Thomas Surrey

The Francis Crick Institute, Londinium, England, United Kingdom

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Publications (79)720.18 Total impact

  • Hella Baumann · Thomas Surrey
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    ABSTRACT: In living cells, the architecture of the microtubule cytoskeleton is intimately linked to its function. The principles determining how microtubules arrange in space are, however, still not fully understood. Biochemical activities controlling microtubule nucleation and dynamics as well as mechanochemical activities exerted by molecular motors and the dynamic microtubules themselves are known to be critical for the correct spatial organization of the microtubule cytoskeleton. In vitro reconstitution approaches have revealed the morphogenetic properties of these activities in minimal systems. In most cases, such in vitro experiments were performed in experimental chambers of spatial dimensions that exceeded typical cell sizes by orders of magnitude. Here, we describe a method for the fluorescence microscopic study of the effects of spatial confinement on the self-organization of purified motors and microtubules that are encapsulated in micrometer-sized lipid-monolayered droplets emulsified in oil. In the future, this experimental setup can be extended in several ways. Additional proteins can be added, either to the lumen or to the boundary of the microcontainers, and the droplets can be transformed into liposomes. Such more complex in vitro reconstitutions would be another step closer to mimicking intracellular cytoskeleton organization. Copyright © 2015 Elsevier Inc. All rights reserved.
    No preview · Article · Dec 2015 · Methods in cell biology
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    Full-text · Dataset · Nov 2015
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    Full-text · Dataset · Oct 2015
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    Full-text · Dataset · Oct 2015
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    ABSTRACT: Tracking dynamic microtubule ends in fluorescence microscopy movies provides insight into the statistical properties of microtubule dynamics and is vital for further analysis that requires knowledge of the trajectories of the microtubule ends. Here we analyse the performance of a previously developed automated microtubule end tracking routine; this has been optimized for comparatively low signal-to-noise image sequences that are characteristic of microscopy movies of dynamic microtubules growing in vitro. Sequences of simulated microtubule images were generated assuming a variety of different experimental conditions. The simulated movies were then tracked and the tracking errors were characterized. We found that the growth characteristics of the microtubules within realistic ranges had a negligible effect on the tracking precision. The fluorophore labelling density, the pixel size of the images, and the exposure times were found to be important parameters limiting the tracking precision which could be explained using concepts of single molecule localization microscopy. The signal-to-noise ratio was found to be a good single predictor of the tracking precision: typical experimental signal-to-noise ratios lead to tracking precisions in the range of tens of nanometres, making the tracking program described here a useful tool for dynamic microtubule end tracking with close to molecular precision.
    Full-text · Article · Oct 2015 · Journal of Microscopy
  • Johanna Roostalu · Nicholas I Cade · Thomas Surrey
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    ABSTRACT: Spindle assembly and function require precise control of microtubule nucleation and dynamics. The chromatin-driven spindle assembly pathway exerts such control locally in the vicinity of chromosomes. One of the key targets of this pathway is TPX2. The molecular mechanism of how TPX2 stimulates microtubule nucleation is not understood. Using microscopy-based dynamic in vitro reconstitution assays with purified proteins, we find that human TPX2 directly stabilizes growing microtubule ends and stimulates microtubule nucleation by stabilizing early microtubule nucleation intermediates. Human microtubule polymerase chTOG (XMAP215/Msps/Stu2p/Dis1/Alp14 homologue) only weakly promotes nucleation, but acts synergistically with TPX2. Hence, a combination of distinct and complementary activities is sufficient for efficient microtubule formation in vitro. Importins control the efficiency of the microtubule nucleation by selectively blocking the interaction of TPX2 with microtubule nucleation intermediates. This in vitro reconstitution reveals the molecular mechanism of regulated microtubule formation by a minimal nucleation module essential for chromatin-dependent microtubule nucleation in cells.
    No preview · Article · Sep 2015 · Nature Cell Biology
  • Rupam Jha · Thomas Surrey
