Steve Caplan

University of Nebraska at Omaha, Omaha, Nebraska, United States

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Publications (80)364.54 Total impact

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    ABSTRACT: The endocytic recycling compartment (ERC) is a series of perinuclear tubular and vesicular membranes that regulates recycling to the plasma membrane. Despite evidence that cargo is sorted at the early/sorting endosome (SE), whether cargo mixes downstream at the ERC or remains segregated is still an unanswered question. Herein, we use 3D Structured Illumination Microscopy, dual-channel and 3D direct Stochastic Optical Reconstruction Microscopy (dSTORM) to obtain new information about ERC morphology and cargo segregation. We show that cargo internalized either via clathrin-mediated endocytosis (CME) or independently of clathrin (CIE) remains segregated in the ERC, likely on distinct carriers. This suggests that no further sorting occurs upon cargo exit from SE. Moreover, 3D dSTORM data support a model in which some, but not all ERC vesicles are tethered by contiguous 'membrane bridges.' Furthermore, TRE preferentially traffic CIE cargo, and may originate from SE membranes. These findings support a significantly altered model for endocytic recycling in mammalian cells, in which sorting occurs in peripheral endosomes and segregation is maintained at the ERC.
    No preview · Article · Oct 2015 · Molecular biology of the cell
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    ABSTRACT: Endocytosis, which encompasses the internalization and sorting of plasma membrane (PM) lipids and proteins to distinct membrane-bound intracellular compartments, is a highly regulated and fundamental cellular process by which eukaryotic cells dynamically regulate their PM composition. Indeed, endocytosis is implicated in crucial cellular processes that include proliferation, migration, and cell division as well as maintenance of tissue homeostasis such as apical-basal polarity. Once PM constituents have been taken up into the cell, either via clathrin-dependent endocytosis (CDE) or clathrin-independent endocytosis (CIE), they typically have two fates: degradation through the late-endosomal/lysosomal pathway or returning to the PM via endocytic recycling pathways. In this review, we will detail experimental procedures that allow for both qualitative and quantitative assessment of endocytic recycling of transmembrane proteins internalized by CDE and CIE, using the HeLa cervical cancer cell line as a model system.
    No preview · Article · Sep 2015 · Methods in cell biology
  • Shuwei Xie · Naava Naslavsky · Steve Caplan

    No preview · Article · Aug 2015
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    Full-text · Dataset · Apr 2015
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    Kriti Bahl · Naava Naslavsky · Steve Caplan
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    ABSTRACT: The C-terminal Eps 15 Homology Domain proteins (EHD1-4) play important roles in regulating endocytic trafficking. EHD2 is the only family member whose crystal structure has been solved, and it contains an unstructured loop consisting of two proline-phenylalanine (PF) motifs: KPFRKLNPF. In contrast, despite EHD2 having nearly 70% amino acid identity with its paralogs, EHD1, EHD3 and EHD4, the latter proteins contain a single KPF or RPF motif, but no NPF motif. In this study, we sought to define the precise role of each PF motif in EHD2's homo-dimerization, binding with the protein partners, and subcellular localization. To test the role of the NPF motif, we generated an EHD2 NPF-to-NAF mutant to mimic the homologous sequences of EHD1 and EHD3. We demonstrated that this mutant lost both its ability to dimerize and bind to Syndapin2. However, it continued to localize primarily to the cytosolic face of the plasma membrane. On the other hand, EHD2 NPF-to-APA mutants displayed normal dimerization and Syndapin2 binding, but exhibited markedly increased nuclear localization and reduced association with the plasma membrane. We then hypothesized that the single PF motif of EHD1 (that aligns with the KPF of EHD2) might be responsible for both binding and localization functions of EHD1. Indeed, the EHD1 RPF motif was required for dimerization, interaction with MICAL-L1 and Syndapin2, as well as localization to tubular recycling endosomes. Moreover, recycling assays demonstrated that EHD1 RPF-to-APA was incapable of supporting normal receptor recycling. Overall, our data suggest that the EHD2 NPF phenylalanine residue is crucial for EHD2 localization to the plasma membrane, whereas the proline residue is essential for EHD2 dimerization and binding. These studies support the recently proposed model in which the EHD2 N-terminal region may regulate the availability of the unstructured loop for interactions with neighboring EHD2 dimers, thus promoting oligomerization.
