Derek Toomre

Yale-New Haven Hospital, New Haven, Connecticut, United States

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Publications (79)701.09 Total impact

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    ABSTRACT: We report a lipid-based strategy to visualize Golgi structure and dynamics at super-resolution in live cells. The method is based on two novel reagents: a trans-cyclooctene-containing ceramide lipid (Cer-TCO) and a highly reactive, tetrazine-tagged near-IR dye (SiR-Tz). These reagents assemble via an extremely rapid “tetrazine-click” reaction into Cer-SiR, a highly photostable “vital dye” that enables prolonged live-cell imaging of the Golgi apparatus by 3D confocal and STED microscopy. Cer-SiR is nontoxic at concentrations as high as 2 μM and does not perturb the mobility of Golgi-resident enzymes or the traffic of cargo from the endoplasmic reticulum through the Golgi and to the plasma membrane.
    Angewandte Chemie International Edition 09/2014; 53(38). DOI:10.1002/anie.201403349 · 11.26 Impact Factor
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    ABSTRACT: Wir berichten über eine Lipid-basierte Strategie zur Visualisierung von Struktur und Dynamik des Golgi-Apparats in lebenden Zellen mithilfe hochauflösender Mikroskopie. Die Methode basiert auf zwei neuen Reagentien: einem trans-Cycloocten enthaltenden Ceramid-Lipid (Cer-TCO) und einem hoch reaktiven, Tetrazin-markierten Nah-IR-Farbstoff (SiR-Tz). Diese beiden Komponenten reagieren in einer extrem schnellen Tetrazin-Klick-Reaktion zu Cer-SiR, einer sehr photostabilen Verbindung, welche die Visualisierung des Golgi-Apparats sowohl mit 3D-Konfokalmikroskopie als auch mit hochauflösender Mikroskopie über eine längere Zeitspanne ermöglicht. Cer-SiR ist nicht toxisch bis zu einer Konzentration von 2 μM und stört weder die Mobilität von Enzymen innerhalb des Golgi-Apparats noch den Transport von Fracht vom Endoplasmatischen Retikulum durch den Golgi-Apparat zur Plasmamembran.
    Angewandte Chemie 09/2014; 126(38). DOI:10.1002/ange.201403349
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    ABSTRACT: Despite recent advances in understanding store-operated calcium entry (SOCE) regulation, the fundamental question of how ER morphology affects this process remains unanswered. Here we show that the loss of a single isoform of RTN4, RTN4b, is sufficient to alter ER morphology and severely compromise SOCE. Mechanistically, we show this to be the result of defective STIM1-Orai1 coupling due to loss of ER tubulation and redistribution of STIM1 to ER sheets. As a functional consequence, RTN4b depleted cells fail to sustain elevated cytoplasmic Ca2+ levels via SOCE and therefor are less susceptible to Ca2+ overload induced apoptosis. Thus, for the first time, our results show a direct correlation between ER morphology and SOCE and highlight the importance of RTN4b in cellular Ca2+ homeostasis.
    Journal of Biological Chemistry 02/2014; 289(13). DOI:10.1074/jbc.M114.548602 · 4.60 Impact Factor
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    ABSTRACT: Neutrophil degranulation plays an important role in acute innate immune responses and is tightly regulated because the granule contents can cause tissue damage. However, this regulation remains poorly understood. Here, we identify the complex of STK24 and CCM3 as being an important regulator of neutrophil degranulation. Lack of either STK24 or CCM3 increases the release of a specific granule pool without affecting other neutrophil functions. STK24 appears to suppress exocytosis by interacting and competing with UNC13D C2B domain for lipid binding, whereas CCM3 has dual roles in exocytosis regulation. Although CCM3 stabilizes STK24, it counteracts STK24-mediated inhibition of exocytosis by recruiting STK24 away from the C2B domain through its Ca(2+)-sensitive interaction with UNC13D C2A domain. This STK24/CCM3-regulated exocytosis plays an important role in the protection of kidneys from ischemia-reperfusion injury. Together, these findings reveal a function of the STK24 and CCM3 complex in the regulation of ligand-stimulated exocytosis.