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    ABSTRACT: The cytoplasmic dynein complex is the major minus-end-directed microtubule motor. Although its directionality is evolutionary well conserved, differences exist among cytoplasmic dyneins from different species in their stepping behaviour, maximum velocity and force production. Recent experiments also suggest differences in processivity regulation. In the present article, we give an overview of dynein's motile properties, with a special emphasis on processivity and its regulation. Furthermore, we summarize recent findings of different pathways for microtubule plus-end loading of dynein. The present review highlights how distinct functions in different cell types or organisms appear to require different mechanochemical dynein properties and localization pathways.
    No preview · Article · Feb 2015 · Biochemical Society Transactions
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    ABSTRACT: Water-in-oil emulsion droplets created in droplet-based microfluidic devices have been tested and used recently as well-defined picoliter-sized 3D compartments for various biochemical and biomedical applications. In many of these applications, fluorescence measurements are applied to reveal the protein content, spatial distribution, and dynamics in the droplets. However, emulsion droplets do not always provide entirely sealed compartments, and partitioning of dyes or labeled molecules to the oil phase is frequently observed. Therefore, stable molecular retention in the droplets represents a challenge, and many physical and chemical key factors of microfluidic system components have to be considered. In this study, we investigated the retention of 12 commonly used water-soluble dyes in droplets having six different aqueous phase conditions. We demonstrate that the physicochemical properties of the dyes have a major influence on the retention level. In particular, hydrophilicity has a strong influence on retention, with highly hydrophilic dyes (LogD < -7) showing stable, buffer/medium independent retention. In the case of less hydrophilic dyes, we showed that retention can be improved by adjusting the surfactants physical properties, such as geometry, length, and concentration. Furthermore, we analyzed the retention stability of labeled biomolecules such as antibodies, streptavidin, and tubulin proteins and showed that stable retention can be strongly dependent on dye and surfactants selection.
    No preview · Article · Jan 2015 · Analytical Chemistry
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    ABSTRACT: During mitosis, chromosome alignment depends on the regulated dynamics of microtubules and on motor protein activities. At the kinetochore, the interplay between microtubule-binding proteins, motors, and kinases is poorly understood. Cenp-E is a kinetochore-associated kinesin involved in chromosome congression, but the mechanism by which this is achieved is unclear. Here, we present a study of the regulation of Cenp-E motility by using purified full-length (FL) Xenopus Cenp-E protein, which demonstrates that FL Cenp-E is a genuine plus-end-directed motor. Furthermore, we find that the Cenp-E tail completely blocks the motility of Cenp-E in vitro. This is achieved through direct interaction between its motor and tail domains. Finally, we show that Cenp-E autoinhibition is reversed by MPS1- or CDK1-cyclin B-mediated phosphorylation of the Cenp-E tail. This suggests a model of dynamic control of Cenp-E motility, and hence chromosome congression, dependent upon phosphorylation at the kinetochore.
    Full-text · Dataset · Dec 2014
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    ABSTRACT: Growing microtubule end regions recruit a variety of proteins collectively termed +TIPs, which confer local functions to the microtubule cytoskeleton. +TIPs form dynamic interaction networks whose behaviour depends on a number of potentially competitive and hierarchical interaction modes. The rules that determine which of the various +TIPs are recruited to the limited number of available binding sites at microtubule ends remain poorly understood. Here we examined how the human dynein complex, the main minus-end-directed motor and an important +TIP (refs , , ), is targeted to growing microtubule ends in the presence of different +TIP competitors. Using a total internal reflection fluorescence microscopy-based reconstitution assay, we found that a hierarchical recruitment mode targets the large dynactin subunit p150Glued to growing microtubule ends via EB1 and CLIP-170 in the presence of competing SxIP-motif-containing peptides. We further show that the human dynein complex is targeted to growing microtubule ends through an interaction of the tail domain of dynein with p150Glued. Our results highlight how the connectivity and hierarchy within dynamic +TIP networks are orchestrated.
    No preview · Article · Jul 2014 · Nature Cell Biology