    Preview · Article · Apr 2015 · PLoS ONE
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    ABSTRACT: Hyaluronan turnover accelerates metastatic progression of prostate cancer partly by increasing rates of tumor cell proliferation and motility. To determine the mechanism, we overexpressed Hyal1 as a fluorescent fusion protein and examined its impact on endocytosis and vesicular trafficking. Overexpression of Hyal1 led to increased rates of internalization of hyaluronan and the endocytic recycling marker transferrin. Live imaging of Hyal1, sucrose gradient centrifugation, and specific co-localization of Rab GTPases defined the subcellular distribution of Hyal1 as early and late endosomes, lysosomes, and recycling vesicles. Manipulation of vesicular trafficking by chemical inhibitors or with constitutively active and dominant negative Rab expression constructs, caused atypical localization of Hyal1. Using the catalytically inactive point mutant Hyal1-E131Q, we found enzymatic activity of Hyal1 was necessary for normal localization within the cell, as Hyal1-E131Q was mainly found within the ER. Expression of a hyaluronan-binding point mutant Hyal1-Y202F revealed that secretion of Hyal1 and concurrent reuptake from the extracellular space is critical for rapid hyaluronan internalization and cell proliferation. Overall, excess Hyal1 secretion accelerates endocytic vesicle trafficking in a substrate-dependent manner, which promotes aggressive tumor cell behavior. Copyright © 2015, The American Society for Biochemistry and Molecular Biology.
    No preview · Article · Apr 2015 · Journal of Biological Chemistry
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    ABSTRACT: Membrane association with mother centriole (M-centriole) distal appendages is critical for ciliogenesis initiation. How the Rab GTPase Rab11-Rab8 cascade functions in early ciliary membrane assembly is unknown. Here, we show that the membrane shaping proteins EHD1 and EHD3, in association with the Rab11-Rab8 cascade, function in early ciliogenesis. EHD1 and EHD3 localize to preciliary membranes and the ciliary pocket. EHD-dependent membrane tubulation is essential for ciliary vesicle formation from smaller distal appendage vesicles (DAVs). Importantly, this step functions in M-centriole to basal body transformation and recruitment of transition zone proteins and IFT20. SNAP29, a SNARE membrane fusion regulator and EHD1-binding protein, is also required for DAV-mediated ciliary vesicle assembly. Interestingly, only after ciliary vesicle assembly is Rab8 activated for ciliary growth. Our studies uncover molecular mechanisms informing a previously uncharacterized ciliogenesis step, whereby EHD1 and EHD3 reorganize the M-centriole and associated DAVs before coordinated ciliary membrane and axoneme growth.
    Full-text · Article · Feb 2015 · Nature Cell Biology
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    ABSTRACT: Amyloid precursor-like protein 2 (APLP2) is aberrantly expressed in pancreatic cancer. Here we showed that APLP2 is increased in pancreatic cancer metastases, particularly in metastatic lesions found in the diaphragm and intestine. Examination of matched human primary tumor-liver metastasis pairs showed that 38.1% of the patients had positive APLP2 expression in both the primary tumor and the corresponding liver metastasis. Stable knock-down of APLP2 expression (with inducible shRNA) in pancreatic cancer cells reduced the ability of these cells to migrate and invade. Loss of APLP2 decreased cortical actin and increased intracellular actin filaments in pancreatic cancer cells. Down-regulation of APLP2 decreased the weight and metastasis of orthotopically transplanted pancreatic tumors in nude mice.
    Full-text · Article · Dec 2014 · Oncotarget
  • Shuwei Xie · Naava Naslavsky · Steve Caplan
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    ABSTRACT: Major histocompatibility complex class I (MHC I) presents intracellular-derived peptides to cytotoxic T lymphocytes and its subcellular itinerary is important in regulating the immune response. While a number of diacylglycerol kinase isoforms have been implicated in clathrin-dependent internalization, MHC I lacks the typical motifs known to mediate clathrin-dependent endocytosis. Here we show that depletion of diacylglycerol kinase α (DGKα), a kinase devoid of a clathrin-dependent adaptor protein complex 2 binding site, caused a delay in MHC I recycling to the plasma membrane without affecting the rate of MHC I internalization. We demonstrate that DGKα knock-down causes accumulation of intracellular and surface MHC I, resulting from decreased degradation. Furthermore, we provide evidence that DGKα is required for the generation of phosphatidic acid required for tubular recycling endosome (TRE) biogenesis. Moreover, we show that DGKα forms a complex with the TRE hub protein, MICAL-L1. Given that MICAL-L1 and the F-BAR-containing membrane-tubulating protein Syndapin2 associate selectively with phosphatidic acid, we propose a positive feedback loop in which DGKα generates phosphatidic acid to drive its own recruitment to TRE via its interaction with MICAL-L1. Our data support a novel role for the involvement of DGKα in TRE biogenesis and MHC I recycling.
