Publications by authors named "Jonathan A Cooper"

63 Publications

Fbxo45 Binds SPRY Motifs in the Extracellular Domain of N-Cadherin and Regulates Neuron Migration during Brain Development.

Mol Cell Biol 2020 06 29;40(14). Epub 2020 Jun 29.

Basic Sciences Division, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA

Several events during the normal development of the mammalian neocortex depend on N-cadherin, including the radial migration of immature projection neurons into the cortical plate. Remarkably, radial migration requires the N-cadherin extracellular domain but not N-cadherin-dependent homophilic cell-cell adhesion, suggesting that other N-cadherin-binding proteins may be involved. We used proximity ligation and affinity purification proteomics to identify N-cadherin-binding proteins. Both screens detected MycBP2 and SPRY domain protein Fbxo45, two components of an intracellular E3 ubiquitin ligase. Fbxo45 appears to be secreted by a nonclassical mechanism, not involving a signal peptide and not requiring transport from the endoplasmic reticulum to the Golgi apparatus. Fbxo45 binding requires N-cadherin SPRY motifs that are not involved in cell-cell adhesion. SPRY mutant N-cadherin does not support radial migration Radial migration was similarly inhibited when Fbxo45 expression was suppressed. The results suggest that projection neuron migration requires both Fbxo45 and the binding of Fbxo45 or another protein to SPRY motifs in the extracellular domain of N-cadherin.
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http://dx.doi.org/10.1128/MCB.00539-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7324847PMC
June 2020

N-cadherin-regulated FGFR ubiquitination and degradation control mammalian neocortical projection neuron migration.

Elife 2019 10 2;8. Epub 2019 Oct 2.

Laboratory of Mammalian Development & Cell Biology, Institute of Neuroscience, Université Catholique de Louvain, Brussels, Belgium.

The functions of FGF receptors (FGFRs) in early development of the cerebral cortex are well established. Their functions in the migration of neocortical projection neurons, however, are unclear. We have found that FGFRs regulate multipolar neuron orientation and the morphological change into bipolar cells necessary to enter the cortical plate. Mechanistically, our results suggest that FGFRs are activated by N-Cadherin. N-Cadherin cell-autonomously binds FGFRs and inhibits FGFR K27- and K29-linked polyubiquitination and lysosomal degradation. Accordingly, FGFRs accumulate and stimulate prolonged Erk1/2 phosphorylation. Neurons inhibited for Erk1/2 are stalled in the multipolar zone. Moreover, Reelin, a secreted protein regulating neuronal positioning, prevents FGFR degradation through N-Cadherin, causing Erk1/2 phosphorylation. These findings reveal novel functions for FGFRs in cortical projection neuron migration, suggest a physiological role for FGFR and N-Cadherin interaction in vivo and identify Reelin as an extracellular upstream regulator and Erk1/2 as downstream effectors of FGFRs during neuron migration.
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http://dx.doi.org/10.7554/eLife.47673DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6786859PMC
October 2019

Comparative Analysis of cul5 and rbx2 Expression in the Developing and Adult Murine Brain and Their Essentiality During Mouse Embryogenesis.

Dev Dyn 2018 11 7;247(11):1227-1236. Epub 2018 Nov 7.

Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington.

Background: The E3 Cullin 5-RING ubiquitin ligase (CRL5) is a multiprotein complex that has recently been highlighted as a major regulator of central nervous system development. Cullin 5 (Cul5) and the RING finger protein Rbx2 are two CRL5 core components required for CRL5 function in the brain, but their full expression patterns and developmental functions have not been described in detail.

Results: Using a gene-trap mouse model for Cul5 and a knock-in-knockout mouse model for Rbx2, we show that lack of Cul5, but not Rbx2, disrupts blastocyst formation. However, Rbx2 is required for embryo survival at later embryonic stages. We also show that cul5 is expressed in the embryo proper as early as E7.5 and its expression is mostly restricted to the central nervous system and limbs at later time points. Finally, we show that rbx2 and cul5 are co-expressed in most areas of the brain during development and in the adult.

Conclusions: Our results show that Cul5, but not Rbx2, is required during early embryogenesis and suggests that Cul5 has Rbx2-independent functions in early development. In the brain, Cul5 and Rbx2 are expressed in a similar fashion, allowing the nucleation of an active CRL5 complex. Developmental Dynamics 247:1227-1236, 2018. © 2018 Wiley Periodicals, Inc.
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http://dx.doi.org/10.1002/dvdy.24675DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6639038PMC
November 2018

Mutations in the Epithelial Cadherin-p120-Catenin Complex Cause Mendelian Non-Syndromic Cleft Lip with or without Cleft Palate.