    Developmental Cell 10/2013; 27(2):215-26. DOI:10.1016/j.devcel.2013.09.021 · 10.37 Impact Factor
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    ABSTRACT: Newly developed scientific complementary metal-oxide semiconductor (sCMOS) cameras have the potential to dramatically accelerate data acquisition, enlarge the field of view and increase the effective quantum efficiency in single-molecule switching nanoscopy. However, sCMOS-intrinsic pixel-dependent readout noise substantially lowers the localization precision and introduces localization artifacts. We present algorithms that overcome these limitations and that provide unbiased, precise localization of single molecules at the theoretical limit. Using these in combination with a multi-emitter fitting algorithm, we demonstrate single-molecule localization super-resolution imaging at rates of up to 32 reconstructed images per second in fixed and living cells.
    Nature Methods 05/2013; 10(7). DOI:10.1038/nmeth.2488 · 25.95 Impact Factor
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    Felix Rivera-Molina, Derek Toomre
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    ABSTRACT: Tethers play ubiquitous roles in membrane trafficking and influence the specificity of vesicle attachment. Unlike soluble N-ethyl-maleimide-sensitive fusion attachment protein receptors (SNAREs), the spatiotemporal dynamics of tethers relative to vesicle fusion are poorly characterized. The most extensively studied tethering complex is the exocyst, which spatially targets vesicles to sites on the plasma membrane. By using a mammalian genetic replacement strategy, we were able to assemble fluorescently tagged Sec8 into the exocyst complex, which was shown to be functional by biochemical, trafficking, and morphological criteria. Ultrasensitive live-cell imaging revealed that Sec8-TagRFP moved to the cell cortex on vesicles, which preferentially originated from the endocytic recycling compartment. Surprisingly, Sec8 remained with vesicles until full dilation of the fusion pore, supporting potential coupling with SNARE fusion machinery. Fluorescence recovery after photobleaching analysis of Sec8 at cell protrusions revealed that a significant fraction was immobile. Additionally, Sec8 dynamically repositioned to the site of membrane expansion, suggesting that it may respond to local cues during early cell polarization.
    The Journal of Cell Biology 05/2013; 201(5). DOI:10.1083/jcb.201212103 · 9.69 Impact Factor
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    ABSTRACT: Neuropilin 1 (NRP1) plays an important but ill-defined role in VEGF-A signaling and vascular morphogenesis. We show that mice with a knockin mutation that ablates the NRP1 cytoplasmic tail (Nrp1cyto) have normal angiogenesis but impaired developmental and adult arteriogenesis. The arteriogenic defect was traced to the absence of a PDZ-dependent interaction between NRP1 and VEGF receptor 2 (VEGFR2) complex and synectin, which delayed trafficking of endocytosed VEGFR2 from Rab5+ to EAA1+ endosomes. This led to increased PTPN1 (PTP1b)-mediated dephosphorylation of VEGFR2 at Y1175, the site involved in activating ERK signaling. The Nrp1cyto mutation also impaired endothelial tubulogenesis in vitro, which could be rescued by expressing full-length NRP1 or constitutively active ERK. These results demonstrate that the NRP1 cytoplasmic domain promotes VEGFR2 trafficking in a PDZ-dependent manner to regulate arteriogenic ERK signaling and establish a role for NRP1 in VEGF-A signaling during vascular morphogenesis.