    No preview · Article · Sep 2014 · Journal of Biological Chemistry
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    ABSTRACT: During interphase, recycling endosomes mediate the transport of internalized cargo back to the plasma membrane. However, in mitotic cells, recycling endosomes are essential for the completion of cytokinesis, the last phase of mitosis that promotes the physical separation the two daughter cells. Despite recent advances, our understanding of the molecular determinants that regulate recycling endosome dynamics during cytokinesis remains incomplete. We have previously demonstrated that Molecule Interacting with CasL Like-1 (MICAL-L1) and C-terminal Eps15 Homology Domain protein 1 (EHD1) coordinately regulate receptor transport from tubular recycling endosomes during interphase. However, their potential roles in controlling cytokinesis had not been addressed. In this study, we show that MICAL-L1 and EHD1 regulate mitosis. Depletion of either protein resulted in increased numbers of bi-nucleated cells. We provide evidence that bi-nucleation in MICAL-L1- and EHD1-depleted cells is a consequence of impaired recycling endosome transport during late cytokinesis. However, depletion of MICAL-L1, but not EHD1, resulted in aberrant chromosome alignment and lagging chromosomes, suggesting an EHD1-independent function for MICAL-L1 earlier in mitosis. Moreover, we provide evidence that MICAL-L1 and EHD1 differentially influence microtubule dynamics during early and late mitosis. Collectively, our new data suggest several unanticipated roles for MICAL-L1 and EHD1 during the cell cycle.
    No preview · Article · Sep 2014 · Traffic
  • James Reinecke · Steve Caplan
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    ABSTRACT: Abstract The regulated intracellular transport of nutrient, adhesion, and growth factor receptors is crucial for maintaining cell and tissue homeostasis. Endocytosis, or endocytic membrane trafficking, involves the steps of intracellular transport that include, but are not limited to, internalization from the plasma membrane, sorting in early endosomes, transport to late endosomes/lysosomes followed by degradation, and/or recycling back to the plasma membrane through tubular recycling endosomes. In addition to regulating the localization of transmembrane receptor proteins, the endocytic pathway also controls the localization of non-receptor molecules. The non-receptor tyrosine kinase c-Src (Src) and its closely related family members Yes and Fyn represent three proteins whose localization and signaling activities are tightly regulated by endocytic trafficking. Here, we provide a brief overview of endocytosis, Src function and its biochemical regulation. We will then concentrate on recent advances in understanding how Src intracellular localization is regulated and how its subcellular localization ultimately dictates downstream functioning. As Src kinases are hyperactive in many cancers, it is essential to decipher the spatiotemporal regulation of this important family of tyrosine kinases.
    No preview · Article · May 2014 · Biomolecular concepts
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    Bishuang Cai · Shuwei Xie · Steve Caplan · Naava Naslavsky
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    ABSTRACT: The biogenesis of tubular recycling endosomes (TREs) and their subsequent vesiculation after cargo-sorting has occurred, is essential for receptor and lipid recycling to the plasma membrane. Although recent studies have implicated the C-terminal Eps15 Homology Domain (EHD) protein, EHD1, as a key regulator of TRE vesiculation, additional proteins involved in this process have been largely uncharacterized. In the present study, we identify the GTPase Regulator Associated with Focal adhesion kinase-1 (GRAF1) protein in a complex with EHD1 and the TRE hub protein, Molecules Interacting with CasL-Like1 (MICAL-L1). Over-expression of GRAF1 caused vesiculation of MICAL-L1-containing TRE, whereas GRAF1-depletion led to impaired TRE vesiculation and delayed receptor recycling. Moreover, co-addition of purified EHD1 and GRAF1 in a semi-permeabilized cell vesiculation assay produced synergistic TRE vesiculation. Overall, based on our data, we suggest that in addition to its roles in clathrin-independent endocytosis, GRAF1 synergizes with EHD1 to support TRE vesiculation.