Am J Hum Genet 2018 06 24;102(6):1143-1157. Epub 2018 May 24.

Centre for Clinical Genetics, Sydney Children's Hospital, Randwick, Sydney, NSW 2031, Australia; Prince of Wales Clinical School, University of New South Wales, Randwick, NSW 2031, Australia; Neuroscience Research Australia, Sydney, NSW 2031, Australia. Electronic address:

Non-syndromic cleft lip with or without cleft palate (NS-CL/P) is one of the most common human birth defects and is generally considered a complex trait. Despite numerous loci identified by genome-wide association studies, the effect sizes of common variants are relatively small, with much of the presumed genetic contribution remaining elusive. We report exome-sequencing results in 209 people from 72 multi-affected families with pedigree structures consistent with autosomal-dominant inheritance and variable penetrance. Herein, pathogenic variants are described in four genes encoding components of the p120-catenin complex (CTNND1, PLEKHA7, PLEKHA5) and an epithelial splicing regulator (ESRP2), in addition to the known CL/P-associated gene, CDH1, which encodes E-cadherin. The findings were also validated in a second cohort of 497 people with NS-CL/P, comprising small families and singletons with pathogenic variants in these genes identified in 14% of multi-affected families and 2% of the replication cohort of smaller families. Enriched expression of each gene/protein in human and mouse embryonic oro-palatal epithelia, demonstration of functional impact of CTNND1 and ESRP2 variants, and recapitulation of the CL/P spectrum in Ctnnd1 knockout mice support a causative role in CL/P pathogenesis. These data show that primary defects in regulators of epithelial cell adhesion are the most significant contributors to NS-CL/P identified to date and that inherited and de novo single gene variants explain a substantial proportion of NS-CL/P.
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http://dx.doi.org/10.1016/j.ajhg.2018.04.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5992119PMC
June 2018

Paracrine Fibroblast Growth Factor Initiates Oncogenic Synergy with Epithelial FGFR/Src Transformation in Prostate Tumor Progression.

Neoplasia 2018 03 11;20(3):233-243. Epub 2018 Feb 11.

Department of Pharmaceutical and Biomedical Sciences, College of Pharmacy, University of Georgia, Athens, Georgia 30602. Electronic address:

Cross talk of stromal-epithelial cells plays an essential role in both normal development and tumor initiation and progression. Fibroblast growth factor (FGF)-FGF receptor (FGFR)-Src kinase axis is one of the major signal transduction pathways to mediate this cross talk. Numerous genomic studies have demonstrated that expression levels of FGFR/Src are deregulated in a variety of cancers including prostate cancer; however, the role that paracrine FGF (from stromal cells) plays in dysregulated expression of epithelial FGFRs/Src and tumor progression in vivo is not well evaluated. In this study, we demonstrate that ectopic expression of wild-type FGFR1/2 or Src kinase in epithelial cells was not sufficient to initiate prostate tumorigenesis under a normal stromal microenvironment in vivo. However, paracrine FGF10 synergized with ectopic expression of epithelial FGFR1 or FGFR2 to induce epithelial-mesenchymal transition. Additionally, paracrine FGF10 sensitized FGFR2-transformed epithelial cells to initiate prostate tumorigenesis. Next, paracrine FGF10 also synergized with overexpression of epithelial Src kinase to high-grade tumors. But loss of the myristoylation site in Src kinase inhibited paracrine FGF10-induced prostate tumorigenesis. Loss of myristoylation alters Src levels in the cell membrane and inhibited FGF-mediated signaling including inhibition of the phosphotyrosine pattern and FAK phosphorylation. Our study demonstrates the potential tumor progression by simultaneous deregulation of proteins in the FGF/FGFRs/Src signal axis and provides a therapeutic strategy of targeting myristoylation of Src kinase to interfere with the tumorigenic process.
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http://dx.doi.org/10.1016/j.neo.2018.01.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5814375PMC
March 2018

SOCS2 Binds to and Regulates EphA2 through Multiple Mechanisms.

Sci Rep 2017 09 7;7(1):10838. Epub 2017 Sep 7.

Division of Basic Sciences, Fred Hutchinson Cancer Research Center, 1100 Fairview Ave N, Seattle, Washington, 98109, USA.