    Developmental Cell 04/2013; 25(2):156-168. DOI:10.1016/j.devcel.2013.03.019 · 10.37 Impact Factor
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    ABSTRACT: We present the integration of an adaptive optics element into a feedback-driven single particle tracking microscope. Our instrument captures three-dimensional (3D) trajectories with down to 130 ls temporal resolution for dynamic studies on the nanoscale. Our 3D beam steering approach tracks particles over an axial range of >6 lm with �2ms mechanical response times and isolates the sample from any tracking motion. Tracking of transport vesicles containing Alexa488-labeled transferrin glycoprotein in living cells demonstrates the speed and sensitivity of our instrument. VC 2013 Author(s). All article content, except where otherwise noted, is licensed under a Creative Commons Attribution 3.0 Unported License. [http://dx.doi.org/10.1063/1.4803538]
    Applied Physics Letters 04/2013; 102(17). DOI:10.1063/1.4803538 · 3.52 Impact Factor
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    ABSTRACT: Phosphoinositides (PIs) are lipid components of cell membranes that regulate a wide variety of cellular functions. Here we exploited the blue light-induced dimerization between two plant proteins, cryptochrome 2 (CRY2) and the transcription factor CIBN, to control plasma membrane PI levels rapidly, locally, and reversibly. The inositol 5-phosphatase domain of OCRL (5-ptase(OCRL)), which acts on PI(4,5)P(2) and PI(3,4,5)P(3), was fused to the photolyase homology region domain of CRY2, and the CRY2-binding domain, CIBN, was fused to plasma membrane-targeting motifs. Blue-light illumination (458-488 nm) of mammalian cells expressing these constructs resulted in nearly instantaneous recruitment of 5-ptase(OCRL) to the plasma membrane, where it caused rapid (within seconds) and reversible (within minutes) dephosphorylation of its targets as revealed by diverse cellular assays: dissociation of PI(4,5)P(2) and PI(3,4,5)P(3) biosensors, disappearance of endocytic clathrin-coated pits, nearly complete inhibition of KCNQ2/3 channel currents, and loss of membrane ruffling. Focal illumination resulted in local and transient 5-ptase(OCRL) recruitment and PI(4,5)P(2) dephosphorylation, causing not only local collapse and retraction of the cell edge or process but also compensatory accumulation of the PI(4,5)P(2) biosensor and membrane ruffling at the opposite side of the cells. Using the same approach for the recruitment of PI3K, local PI(3,4,5)P(3) synthesis and membrane ruffling could be induced, with corresponding loss of ruffling distally to the illuminated region. This technique provides a powerful tool for dissecting with high spatial-temporal kinetics the cellular functions of various PIs and reversibly controlling the functions of downstream effectors of these signaling lipids.
    Proceedings of the National Academy of Sciences 07/2012; 109(35):E2316-23. DOI:10.1073/pnas.1211305109 · 9.81 Impact Factor
  • Derek Toomre
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    ABSTRACT: Live cell fluorescent microscopy is important in elucidating dynamic cellular processes such as cell signaling, membrane trafficking, and cytoskeleton remodeling. Often, transient intermediate states are revealed only when imaged and quantitated at the single-molecule, vesicle, or organelle level. Such insight depends on the spatiotemporal resolution and sensitivity of a given microscopy method. Confocal microscopes optically section the cell and improve image contrast and axial resolution (>600 nm) compared with conventional epifluorescence microscopes. Another approach, which can selectively excite fluorophores in an even thinner optical plane (<100 nm) is total internal reflection fluorescence microscopy (TIRFM). The key principle of TIRFM is that a thin, exponentially decaying, evanescent field of excitation can be generated at the interface of two mediums of different refractive index (RI) (e.g., the glass coverslip and the biological specimen); as such, TIRFM is ill-suited to deep imaging of cells or tissue. However, for processes near the lower cell cortex, the sensitivity of TIRFM is exquisite. The recent availability of a very high numerical-aperture (NA) objective lens (>1.45) and turnkey TIRFM systems by all the major microscopy manufacturers has made TIRFM increasingly accessible and attractive to biologists, especially when performed in a quantitative manner and complemented with orthogonal genetic and molecular manipulations. This article discusses sample preparation for TIRFM, acquisition of time-lapse movies, and quantitative analysis. It also gives examples of imaging cytoskeleton dynamics and exo- and endocytosis using TIRFM.