    Preview · Article · May 2014 · Frontiers in Cell and Developmental Biology
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    ABSTRACT: Protein kinases have long been reported to regulate connexins, however little is known about the involvement of phosphatases in the modulation of gap junction intercellular communication and subsequent down-stream effects on cellular processes. Here we identified an interaction between the T-Cell Protein Tyrosine Phosphatase (TC-PTP) and the carboxyl terminus of Cx43. NRK cells endogenously expressing Cx43 and a version containing v-Src with temperature sensitive activity were used to demonstrate that EGF and v-Src stimulation, respectively, induced TC-PTP to co-localize with Cx43 at the plasma membrane. Cell biology experiments using phospho-specific antibodies and biophysical assays demonstrated that the interaction is direct and that TC-PTP dephosphorylates Cx43 residues Y247 and Y265, but not v-Src. TC-PTP also indirectly led to dephosphorylation of Cx43 S368 by inactivating PKCα and PKCδ, with no effect on S279 and S282 phosphorylation levels (MAPK target). Dephosphorylation maintained Cx43 gap junctions at the plaque as well as partially reversed channel closure caused by v-Src phosphorylation. Understanding dephosphorylation, along with the well-documented roles of Cx43 phosphorylation, will help build a better foundation to modulate the regulation of gap junction channels to benefit human health.
    Full-text · Article · May 2014 · Journal of Cell Science
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    ABSTRACT: There is increased interest in immune-based monoclonal antibody therapies for different malignancies because of their potential specificity and limited toxicity. The activity of some therapeutic monoclonal antibodies is partially dependent on complement-dependent cytolysis (CDC), in which the immune system surveys for invading pathogens, infected cells and malignant cells and facilitates their destruction. CD59 is a ubiquitously expressed cell-surface glycosyl-phosphatidylinositol-anchored protein that protects cells from CDC. However, in certain tumors, CD59 expression is enhanced, posing a significant obstacle for treatment, by hindering effective monoclonal antibody-induced CDC. In this study, we used non-small lung carcinoma cells to characterize the mechanism of a novel CD59-inhibitor: the 114 amino acid recombinant form of the 4th domain of intermedilysin (rILYd4), a pore forming toxin secreted by Streptococcus intermedius. We compared the rates of internalization of CD59 in the presence of rILYd4 or anti-CD59 antibodies and determined that rILYd4 induces more rapid CD59 uptake at early time points. Most significantly, upon binding to rILYd4, CD59 undergoes massive degradation in lysosomes within minutes. The remaining rILYd4/CD59 complexes recycle to the PM and are shed from the cell. In comparison, upon internalization of CD59 via anti-CD59 antibody binding, the antibody/CD59 complex is recycled via early- and recycling-endosomes, mostly avoiding degradation. Our study supports a novel role for rILYd4 in promoting internalization and rapid degradation of the complement inhibitor CD59, and highlights the potential for improving CDC-based immunotherapy.
    Preview · Article · Mar 2014 · Journal of Biological Chemistry
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    ABSTRACT: Localization of the non-receptor tyrosine kinase c-Src (Src) to the cell periphery is required for its activation and to mediate focal adhesion turnover, cell spreading and migration. Inactive Src localizes to a perinuclear compartment and endocytic transport mediates Src movement to the plasma membrane. However, the precise pathways and regulatory proteins responsible for Src transport are incompletely understood. Here we demonstrate that Src partially co-localizes with the endocytic regulatory protein Molecule Interacting with CasL-Like1 (MICAL-L1) in mammalian cells. Furthermore, MICAL-L1 is required for growth factor and integrin-induced Src activation and transport to the cell periphery in HeLa cells and human fibroblasts. Accordingly, MICAL-L1 depletion impairs focal adhesion turnover, cell spreading and cell migration. Interestingly, we find that the MICAL-L1 interaction partner Eps15 Homology Domain-containing protein 1 (EHD1) is also required for Src activation and transport. Moreover, EHD1 recruitment to Src-containing recycling endosomes by MICAL-L1 is required for Src release from the perinuclear endocytic recycling compartment in response to growth factor stimulation. Our study sheds new light on the mode by which Src is transported to the plasma membrane and activated, and provides a new function for MICAL-L1 and EHD1 in the regulation of intracellular non-receptor tyrosine kinases.