Suppressors of cytokine signaling (SOCS) proteins inhibit signaling by serving as substrate receptors for the Cullin5-RING E3 ubiquitin ligase (CRL5) and through a variety of CRL5-independent mechanisms. CRL5, SOCS2 and SOCS6 are implicated in suppressing transformation of epithelial cells. We identified cell proteins that interact with SOCS2 and SOCS6 using two parallel proteomics techniques: BioID and Flag affinity purification mass spectrometry. The receptor tyrosine kinase ephrin type-A receptor 2 (EphA2) was identified as a SOCS2-interacting protein. SOCS2-EphA2 binding requires the SOCS2 SH2 domain and EphA2 activation loop autophosphorylation, which is stimulated by Ephrin A1 (EfnA1) or by phosphotyrosine phosphatase inhibition. Surprisingly, EfnA1-stimulated EphA2-SOCS2 binding is delayed until EphA2 has been internalized into endosomes. This suggests that SOCS2 binds to EphA2 in the context of endosomal membranes. We also found that SOCS2 overexpression decreases steady state levels of EphA2, consistent with increased EphA2 degradation. This effect is indirect: SOCS2 induces EfnA1 expression, and EfnA1 induces EphA2 down-regulation. Other RTKs have been reported to bind, and be regulated by, over-expressed SOCS proteins. Our data suggest that SOCS protein over-expression may regulate receptor tyrosine kinases through indirect and direct mechanisms.
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http://dx.doi.org/10.1038/s41598-017-11040-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5589800PMC
September 2017

Llgl1 Connects Cell Polarity with Cell-Cell Adhesion in Embryonic Neural Stem Cells.

Dev Cell 2017 06 25;41(5):481-495.e5. Epub 2017 May 25.

Division of Human Biology, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA; Molecular and Cellular Biology Program, University of Washington, Seattle, WA 98195, USA; Department of Pathology, Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, WA 98195, USA. Electronic address:

Malformations of the cerebral cortex (MCCs) are devastating developmental disorders. We report here that mice with embryonic neural stem-cell-specific deletion of Llgl1 (Nestin-Cre/Llgl1), a mammalian ortholog of the Drosophila cell polarity gene lgl, exhibit MCCs resembling severe periventricular heterotopia (PH). Immunohistochemical analyses and live cortical imaging of PH formation revealed that disruption of apical junctional complexes (AJCs) was responsible for PH in Nestin-Cre/Llgl1 brains. While it is well known that cell polarity proteins govern the formation of AJCs, the exact mechanisms remain unclear. We show that LLGL1 directly binds to and promotes internalization of N-cadherin, and N-cadherin/LLGL1 interaction is inhibited by atypical protein kinase C-mediated phosphorylation of LLGL1, restricting the accumulation of AJCs to the basolateral-apical boundary. Disruption of the N-cadherin-LLGL1 interaction during cortical development in vivo is sufficient for PH. These findings reveal a mechanism responsible for the physical and functional connection between cell polarity and cell-cell adhesion machineries in mammalian cells.
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http://dx.doi.org/10.1016/j.devcel.2017.05.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5519327PMC
June 2017

Optogenetic control of the Dab1 signaling pathway.

Sci Rep 2017 03 8;7:43760. Epub 2017 Mar 8.

Division of Basic Sciences, Fred Hutchinson Cancer Research Center, 1100 Fairview Ave N, Seattle, Washington 98109, USA.

The Reelin-Dab1 signaling pathway regulates development of the mammalian brain, including neuron migrations in various brain regions, as well as learning and memory in adults. Extracellular Reelin binds to cell surface receptors and activates phosphorylation of the intracellular Dab1 protein. Dab1 is required for most effects of Reelin, but Dab1-independent pathways may contribute. Here we developed a single-component, photoactivatable Dab1 (opto-Dab1) by using the blue light-sensitive dimerization/oligomerization property of A. thaliana Cryptochrome 2 (Cry2). Opto-Dab1 can activate downstream signals rapidly, locally, and reversibly upon blue light illumination. The high spatiotemporal resolution of the opto-Dab1 probe also allows us to control membrane protrusion, retraction and ruffling by local illumination in both COS7 cells and in primary neurons. This shows that Dab1 activation is sufficient to orient cell movement in the absence of other signals. Opto-Dab1 may be useful to study the biological functions of the Reelin-Dab1 signaling pathway both in vitro and in vivo.
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http://dx.doi.org/10.1038/srep43760DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5363252PMC
March 2017

The ubiquitin-proteasome system regulates focal adhesions at the leading edge of migrating cells.

Elife 2016 09 22;5. Epub 2016 Sep 22.

Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, United States.