    Cold Spring Harbor Protocols 04/2012; 2012(4):439-46. DOI:10.1101/pdb.ip068676 · 4.63 Impact Factor
  • Derek Toomre
    [Show abstract] [Hide abstract]
    ABSTRACT: Live cell fluorescent microscopy is important in elucidating dynamic cellular processes such as cell signaling, membrane trafficking, and cytoskeleton remodeling. Often, transient intermediate states are revealed only when imaged and quantitated at the single-molecule, vesicle, or organelle level. Such insight depends on the spatiotemporal resolution and sensitivity of a given microscopy method. Confocal microscopes optically section the cell and improve image contrast and axial resolution (>600 nm) compared with conventional epifluorescence microscopes. Another approach, which can selectively excite fluorophores in an even thinner optical plane (<100 nm) is total internal reflection fluorescence microscopy (TIRFM). The key principle of TIRFM is that a thin, exponentially decaying, evanescent field of excitation can be generated at the interface of two mediums of different refractive index (RI) (e.g., the glass coverslip and the biological specimen); as such, TIRFM is ill-suited to deep imaging of cells or tissue. However, for processes near the lower cell cortex, the sensitivity of TIRFM is exquisite. The recent availability of a very high numerical-aperture (NA) objective lens (>1.45) and turnkey TIRFM systems by all the major microscopy manufacturers has made TIRFM increasingly accessible and attractive to biologists, especially when performed in a quantitative manner and complemented with orthogonal genetic and molecular manipulations. This article discusses the optical principles of TIRFM (including a sample calculation of penetration depth), the components of a TIRFM setup, and the use of TIRFM in combination with other imaging modalities.
    Cold Spring Harbor Protocols 04/2012; 2012(4):414-24. DOI:10.1101/pdb.top068650 · 4.63 Impact Factor
  • Derek Toomre
    [Show abstract] [Hide abstract]
    ABSTRACT: Live cell fluorescent microscopy is important in elucidating dynamic cellular processes such as cell signaling, membrane trafficking, and cytoskeleton remodeling. Often, transient intermediate states are revealed only when imaged and quantitated at the single-molecule, vesicle, or organelle level. Such insight depends on the spatiotemporal resolution and sensitivity of a given microscopy method. Confocal microscopes optically section the cell and improve image contrast and axial resolution (>600 nm) compared with conventional epifluorescence microscopes. Another approach, which can selectively excite fluorophores in an even thinner optical plane (<100 nm) is total internal reflection fluorescence microscopy (TIRFM). The key principle of TIRFM is that a thin, exponentially decaying, evanescent field of excitation can be generated at the interface of two mediums of different refractive index (RI) (e.g., the glass coverslip and the biological specimen); as such, TIRFM is ill-suited to deep imaging of cells or tissue. However, for processes near the lower cell cortex, the sensitivity of TIRFM is exquisite. The recent availability of a very high numerical-aperture (NA) objective lens (>1.45) and turnkey TIRFM systems by all the major microscopy manufacturers has made TIRFM increasingly accessible and attractive to biologists, especially when performed in a quantitative manner and complemented with orthogonal genetic and molecular manipulations. This protocol describes the procedure for alignment and calibration of TIRFM systems using standard cellular samples. The goal is to correctly collimate and align the TIRF illuminator vis-à-vis the downstream optics. For illustration, a 488-nm laser and green fluorescent protein (GFP) filter cube are used.
    Cold Spring Harbor Protocols 04/2012; 2012(4):504-9. DOI:10.1101/pdb.prot068668 · 4.63 Impact Factor
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    ABSTRACT: PV1 protein is an essential component of stomatal and fenestral diaphragms, which are formed at the plasma membrane of endothelial cells (ECs), on structures such as caveolae, fenestrae and transendothelial channels. Knockout of PV1 in mice results in in utero and perinatal mortality. To be able to interpret the complex PV1 knockout phenotype, it is critical to determine whether the formation of diaphragms is the only cellular role of PV1. We addressed this question by measuring the effect of complete and partial removal of structures capable of forming diaphragms on PV1 protein level. Removal of caveolae in mice by knocking out caveolin-1 or cavin-1 resulted in a dramatic reduction of PV1 protein level in lungs but not kidneys. The magnitude of PV1 reduction correlated with the abundance of structures capable of forming diaphragms in the microvasculature of these organs. The absence of caveolae in the lung ECs did not affect the transcription or translation of PV1, but it caused a sharp increase in PV1 protein internalization rate via a clathrin- and dynamin-independent pathway followed by degradation in lysosomes. Thus, PV1 is retained on the cell surface of ECs by structures capable of forming diaphragms, but undergoes rapid internalization and degradation in the absence of these structures, suggesting that formation of diaphragms is the only role of PV1.
