Jeffrey H Stear

Humboldt University of Berlin, Berlin, Land Berlin, Germany

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Publications (7)65.09 Total impact

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    ABSTRACT: XMAP215/Dis1 family proteins positively regulate microtubule growth. Repeats at their N termini, called TOG domains, are important for this function. While TOG domains directly bind tubulin dimers, it is unclear how this interaction translates to polymerase activity. Understanding the functional roles of TOG domains is further complicated by the fact that the number of these domains present in the proteins of different species varies. Here, we take advantage of a recent crystal structure of the third TOG domain from Caenorhabditis elegans, Zyg9, and mutate key residues in each TOG domain of XMAP215 that are predicted to be important for interaction with the tubulin heterodimer. We determined the contributions of the individual TOG domains to microtubule growth. We show that the TOG domains are absolutely required to bind free tubulin and that the domains differentially contribute to XMAP215's overall affinity for free tubulin. The mutants' overall affinity for free tubulin correlates well with polymerase activity. Furthermore, we demonstrate that an additional basic region is important for targeting to the microtubule lattice and is critical for XMAP215 to function at physiological concentrations. Using this information, we have engineered a "bonsai" protein, with two TOG domains and a basic region, that has almost full polymerase activity.
    Proceedings of the National Academy of Sciences 02/2011; 108(7):2741-6. DOI:10.1073/pnas.1016498108
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    ABSTRACT: In vitro assays that reconstitute the dynamic behavior of microtubules provide insight into the roles of microtubule-associated proteins (MAPs) in regulating the growth, shrinkage, and catastrophe of microtubules. The use of total internal reflection fluorescence microscopy with fluorescently labeled tubulin and MAPs has allowed us to study microtubule dynamics at the resolution of single molecules. In this chapter we present a practical overview of how these assays are performed in our laboratory: fluorescent labeling methods, strategies to prolong the time to photo-bleaching, preparation of stabilized microtubules, flow-cells, microtubule immobilization, and finally an overview of the workflow that we follow when performing the experiments. At all stages, we focus on practical tips and highlight potential stumbling blocks.
    Methods in cell biology 01/2010; 95:221-45. DOI:10.1016/S0091-679X(10)95013-9
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    ABSTRACT: Plus-end-tracking proteins (+TIPs) are localized at the fast-growing, or plus end, of microtubules, and link microtubule ends to cellular structures. One of the best studied +TIPs is EB1, which forms comet-like structures at the tips of growing microtubules. The molecular mechanisms by which EB1 recognizes and tracks growing microtubule ends are largely unknown. However, one clue is that EB1 can bind directly to a microtubule end in the absence of other proteins. Here we use an in vitro assay for dynamic microtubule growth with two-color total-internal-reflection-fluorescence imaging to investigate binding of mammalian EB1 to both stabilized and dynamic microtubules. We find that under conditions of microtubule growth, EB1 not only tip tracks, as previously shown, but also preferentially recognizes the GMPCPP microtubule lattice as opposed to the GDP lattice. The interaction of EB1 with the GMPCPP microtubule lattice depends on the E-hook of tubulin, as well as the amount of salt in solution. The ability to distinguish different nucleotide states of tubulin in microtubule lattice may contribute to the end-tracking mechanism of EB1.
    PLoS ONE 10/2009; 4(10):e7585. DOI:10.1371/journal.pone.0007585
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    ABSTRACT: Fast growth of microtubules is essential for rapid assembly of the microtubule cytoskeleton during cell proliferation and differentiation. XMAP215 belongs to a conserved family of proteins that promote microtubule growth. To determine how XMAP215 accelerates growth, we developed a single-molecule assay to visualize directly XMAP215-GFP interacting with dynamic microtubules. XMAP215 binds free tubulin in a 1:1 complex that interacts with the microtubule lattice and targets the ends by a diffusion-facilitated mechanism. XMAP215 persists at the plus end for many rounds of tubulin subunit addition in a form of "tip tracking." These results show that XMAP215 is a processive polymerase that directly catalyzes the addition of up to 25 tubulin dimers to the growing plus end. Under some circumstances XMAP215 can also catalyze the reverse reaction, namely microtubule shrinkage. The similarities between XMAP215 and formins, actin polymerases, suggest that processive tip tracking is a common mechanism for stimulating the growth of cytoskeletal polymers.
    Cell 02/2008; 132(1):79-88. DOI:10.1016/j.cell.2007.11.043
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    Jeffrey H Stear, Mark B Roth
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    ABSTRACT: Previous studies of the kinetochore in mammalian systems have demonstrated that this structure undergoes reorganizations after microtubule attachment or in response to activation of the spindle checkpoint. Here, we show that the Caenorhabditis elegans kinetochore displays analogous rearrangements at prometaphase, when microtubule/chromosome interactions are being established, and after exposure to checkpoint stimuli such as nocodazole or anoxia. These reorganizations are characterized by a dissociation of several kinetochore proteins, including HCP-1/CeCENP-F, HIM-10/CeNuf2, SAN-1/CeMad3, and CeBUB-1, from the centromere. We further demonstrate that at metaphase, despite having dissociated from the centromere, these reorganized kinetochore proteins maintain their associations with the metaphase plate. After checkpoint activation, these proteins are detectable as large "flares" that project out laterally from the metaphase plate. Disrupting these gene products via RNA interference results in sensitivity to checkpoint stimuli, as well as defects in the organization of chromosomes at metaphase. These phenotypes suggest that these proteins, and by extension their reorganization during mitosis, are important for mediating the checkpoint response as well as directing the assembly of the metaphase plate.
    Molecular Biology of the Cell 12/2004; 15(11):5187-96. DOI:10.1091/mbc.E04-06-0486
  • Jeffrey H Stear, Mark B Roth
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    ABSTRACT: Previous studies of mitosis show that capture of single kinetochores by microtubules from both centrosomes (merotelic orientation) is a major cause of aneuploidy. We have characterized hcp-6, a temperature-sensitive chromosome segregation mutant in C. elegans that exhibits chromosomes attached to both poles via a single sister kinetochore. We demonstrate that the primary defect in this mutant is a failure to fully condense chromosomes during prophase. Although centromere formation and sister centromere resolution remain unaffected in hcp-6, the chromosomes lack the rigidity of wild-type chromosomes and twist around the long axis of the chromosome. As such, they are unable to establish a proper orientation at prometaphase, allowing individual kinetochores to be captured by microtubules from both poles. We therefore propose that chromosome rigidity plays an essential role in maintaining chromosome orientation to prevent merotelic capture.
    Genes & Development 07/2002; 16(12):1498-508. DOI:10.1101/gad.989102
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    Jeffrey H. Stear, Mark B. Roth

Publication Stats

413 Citations
65.09 Total Impact Points

Institutions

  • 2010–2011
    • Humboldt University of Berlin
      • Department of Biology
      Berlin, Land Berlin, Germany
  • 2008–2009
    • Max Planck Institute of Molecular Cell Biology and Genetics
      Dresden, Saxony, Germany
  • 2002–2004
    • Fred Hutchinson Cancer Research Center
      • Division of Basic Sciences
      Seattle, Washington, United States