Cell migration requires the cyclical assembly and disassembly of focal adhesions. Adhesion induces phosphorylation of focal adhesion proteins, including Cas (Crk-associated substrate/p130Cas/BCAR1). However, Cas phosphorylation stimulates adhesion turnover. This raises the question of how adhesion assembly occurs against opposition from phospho-Cas. Here we show that suppressor of cytokine signaling 6 (SOCS6) and Cullin 5, two components of the CRL5 ubiquitin ligase, inhibit Cas-dependent focal adhesion turnover at the front but not rear of migrating epithelial cells. The front focal adhesions contain phospho-Cas which recruits SOCS6. If SOCS6 cannot access focal adhesions, or if cullins or the proteasome are inhibited, adhesion disassembly is stimulated. This suggests that the localized targeting of phospho-Cas within adhesions by CRL5 and concurrent cullin and proteasome activity provide a negative feedback loop, ensuring that adhesion assembly predominates over disassembly at the leading edge. By this mechanism, ubiquitination provides a new level of spatio-temporal control over cell migration.
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http://dx.doi.org/10.7554/eLife.17440DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5092051PMC
September 2016

Disabled homolog 2 controls macrophage phenotypic polarization and adipose tissue inflammation.

J Clin Invest 2016 Apr 29;126(4):1311-22. Epub 2016 Feb 29.

Acute and chronic tissue injury results in the generation of a myriad of environmental cues that macrophages respond to by changing their phenotype and function. This phenotypic regulation is critical for controlling tissue inflammation and resolution. Here, we have identified the adaptor protein disabled homolog 2 (DAB2) as a regulator of phenotypic switching in macrophages. Dab2 expression was upregulated in M2 macrophages and suppressed in M1 macrophages isolated from both mice and humans, and genetic deletion of Dab2 predisposed macrophages to adopt a proinflammatory M1 phenotype. In mice with myeloid cell-specific deletion of Dab2 (Dab2fl/fl Lysm-Cre), treatment with sublethal doses of LPS resulted in increased proinflammatory gene expression and macrophage activation. Moreover, chronic high-fat feeding exacerbated adipose tissue inflammation, M1 polarization of adipose tissue macrophages, and the development of insulin resistance in DAB2-deficient animals compared with controls. Mutational analyses revealed that DAB2 interacts with TNF receptor-associated factor 6 (TRAF6) and attenuates IκB kinase β-dependent (IKKβ-dependent) phosphorylation of Ser536 in the transactivation domain of NF-κB p65. Together, these findings reveal that DAB2 is critical for controlling inflammatory signaling during phenotypic polarization of macrophages and suggest that manipulation of DAB2 expression and function may hold therapeutic potential for the treatment of acute and chronic inflammatory disorders.
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http://dx.doi.org/10.1172/JCI79590DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4811113PMC
April 2016

Cell regulation by phosphotyrosine-targeted ubiquitin ligases.

Mol Cell Biol 2015 Jun 16;35(11):1886-97. Epub 2015 Mar 16.

Department of Biochemistry, Western University, London, Ontario, Canada.

Three classes of E3 ubiquitin ligases, members of the Cbl, Hakai, and SOCS-Cul5-RING ligase families, stimulate the ubiquitination of phosphotyrosine-containing proteins, including receptor and nonreceptor tyrosine kinases and their phosphorylated substrates. Because ubiquitination frequently routes proteins for degradation by the lysosome or proteasome, these E3 ligases are able to potently inhibit tyrosine kinase signaling. Their loss or mutational inactivation can contribute to cancer, autoimmunity, or endocrine disorders, such as diabetes. However, these ligases also have biological functions that are independent of their ubiquitination activity. Here we review relevant literature and then focus on more-recent developments in understanding the structures, substrates, and pathways through which the phosphotyrosine-specific ubiquitin ligases regulate diverse aspects of cell biology.
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http://dx.doi.org/10.1128/MCB.00098-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4420928PMC
June 2015

Molecules and mechanisms that regulate multipolar migration in the intermediate zone.

Front Cell Neurosci 2014 14;8:386. Epub 2014 Nov 14.

Fred Hutchinson Cancer Research Center, Division of Basic Sciences Seattle, Washington, USA.