    PLoS ONE 03/2012; 7(3):e32655. DOI:10.1371/journal.pone.0032655 · 3.53 Impact Factor
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    Kohei Arasaki, Derek K Toomre, Craig R Roy
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    ABSTRACT: The intracellular bacterial pathogen Legionella pneumophila subverts host membrane transport pathways to promote fusion of vesicles exiting the endoplasmic reticulum (ER) with the pathogen-containing vacuole. During infection there is noncanonical pairing of the SNARE protein Sec22b on ER-derived vesicles with plasma membrane (PM)-localized syntaxin proteins on the vacuole. We show that the L. pneumophila Rab1-targeting effector DrrA is sufficient to stimulate this noncanonical SNARE association and promote membrane fusion. DrrA activation of the Rab1 GTPase on PM-derived organelles stimulated the tethering of ER-derived vesicles with the PM-derived organelle, resulting in vesicle fusion through the pairing of Sec22b with the PM syntaxin proteins. Thus, the effector protein DrrA stimulates a host membrane transport pathway that enables ER-derived vesicles to remodel a PM-derived organelle, suggesting that Rab1 activation at the PM is sufficient to promote the recruitment and fusion of ER-derived vesicles.
    Cell host & microbe 01/2012; 11(1):46-57. DOI:10.1016/j.chom.2011.11.009 · 12.19 Impact Factor
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    ABSTRACT: Recent advancement in live cell fluorescence microscopy has enabled image acquisition at single particle resolution, through which biologists can investigate the underlying mechanisms of cellular processes. In this paper, we present a method to automatically detect the features of sub-cellular particles in 2D fluorescence images, including x-y positions, fluorescence intensities, and relative sizes. The method consists of two parts. One is an initial detection method, which finds particle candidates in the images using image filters and clustering algorithms. The other is a MAP-Bayesian based estimation method, which provides the optimal estimations of particle features. The method is evaluated on synthetic data and results show that it has high accuracy. The results on real data confirmed by human expert cell biologists are also presented.
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    ABSTRACT: Multi-angle total internal reflection fluorescence microscopy (MA-TIRFM) is a relatively new and powerful tool to study subcellular particles near cell membrane due to its unique illumination mechanism. We present a MAP-Bayesian method to automatically estimate features of individual particles in MA-TIRF images, including 3D positions, relative sizes, and relative amount of fluorophores. Using the MAP criterion, the optimal values of the features can be obtained by maximizing a nonlinear functional. Initial feature values are estimated by using image filters and clustering algorithms. The method is evaluated on synthetic data and results show that it has high accuracy. The result on real data from our initial experiments is also presented.
    Proceedings / IEEE International Symposium on Biomedical Imaging: from nano to macro. IEEE International Symposium on Biomedical Imaging 01/2012; DOI:10.1109/ISBI.2012.6235722
  • Yingke Xu, Thomas J Melia, Derek K Toomre
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    ABSTRACT: Cellular compartmentalization into discrete organelles is maintained by membrane trafficking including vesiculation and tubulation. Recent advances in superresolution imaging have begun to bring these small and dynamic events into focus. Most nanoscopes exploit, and are limited by, switching dyes ON and OFF. Using ground state depletion to switch dyes into long-lived dark states can exploit specific photophysical properties of dyes, such as redox potential or pK(a), and expand the repertoire of nanoscopy probes for multicolor imaging. Seeing is not enough, and new technologies based on homodimerization, heterodimerization and selective release can manipulate membrane trafficking in pulse-chase and light-controlled ways. Herein we highlight the utility and promise of these strategies and discuss their current limitations.