  • No preview · Conference Paper · Jul 2014
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    Hella Baumann · Thomas Surrey
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    ABSTRACT: The correct spatial organisation of microtubules is of crucial importance for determining the internal architecture of eukaryotic cells. Microtubules are arranged in space by a multitude of biochemical activities and by spatial constraints imposed by the cell boundary. The principles underlying the establishment of distinct intracellular architectures are only poorly understood. Here, we have studied the effect of spatial confinement on the self-organisation of purified motors and microtubules that are encapsulated in lipid-monolayered droplets in oil, varying in diameter from five to hundred micrometres, which covers the size range of typical cell bodies. We found that droplet size alone had a major organising influence. The presence of a microtubule-crosslinking motor protein decreased the number of accessible types of microtubule organisations. Depending on the degree of spatial confinement, the presence of the motor caused either the formation of a cortical array of bent microtubule bundles or the generation of single microtubule asters in the droplets. These are two of the most prominent forms of microtubule arrangements in plant and metazoan cells. These results provide insight into the combined organising influence of spatial constraints and crosslinking motor activities determining distinct microtubule architectures in a minimal biomimetic system. In the future, this simple lipid monolayered droplet system characterised here, can be readily expanded to include further biochemical activities or used as the starting point for the investigation of motor-mediated microtubule organisation inside liposomes surrounded by a deformable lipid bilayer.
    Full-text · Article · Jun 2014 · Journal of Biological Chemistry
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    ABSTRACT: Interactions between antiparallel microtubules are essential for the organization of spindles in dividing cells. The ability to form immobilized antiparallel microtubule pairs in vitro, combined with the ability to image them via TIRF microscopy, permits detailed biochemical characterization of microtubule cross-linking proteins and their effects on microtubule dynamics. Here, we describe methods for chemical micropatterning of microtubule seeds on glass surfaces in configurations that specifically promote the formation of antiparallel microtubule overlaps in vitro. We demonstrate that this assay is especially well suited for reconstitution of minimal midzone overlaps stabilized by the antiparallel microtubule cross-linking protein PRC1 and its binding partners. The micropatterning method is suitable for use with a broad range of proteins, and the assay is generally applicable to any microtubule cross-linking protein.
    No preview · Article · Mar 2014 · Methods in enzymology
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    ABSTRACT: End-binding proteins (EBs) have the ability to autonomously track the ends of growing microtubules, where they recruit several proteins that control various aspects of microtubule cytoskeleton organization and function. The structural nature of the binding site recognized by EBs at growing microtubule ends has been a subject of debate. Recently, a fluorescence microscopy assay used for the study of dynamic end tracking in vitro was adapted for cryoelectron microscopy (cryo-EM). In combination with single-particle reconstruction methods, this modified assay was used to produce the first subnanometer-resolution model of how the microtubule-binding domain of EBs binds to microtubules grown in the presence of GTPγS. A GTPγS microtubule can be considered a static mimic of the transiently existing binding region of EBs at a microtubule end growing in the presence of GTP. Here we describe in detail the procedure used to generate these samples. It relies on the polymerization of microtubules from preformed stabilized and quantum dot-labeled microtubule seeds. This allows the cryo-EM analysis of proteins bound to paclitaxel-free microtubules. It provides freedom for using different GTP analogues during microtubule elongation independent of their nucleation properties. This assay could also be useful for the cryo-EM analysis of other microtubule-associated proteins.
    No preview · Article · Mar 2014 · Methods in molecular biology (Clifton, N.J.)
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    ABSTRACT: The dynamic properties of microtubules depend on complex nanoscale structural rearrangements in their end regions. Members of the EB1 and XMAP215 protein families interact autonomously with microtubule ends. EB1 recruits several other proteins to growing microtubule ends and has seemingly antagonistic effects on microtubule dynamics: it induces catastrophes, and it increases growth velocity, as does the polymerase XMAP215. RESULTS: Using a combination of in vitro reconstitution, time-lapse fluorescence microscopy, and subpixel-precision image analysis and convolved model fitting, we have studied the effects of EB1 on conformational transitions in growing microtubule ends and on the time course of catastrophes. EB1 density distributions at growing microtubule ends reveal two consecutive conformational transitions in the microtubule end region, which have growth-velocity-independent kinetics. EB1 binds to the microtubule after the first and before the second conformational transition has occurred, positioning it several tens of nanometers behind XMAP215, which binds to the extreme microtubule end. EB1 binding accelerates conformational maturation in the microtubule, most likely by promoting lateral protofilament interactions and by accelerating reactions of the guanosine triphosphate (GTP) hydrolysis cycle. The microtubule maturation time is directly linked to the duration of a growth pause just before microtubule depolymerization, indicating an important role of the maturation time for the control of dynamic instability. CONCLUSIONS: These activities establish EB1 as a microtubule maturation factor and provide a mechanistic explanation for its effects on microtubule growth and catastrophe frequency, which cause microtubules to be more dynamic.
    Full-text · Article · Feb 2014 · Current Biology
  • Johanna Roostalu · Thomas Surrey
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    ABSTRACT: Multiple activities cooperate to determine the architecture of the mitotic spindle. Kip3 is a kinesin-8 motor protein in budding yeast that acts as a microtubule depolymerase. Now Kip3 is shown to also crosslink and slide antiparallel microtubules, providing additional insights into how kinesin-8 motors control spindle integrity.
    No preview · Article · Aug 2013 · Nature Cell Biology
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    Ivo A Telley · Imre Gáspár · Anne Ephrussi · Thomas Surrey