Most neurons migrate with an elongated, "bipolar" morphology, extending a long leading process that explores the environment. However, when immature projection neurons enter the intermediate zone (IZ) of the neocortex they become "multipolar". Multipolar cells extend and retract cytoplasmic processes in different directions and move erratically-sideways, up and down. Multipolar cells extend axons while they are in the lower half of the IZ. Remarkably, the cells then resume radial migration: they reorient their centrosome and Golgi apparatus towards the pia, transform back to bipolar morphology, and commence locomotion along radial glia (RG) fibers. This reorientation implies the existence of directional signals in the IZ that are ignored during the multipolar stage but sensed after axonogenesis. In vivo genetic manipulation has implicated a variety of candidate directional signals, cell surface receptors, and signaling pathways, that may be involved in polarizing multipolar cells and stabilizing a pia-directed leading process for radial migration. Other signals are implicated in starting multipolar migration and triggering axon outgrowth. Here we review the molecules and mechanisms that regulate multipolar migration, and also discuss how multipolar migration affects the orderly arrangement of neurons in layers and columns in the developing neocortex.
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http://dx.doi.org/10.3389/fncel.2014.00386DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4231986PMC
December 2014

Disabled-2 is required for efficient hemostasis and platelet activation by thrombin in mice.

Arterioscler Thromb Vasc Biol 2014 Nov 11;34(11):2404-12. Epub 2014 Sep 11.

From the Graduate Institute of Biomedical Sciences (H.-J.T., Y.-W.C., C.-P.T.), and Department of Medical Biotechnology and Laboratory Science (C.-L.H., D.-Y.H., C.-C.L., C.-P.T.), College of Medicine, Chang Gung University, Taoyuan, Taiwan; Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, WA (J.A.C.); Department of Medical Laboratory Science and Biotechnology, China Medical University, Taichung, Taiwan (J.-C.C.); Molecular Medicine Research Center, Chang Gung University (C.-P.T.) and Department of Family Medicine (C.-P.T.), Chang Gung Memorial Hospital, Taoyuan, Taiwan.

Objective: The essential role of platelet activation in hemostasis and thrombotic diseases focuses attention on unveiling the underlying intracellular signals of platelet activation. Disabled-2 (Dab2) has been implicated in platelet aggregation and in the control of clotting responses. However, there is not yet any in vivo study to provide direct evidence for the role of Dab2 in hemostasis and platelet activation.

Approach And Results: Megakaryocyte lineage-restricted Dab2 knockout (Dab2(-/-)) mice were generated to delineate in vivo functions of Dab2 in platelets. Dab2(-/-) mice appeared normal in size with prolonged bleeding time and impaired thrombus formation. Although normal in platelet production and granule biogenesis, Dab2(-/-) platelets elicited a selective defect in platelet aggregation and spreading on fibrinogen in response to low concentrations of thrombin, but not other soluble agonists. Investigation of the role of Dab2 in thrombin signaling revealed that Dab2 has no effect on the expression of thrombin receptors and the outside-in signaling. Dab2(-/-) platelets stimulated by low concentrations of thrombin were normal in Gαq-mediated calcium mobilization and protein kinase C activation, but were defective in Gα₁₂/₁₃-mediated RhoA-ROCKII activation. The attenuated Gα₁₂/₁₃ signaling led to impaired ADP release, Akt-mammalian target of rapamycin and integrin αIIbβ3 activation, fibrinogen binding, and clot retraction. The defective responses of Dab2(-/-) platelets to low concentrations of thrombin stimulation may contribute to the impaired hemostasis and thrombosis of Dab2(-/-) mice.

Conclusions: This study sheds new insight in platelet biology and represents the first report demonstrating that Dab2 is a key regulator of hemostasis and thrombosis by functional interplay with Gα₁₂/₁₃-mediated thrombin signaling.
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http://dx.doi.org/10.1161/ATVBAHA.114.302602DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4199904PMC
November 2014

Cullin 5 destabilizes Cas to inhibit Src-dependent cell transformation.

J Cell Sci 2014 Feb 27;127(Pt 3):509-20. Epub 2013 Nov 27.

Division of Basic Sciences, Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue N, Seattle, WA 98109, USA.

Phosphorylation-dependent protein ubiquitylation and degradation provides an irreversible mechanism to terminate protein kinase signaling. Here, we report that mammary epithelial cells require cullin-5-RING-E3-ubiquitin-ligase complexes (Cul5-CRLs) to prevent transformation by a Src-Cas signaling pathway. Removal of Cul5 stimulates growth-factor-independent growth and migration, membrane dynamics and colony dysmorphogenesis, which are all dependent on the endogenous tyrosine kinase Src. Src is activated in Cul5-deficient cells, but Src activation alone is not sufficient to cause transformation. We found that Cul5 and Src together stimulate degradation of the Src substrate p130Cas (Crk-associated substrate). Phosphorylation stimulates Cas binding to the Cul5-CRL adaptor protein SOCS6 and consequent proteasome-dependent degradation. Cas is necessary for the transformation of Cul5-deficient cells. Either knockdown of SOCS6 or use of a degradation-resistant Cas mutant stimulates membrane ruffling, but not other aspects of transformation. Our results show that endogenous Cul5 suppresses epithelial cell transformation by several pathways, including inhibition of Src-Cas-induced ruffling through SOCS6.
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http://dx.doi.org/10.1242/jcs.127829DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4007763PMC
February 2014

Rbx2 regulates neuronal migration through different cullin 5-RING ligase adaptors.