    Preview · Article · Jan 2014 · Journal of Cell Science
  • Laura C Simone · Naava Naslavsky · Steve Caplan
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    ABSTRACT: The C-terminal Eps15 homology domain-containing (EHD) proteins participate in multiple aspects of endocytic membrane trafficking. Of the four mammalian EHD proteins, EHD2 appears to be the most disparate, both in terms of sequence homology, and in subcellular localization/function. Since its initial description as a plasma membrane-associated protein, the precise function of EHD2 has remained enigmatic. Various reports have suggested roles for EHD2 at the plasma membrane, within the endocytic transport system, and even in the nucleus. For example, EHD2 facilitates membrane fusion/repair in muscle cells. Recently the focus has shifted to the role of EHD2 in regulating caveolae. Indeed, EHD2 is highly expressed in tissues rich in caveolae, including fat, muscle and blood vessels. This review highlights cumulative evidence linking EHD2 to actin-rich structures at the plasma membrane, where the plasma membrane-associated phospholipid phosphatidylinositol 4,5-bisphosphate controls EHD2 recruitment. Herein we examine the key pathways where EHD2 might function, and address its potential involvement in these processes.
    No preview · Article · Dec 2013 · Histology and histopathology
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    Laura C Simone · Steve Caplan · Naava Naslavsky
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    ABSTRACT: The four mammalian C-terminal Eps15 homology domain-containing proteins (EHD1-EHD4) play pivotal roles in endocytic membrane trafficking. While EHD1, EHD3 and EHD4 associate with intracellular tubular/vesicular membranes, EHD2 localizes to the inner leaflet of the plasma membrane. Currently, little is known about the regulation of EHD2. Thus, we sought to define the factors responsible for EHD2's association with the plasma membrane. The subcellular localization of endogenous EHD2 was examined in HeLa cells using confocal microscopy. Although EHD partner proteins typically mediate EHD membrane recruitment, EHD2 was targeted to the plasma membrane independent of two well-characterized binding proteins, syndapin2 and EHBP1. Additionally, the EH domain of EHD2, which facilitates canonical EHD protein interactions, was not required to direct overexpressed EHD2 to the cell surface. On the other hand, several lines of evidence indicate that the plasma membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2) plays a crucial role in regulating EHD2 subcellular localization. Pharmacologic perturbation of PIP2 metabolism altered PIP2 plasma membrane distribution (as assessed by confocal microscopy), and caused EHD2 to redistribute away from the plasma membrane. Furthermore, overexpressed EHD2 localized to PIP2-enriched vacuoles generated by active Arf6. Finally, we show that although cytochalasin D caused actin microfilaments to collapse, EHD2 was nevertheless maintained at the plasma membrane. Intriguingly, cytochalasin D induced relocalization of both PIP2 and EHD2 to actin aggregates, supporting a role of PIP2 in controlling EHD2 subcellular localization. Altogether, these studies emphasize the significance of membrane lipid composition for EHD2 subcellular distribution and offer new insights into the regulation of this important endocytic protein.
    Preview · Article · Sep 2013 · PLoS ONE
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    ABSTRACT: Endocytic recycling involves the return of membranes and receptors to the plasma membrane following their internalization into the cell. Recycling generally occurs from a series of vesicular and tubular membranes localized to the perinuclear region, collectively known as the endocytic recycling compartment. Within this compartment, receptors are sorted into tubular extensions that later undergo vesiculation, allowing transport vesicles to move along microtubules and return to the cell surface where they ultimately undergo fusion with the plasma membrane. Recent studies have led to the hypothesis that the C-terminal Eps15 Homology Domain ATPase proteins are involved in the vesiculation process. Herein, we address the functional roles of the four EHD proteins. We developed a novel semi-permeabilized cell system in which addition of purified EHD proteins to reconstitute vesiculation allows us to assess each protein's ability to vesiculate MICAL-L1-decorated tubular recycling endosomes (TRE). Using this assay, we show that EHD1 vesiculates membranes, consistent with enhanced TRE generation observed upon EHD1-depletion. EHD4 serves a similar role to EHD1 in TRE vesiculation, whereas EHD2, despite being capable of vesiculating TRE in the semi-permeabilized cells, fails do so in vivo. Surprisingly, the addition of EHD3 causes tubulation of endocytic membranes in our semi-permeabilized cell system, consistent with the lack of tubulation observed upon EHD3-depletion. Our novel vesiculation assay and in vitro electron microscopy analysis, combined with in vivo data, provide convincing evidence that the functions of both EHD1 and EHD4 are primarily in TRE membrane vesiculation, whereas EHD3 is a membrane tubulating protein.
