of tubulin washout, showing a correlation between instantaneous microtubule stability and protective cap size. (Click to view larger image)

Local regulation of microtubule nucleation

Microtubule cytoskeleton function requires precise control of microtubule nucleation and dynamics. Compared to the regulation of microtubule growth, the molecular mechanism underlying the regulation microtubule nucleation is poorly understood. In mitosis and meiosis, the chromatin-driven spindle assembly pathway exerts control of microtubule nucleation locally in the vicinity of chromosomes. One of the key targets is the multifunctional protein TPX2. Using a novel TIRF microscopy-based in vitro reconstitution assay, we found that TPX2 directly stabilises growing microtubule ends and stimulates microtubule nucleation by stabilising early microtubule nucleation intermediates and acts synergistically with the microtubule polymerase chTOG (XMAP215 homolog) to promote microtubule nucleation (Roostalu et al., Nat Cell Biol., 2015). Importins control the efficiency of microtubule nucleation by selectively blocking TPX2's interaction with microtubule nucleation intermediates.. Currently we are investigating role of other nucleation factor using TIRF microsopy assays.

Microtubule nucleation

Time course of TPX2 and chTOG stimulated microtubule nucleation in the absence (top) and presence (bottom) of importins, showing that regulation of microtubule nucleation is regulated by importin in vitro (Roostalu et al., Nat. Cell Biol., 2015). (Click to view larger image)

Dynein

Cytoplasmic dynein is the main minus end directed motor in human cells, being involved in a multitude of essential cellular functions ranging from trafficking in interphase to spindle organisation and positioning during mitosis. One major bottleneck in dynein research was the unavailability of recombinant, fluorescently labelled human dynein. We succeeded in producing such a recombinant human dynein complex and demonstrated in single molecule TIRF microscopy experiments that human dynein by itself was unexpectedly non-processive (Troker et al., PNAS, 2012), different from the previously established view, showing that human dynein differs from intrinsically processive yeast dynein.

In addition to being an active minus motor, dynein is also found to track growing microtubule plus ends to facilitate dynein mediated interactions between microtubule ends and intracellular target structures and to initiate cargo transport. The combinatorial action of several regulators control dynein's activity. However, their molecular mechanism of action is still poorly understood. To understand the requirements for dynein end tracking, we reconstituted the recruitment of human dynein to growing microtubule ends. We showed that the existence of a hieararchical recruitment mode (EB1 - CLIP-170 - p150 Glued - dynein) allows human dynein to be recruited to microtubule ends in the presence of competing end tracking proteins (Duellberg et al., NCB, 2014). These results highlight how the connectivity and hierarchy within dynamic +TIP networks are orchestrated.

Topology, shape, size

Here we adopt an engineering perspective to determine the minimal composition of a system of mutually interacting proteins able to generate specific microtubule architectures.

In dividing cells bipolar spindles have two spindle poles and the centre of the spindle consists of a crucial topological feature: stable antiparallel microtubule overlaps. In an in vitro study with purified motor proteins (plus-directed kinesin-5 and minus-directed kinesin-14), we have shown that mitotic motors promote microtubule sorting and organisation:  microtubule minus ends are focussed into poles (as in mitosis) and microtubule plus pole formation is prevented ensuring that stabilizing microtubule overlaps can form in the spindle centre (Hentrich & Surrey, J. Cell Biol., 2010)

Presently, a major unanswered question is exactly what are the exact biophysical determinants governing spindle morphology in metaphase?

Figure

Microtubule (green) asters organised by cross-linking motors (red) in vitro. (Image taken by Christian Hentrich).

The centre of anaphase spindles is topologically simpler than that of metaphase spindles due to the absence of chromosomes, which have already been pulled toward the spindle poles. It is known that a unique set of proteins localises to the spindle centre at anaphase onset caused by a global change in phosphorylation activities at this stage of the cell cycle. One of these proteins is PRC1; using TIRF microscopy imaging, we have shown that PRC1 binds selectively to antiparallel microtubules in vitro, and recruits a molecular motor (kinesin-4) which inhibits microtubule growth.

The combination of these activities represents a minimal system capable of generating a microtubule overlap with defined length by self-regulation, as we have shown by in vitro reconstitution of overlaps between the two individual microtubules (Bieling et al., Cell, 2010). Currently we investigate larger scale assemblies of anaphase-like spindles using synthetic biochemistry approaches.

Figure

Snapshots of a single microtubule (blue) overlap forming in the presence of PRC1 (red) and kinesin-4 (green). Time in min:sec. Scale bar 10 µm. (From Bieling et al., Cell, 2010).

Thomas Surrey

thomas.surrey@crick.ac.uk
+44 (0)20 379 62044

  • Qualifications and history
  • 1995 PhD, University of Tuebingen and Max-Planck Institute for Biology, Germany
  • 1995 Postdoctoral Fellow, Princeton University, USA
  • 1998  Postdoctoral fellow and staff scientist,  European Molecular Biology Laboratory, Germany
  • 2002  Team and group leader, European Molecular Biology Laboratory, Germany
  • 2011 Established lab at the London Research Institute, Cancer Research UK
  • 2015 Group Leader, the Francis Crick Institute, London, UK