Dev Cell 2013 Nov 7;27(4):399-411. Epub 2013 Nov 7.

Division of Basic Sciences, Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue North, Seattle, WA 98109, USA.

Morphogenesis requires the proper migration and positioning of different cell types in the embryo. Much more is known about how cells start and guide their migrations than about how they stop when they reach their destinations. Here we provide evidence that Rbx2, a subunit of the Cullin 5-RING E3 ubiquitin ligase (CRL5) complex, stops neocortical projection neurons at their target layers. Rbx2 mutation causes neocortical and cerebellar ectopias dependent on Dab1, a key signaling protein in the Reelin pathway. SOCS7, a CRL5 substrate adaptor protein, is also required for neocortical layering. SOCS7-CRL5 complexes stimulate the ubiquitylation and turnover of Dab1. SOCS7 is upregulated during projection neuron migration, and unscheduled SOCS7 expression stops migration prematurely. Cerebellar development requires Rbx2 but not SOCS7, pointing to the importance of other CRL5 adaptors. Our results suggest that CRL5 adaptor expression is spatiotemporally regulated to modulate Reelin signaling and ensure normal neuron positioning in the developing brain.
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http://dx.doi.org/10.1016/j.devcel.2013.09.022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3851519PMC
November 2013

EphA2 and Src regulate equatorial cell morphogenesis during lens development.

Development 2013 Oct 11;140(20):4237-45. Epub 2013 Sep 11.

School of Optometry and Vision Science Program, University of California Berkeley, Berkeley, CA 94720, USA.

High refractive index and transparency of the eye lens require uniformly shaped and precisely aligned lens fiber cells. During lens development, equatorial epithelial cells undergo cell-to-cell alignment to form meridional rows of hexagonal cells. The mechanism that controls this morphogenesis from randomly packed cuboidal epithelial cells to highly organized hexagonal fiber cells remains unknown. In Epha2(-/-) mouse lenses, equatorial epithelial cells fail to form precisely aligned meridional rows; moreover, the lens fulcrum, where the apical tips of elongating epithelial cells constrict to form an anchor point before fiber cell differentiation and elongation at the equator, is disrupted. Phosphorylated Src-Y424 and cortactin-Y466, actin and EphA2 cluster at the vertices of wild-type hexagonal epithelial cells in organized meridional rows. However, phosphorylated Src and phosphorylated cortactin are not detected in disorganized Epha2(-/-) cells with altered F-actin distribution. E-cadherin junctions, which are normally located at the basal-lateral ends of equatorial epithelial cells and are diminished in newly differentiating fiber cells, become widely distributed in the apical, lateral and basal sides of epithelial cells and persist in differentiating fiber cells in Epha2(-/-) lenses. Src(-/-) equatorial epithelial cells also fail to form precisely aligned meridional rows and lens fulcrum. These results indicate that EphA2/Src signaling is essential for the formation of the lens fulcrum. EphA2 also regulates Src/cortactin/F-actin complexes at the vertices of hexagonal equatorial cells for cell-to-cell alignment. This mechanistic information explains how EphA2 mutations lead to disorganized lens cells that subsequently contribute to altered refractive index and cataracts in humans and mice.
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http://dx.doi.org/10.1242/dev.100727DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3787762PMC
October 2013

Cell biology in neuroscience: mechanisms of cell migration in the nervous system.

J Cell Biol 2013 Sep;202(5):725-34

Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA.

Many neurons resemble other cells in developing embryos in migrating long distances before they differentiate. However, despite shared basic machinery, neurons differ from other migrating cells. Most dramatically, migrating neurons have a long and dynamic leading process, and may extend an axon from the rear while they migrate. Neurons must coordinate the extension and branching of their leading processes, cell movement with axon specification and extension, switching between actin and microtubule motors, and attachment and recycling of diverse adhesion proteins. New research is needed to fully understand how migration of such morphologically complicated cells is coordinated over space and time.
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http://dx.doi.org/10.1083/jcb.201305021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3760606PMC
September 2013

Spatial regulation of VEGF receptor endocytosis in angiogenesis.

Nat Cell Biol 2013 Mar 27;15(3):249-60. Epub 2013 Jan 27.