    Current opinion in chemical biology 11/2011; 15(6):822-30. DOI:10.1016/j.cbpa.2011.10.016 · 7.65 Impact Factor
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    ABSTRACT: In addition to enhancing or repressing transcription, steroid hormone receptors rapidly transduce kinase activation signals. On ligand engagement, an N-terminus-truncated splice isoform of estrogen receptor (ER) α, ER46, triggers membrane-initiated signals, resulting in endothelial nitric oxide synthase (eNOS) activation and endothelial NO production. The orientation of ER46 at the plasma membrane is incompletely defined. With the use of ecliptic pHluorin-fused ER46, total internal reflection fluorescence microscopy in live human endothelial cells illustrates that ER46 can topologically conform to a type I transmembrane protein structure. Mutation of isoleucine-386 at the center of ER46's transmembrane hydrophobic core prevents membrane spanning, obscures the N-terminal ectodomain, and effects a marked reduction in membrane-impermeant estrogen binding with diminished rapid eNOS activation and NO production, despite maintained genomic induction of an estrogen response element-luciferase reporter. Thus there exist pools of transmembrane steroid hormone receptors that are efficient signaling molecules and potential novel therapeutic targets.
    Molecular biology of the cell 09/2011; 22(22):4415-23. DOI:10.1091/mbc.E11-05-0416 · 5.98 Impact Factor
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    ABSTRACT: Insulin stimulates translocation of GLUT4 storage vesicles (GSVs) to the surface of adipocytes, but precisely where insulin acts is controversial. Here we quantify the size, dynamics, and frequency of single vesicle exocytosis in 3T3-L1 adipocytes. We use a new GSV reporter, VAMP2-pHluorin, and bypass insulin signaling by disrupting the GLUT4-retention protein TUG. Remarkably, in unstimulated TUG-depleted cells, the exocytic rate is similar to that in insulin-stimulated control cells. In TUG-depleted cells, insulin triggers a transient, twofold burst of exocytosis. Surprisingly, insulin promotes fusion pore expansion, blocked by acute perturbation of phospholipase D, which reflects both properties intrinsic to the mobilized vesicles and a novel regulatory site at the fusion pore itself. Prolonged stimulation causes cargo to switch from approximately 60 nm GSVs to larger exocytic vesicles characteristic of endosomes. Our results support a model whereby insulin promotes exocytic flux primarily by releasing an intracellular brake, but also by accelerating plasma membrane fusion and switching vesicle traffic between two distinct circuits.
    The Journal of Cell Biology 05/2011; 193(4):643-53. DOI:10.1083/jcb.201008135 · 9.69 Impact Factor

Publication Stats

8k Citations
701.09 Total Impact Points

Institutions

  • 2005–2014
    • Yale-New Haven Hospital
      • Department of Pathology
      New Haven, Connecticut, United States
    • Yale University
      • • Department of Cell Biology
      • • Department of Electrical Engineering
      New Haven, Connecticut, United States
  • 2012
    • University of New Haven
      New Haven, Connecticut, United States
  • 2010
    • Howard Hughes Medical Institute
      Ashburn, Virginia, United States
  • 2006–2010
    • University of Valencia
      • Department of Informatic
      Valenza, Valencia, Spain
    • University of Castilla-La Mancha
      Ciudad Real, Castille-La Mancha, Spain
  • 2009
    • Molecular and Cellular Biology Program
      Seattle, Washington, United States
  • 2004–2008
    • Ludwig Boltzmann Institute for Cancer Research
      Wien, Vienna, Austria
  • 2002–2006
    • Ludwig Institute for Cancer Research
      La Jolla, California, United States
  • 2001–2002
    • European Molecular Biology Laboratory
      • Cell Biology and Biophysics Unit (Heidelberg)
      Heidelburg, Baden-Württemberg, Germany
  • 2000
    • Max Planck Institute of Molecular Cell Biology and Genetics
      Dresden, Saxony, Germany