    Full-text · Article · Sep 2013 · Journal of Biological Chemistry
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    ABSTRACT: Significance: The molecules interacting with CasL (MICAL) family members participate in a multitude of activities, including axonal growth cone repulsion, membrane trafficking, apoptosis, and bristle development in flies. An interesting feature of MICAL proteins is the presence of an N-terminal flavo-mono-oxygenase domain. This mono-oxygenase domain generates redox potential with which MICALs can either oxidize proteins or produce reactive oxygen species (ROS). Actin is one such protein that is affected by MICAL function, leading to dramatic cytoskeletal rearrangements. This review describes the MICAL-family members, and discusses their mechanisms of actin-binding and regulation of actin cytoskeleton organization. Recent advances: Recent studies show that MICALs directly induce oxidation of actin molecules, leading to actin depolymerization. ROS production by MICALs also causes oxidation of collapsin response mediator protein-2, a microtubule assembly promoter, which subsequently undergoes phosphorylation. Critical issues: MICAL proteins oxidize proteins through two mechanisms: either directly by oxidizing methionine residues or indirectly via the production of ROS. It remains unclear whether MICAL proteins employ both mechanisms or whether the activity of MICAL-family proteins might vary with different substrates. Future directions: The identification of additional substrates oxidized by MICAL will shed new light on MICAL protein function. Additional directions include expanding studies toward the MICAL-like homologs that lack flavin adenine dinucleotide domains and oxidation activity.
    Full-text · Article · Jul 2013 · Antioxidants & Redox Signaling
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    ABSTRACT: The C-terminal Eps15 homology (EH) domain 3 (EHD3) belongs to a eukaryotic family of endocytic regulatory proteins and is involved in the recycling of various receptors from the early endosome to the endocytic recycling compartment or in retrograde transport from the endosomes to the Golgi. EH domains are highly conserved in the EHD family and function as protein-protein interaction units that bind to Asn-Pro-Phe (NPF) motif-containing proteins. The EH domain of EHD1 was the first C-terminal EH domain from the EHD family to be solved by NMR. The differences observed between this domain and proteins with N-terminal EH domains helped describe a mechanism for the differential binding of NPF-containing proteins. Here, structural studies were expanded to include the EHD3 EH domain. While the EHD1 and EHD3 EH domains are highly homologous, they have different protein partners. A comparison of these structures will help determine the selectivity in protein binding between the EHD family members and lead to a better understanding of their unique roles in endocytic regulation.
    No preview · Article · Jun 2013 · Biomolecular NMR Assignments

Publication Stats

3k Citations
364.54 Total Impact Points

Institutions

  • 2008-2015
    • University of Nebraska at Omaha
      • Department of Biochemistry and Molecular Biology
      Omaha, Nebraska, United States
  • 2004-2015
    • University of Nebraska Medical Center
      • • Department of Pharmaceutical Sciences
      • • Department of Pathology and Microbiology
      • • Department of Pharmacology and Experimental Neuroscience
      • • Department of Oral Biology
      Omaha, Nebraska, United States
  • 2013
    • French National Centre for Scientific Research
      Lutetia Parisorum, Île-de-France, France
  • 2009
    • The Nebraska Medical Center
      Omaha, Nebraska, United States
  • 2001-2002
    • National Institute of Child Health and Human Development
      Maryland, United States
    • Eunice Kennedy Shriver National Institute of Child Health and Human Development
      Роквилл, Maryland, United States
  • 2000
    • National Institutes of Health
      • Cell Biology and Metabolism Program
      Bethesda, MD, United States
  • 1993-1996
    • Hebrew University of Jerusalem
      • Hadassah Medical School
      Yerushalayim, Jerusalem, Israel