Max Planck Institute for Molecular Biomedicine, Department of Tissue Morphogenesis, and University of Muenster, Faculty of Medicine, D-48149 Muenster, Germany.

Activities as diverse as migration, proliferation and patterning occur simultaneously and in a coordinated fashion during tissue morphogenesis. In the growing vasculature, the formation of motile, invasive and filopodia-carrying endothelial sprouts is balanced with the stabilization of blood-transporting vessels. Here, we show that sprouting endothelial cells in the retina have high rates of VEGF uptake, VEGF receptor endocytosis and turnover. These internalization processes are opposed by atypical protein kinase C activity in more stable and mature vessels. aPKC phosphorylates Dab2, a clathrin-associated sorting protein that, together with the transmembrane protein ephrin-B2 and the cell polarity regulator PAR-3, enables VEGF receptor endocytosis and downstream signal transduction. Accordingly, VEGF receptor internalization and the angiogenic growth of vascular beds are defective in loss-of-function mice lacking key components of this regulatory pathway. Our work uncovers how vessel growth is dynamically controlled by local VEGF receptor endocytosis and the activity of cell polarity proteins.
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http://dx.doi.org/10.1038/ncb2679DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3901019PMC
March 2013

Regulation of dendritic branching by Cdc42 GAPs.

Genes Dev 2012 Aug;26(15):1653-8

Fred Hutchinson Cancer Research Center, Seattle, Washington 98109, USA.

Nerve cells form elaborate, highly branched dendritic trees that are optimized for the receipt of synaptic signals. Recent work published in this issue of Genes & Development by Rosario and colleagues (pp. 1743-1757) shows that a Cdc42-specific GTPase-activating protein (NOMA-GAP) regulates the branching of dendrites by neurons in the top layers of the mouse cortex. The results raise interesting questions regarding the specification of arbors in different cortical layers and the mechanisms of dendrite branching.
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http://dx.doi.org/10.1101/gad.199034.112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3418583PMC
August 2012

The clathrin adaptor Dab2 recruits EH domain scaffold proteins to regulate integrin β1 endocytosis.

Mol Biol Cell 2012 Aug 30;23(15):2905-16. Epub 2012 May 30.

Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA.

Endocytic adaptor proteins facilitate cargo recruitment and clathrin-coated pit nucleation. The prototypical clathrin adaptor AP2 mediates cargo recruitment, maturation, and scission of the pit by binding cargo, clathrin, and accessory proteins, including the Eps-homology (EH) domain proteins Eps15 and intersectin. However, clathrin-mediated endocytosis of some cargoes proceeds efficiently in AP2-depleted cells. We found that Dab2, another endocytic adaptor, also binds to Eps15 and intersectin. Depletion of EH domain proteins altered the number and size of clathrin structures and impaired the endocytosis of the Dab2- and AP2-dependent cargoes, integrin β1 and transferrin receptor, respectively. To test the importance of Dab2 binding to EH domain proteins for endocytosis, we mutated the EH domain-binding sites. This mutant localized to clathrin structures with integrin β1, AP2, and reduced amounts of Eps15. Of interest, although integrin β1 endocytosis was impaired, transferrin receptor internalization was unaffected. Surprisingly, whereas clathrin structures contain both Dab2 and AP2, integrin β1 and transferrin localize in separate pits. These data suggest that Dab2-mediated recruitment of EH domain proteins selectively drives the internalization of the Dab2 cargo, integrin β1. We propose that adaptors may need to be bound to their cargo to regulate EH domain proteins and internalize efficiently.
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http://dx.doi.org/10.1091/mbc.E11-12-1007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3408417PMC
August 2012

Identification of Stk25 as a genetic modifier of Tau phosphorylation in Dab1-mutant mice.

PLoS One 2012 15;7(2):e31152. Epub 2012 Feb 15.

Neuroscience and Physiology, State University of New York Upstate Medical University, Syracuse, New York, United States of America.