    Full-text · Dataset · Feb 2013
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    Ivo A Telley · Imre Gáspár · Anne Ephrussi · Thomas Surrey
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    ABSTRACT: Spindle assembly and chromosome segregation rely on a complex interplay of biochemical and mechanical processes. Analysis of this interplay requires precise manipulation of endogenous cellular components and high-resolution visualization. Here we provide a protocol for generating an extract from individual Drosophila syncytial embryos that supports repeated mitotic nuclear divisions with native characteristics. In contrast to the large-scale, metaphase-arrested Xenopus egg extract system, this assay enables the serial generation of extracts from single embryos of a genetically tractable organism, and each extract contains dozens of autonomously dividing nuclei that can be prepared and imaged within 60-90 min after embryo collection. We describe the microscopy setup and micropipette production that facilitate single-embryo manipulation, the preparation of embryos and the steps for making functional extracts that allow time-lapse microscopy of mitotic divisions ex vivo. The assay enables a unique combination of genetic, biochemical, optical and mechanical manipulations of the mitotic machinery.
    Full-text · Article · Jan 2013 · Nature Protocol
  • Surajit Ghosh · Christian Hentrich · Thomas Surrey
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    ABSTRACT: Microtubule organisation in living cells is determined by spatial control of microtubule nucleation, their dynamic properties and transport by molecular motors. Here, we establish a new micro-pattern-guided method for controlling local microtubule nucleation by spatially confined immobilisation of a microtubule polymerase and show that these nucleated microtubules can be transported and organised in space by motor proteins. This assay provides a new platform for deciphering the principles underlying mesoscale microtubule organisation.
    No preview · Article · Jan 2013 · ACS Chemical Biology
  • Marileen Dogterom · Thomas Surrey
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    ABSTRACT: Microtubules organize into a set of distinct patterns with the help of associated molecules that control nucleation, polymerization, crosslinking, and transport. These patterns, alone or in combination with each other, define the functional architecture of the microtubule cytoskeleton in living cells. In vitro experiments of increasing complexity help understand, in combination with theoretical models, the basic mechanisms by which elementary microtubule patterns arise, how they are maintained, and how they position themselves with respect to the confining geometry of living cells.
    No preview · Article · Dec 2012 · Current opinion in cell biology

Publication Stats

3k Citations
720.18 Total Impact Points


  • 2015
    • The Francis Crick Institute
      Londinium, England, United Kingdom
  • 2012-2015
    • London Research Institute
      Londinium, England, United Kingdom
  • 2012-2014
    • Cancer Research UK
      Londinium, England, United Kingdom
  • 2003-2014
    • European Molecular Biology Laboratory
      • • Cell Biology and Biophysics Unit (Heidelberg)
      • • Developmental Biology Unit (Heidelberg)
      Heidelburg, Baden-Württemberg, Germany
  • 2010
    • Keio University
      • Department of Chemistry
      Edo, Tōkyō, Japan
  • 2009
    • Université de Montpellier
      Montpelhièr, Languedoc-Roussillon, France
    • Universität Osnabrück
      Osnabrück, Lower Saxony, Germany
  • 2008
    • The Ohio State University
      • Ohio Agricultural Research and Development Center
      Columbus, Ohio, United States
  • 1998
    • Princeton University
      • Department of Physics
      Princeton, NJ, United States