Hyperphosphorylation of the microtubule binding protein Tau is a feature of a number of neurodegenerative diseases, including Alzheimer's disease. Tau is hyperphosphorylated in the hippocampus of dab1-null mice in a strain-dependent manner; however, it has not been clear if the Tau phosphorylation phenotype is a secondary effect of the morbidity of these mutants. The dab1 gene encodes a docking protein that is required for normal brain lamination and dendritogenesis as part of the Reelin signaling pathway. We show that dab1 gene inactivation after brain development leads to Tau hyperphosphorylation in anatomically normal mice. Genomic regions that regulate the phospho Tau phenotype in dab1 mutants have previously been identified. Using a microarray gene expression comparison between dab1-mutants from the high-phospho Tau expressing and low-phospho Tau expressing strains, we identified Stk25 as a differentially expressed modifier of dab1-mutant phenotypes. Stk25 knockdown reduces Tau phosphorylation in embryonic neurons. Furthermore, Stk25 regulates neuronal polarization and Golgi morphology in an antagonistic manner to Dab1. This work provides insights into the complex regulation of neuronal behavior during brain development and provides insights into the molecular cascades that regulate Tau phosphorylation.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0031152PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3280280PMC
June 2012

FCH domain only-2 organizes clathrin-coated structures and interacts with Disabled-2 for low-density lipoprotein receptor endocytosis.

Mol Biol Cell 2012 Apr 9;23(7):1330-42. Epub 2012 Feb 9.

Molecular and Cellular Biology Program, University of Washington, Seattle, WA 98195, USA.

Clathrin-mediated endocytosis regulates the internalization of many nutrient and signaling receptors. Clathrin and endocytic accessory proteins are recruited to receptors by specific adaptors. The adaptor Disabled-2 (Dab2) recruits its cargoes, including the low-density lipoprotein receptor (LDLR), and mediates endocytosis, even when the major adaptor protein AP2 is depleted. We hypothesized that the accessory proteins normally recruited by AP2 may be recruited by Dab2 if AP2 is absent. We identified one such accessory protein, the F-BAR protein FCH domain only-2 (FCHO2), as a major Dab2-interacting protein. The μ-homology domain (μHD) of FCHO2 binds directly to DPF sequences in Dab2 that also bind AP2. Disrupting the Dab2-FCHO2 interaction inhibited Dab2-mediated LDLR endocytosis in AP2-depleted cells. Depleting FCHO2 reduced the number but increased the size of clathrin structures on the adherent surface of HeLa cells and inhibited LDLR and transferrin receptor clustering. However, LDLR was internalized efficiently by FCHO2-deficient cells when additional time was provided for LDLR to enter the enlarged structures before budding, suggesting that later steps of endocytosis are normal under these conditions. These results indicate FCHO2 regulates the size of clathrin structures, and its interaction with Dab2 is needed for LDLR endocytosis under conditions of low AP2.
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http://dx.doi.org/10.1091/mbc.E11-09-0812DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3315808PMC
April 2012

Reelin, Rap1 and N-cadherin orient the migration of multipolar neurons in the developing neocortex.

Nat Neurosci 2011 Jun 24;14(6):697-703. Epub 2011 Apr 24.

Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA.

Projection neurons migrate from the ventricular zone to the neocortical plate during the development of the mouse brain. Their overall movement is radial, but they become multipolar and move nonradially in the intermediate zone. Here we show that Reelin, the Rap1 GTPase and N-cadherin (NCad) are important for multipolar neurons to polarize their migration toward the cortical plate. Inhibition and rescue experiments indicated that Reelin regulates migration through Rap1 and Akt, and that the Rap1-regulated GTPases RalA, RalB, Rac1 and Cdc42 are also involved. We found that Rap1 regulated the plasma membrane localization of NCad and NCad rescued radial polarization when Rap1 was inhibited. However, inhibition of Rap1 or NCad had little effect on glia-dependent locomotion. We propose a multistep mechanism in which Reelin activates Rap1, Rap1 upregulates NCad, and NCad is needed to orient cell migration.
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http://dx.doi.org/10.1038/nn.2816DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3102785PMC
June 2011

Differential transformation capacity of Src family kinases during the initiation of prostate cancer.

Proc Natl Acad Sci U S A 2011 Apr 4;108(16):6579-84. Epub 2011 Apr 4.

Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, CA 90095, USA.

Src family kinases (SFKs) are pleiotropic activators that are responsible for integrating signal transduction for multiple receptors that regulate cellular proliferation, invasion, and metastasis in a variety of human cancers. Independent groups have identified increased expression of individual SFK members during prostate cancer progression, raising the question of whether SFKs display functional equivalence. Here, we show that Src kinase, followed by Fyn kinase and then Lyn kinase, exhibit ranked tumorigenic potential during both paracrine-induced and cell-autonomous-initiated prostate cancer. This quantitative variation in transformation potential appears to be regulated in part by posttranslational palmitoylation. Our data indicate that development of inhibitors against specific SFK members could provide unique targeted therapeutic strategies.
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http://dx.doi.org/10.1073/pnas.1103904108DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3080985PMC
April 2011

Reelin and stk25 have opposing roles in neuronal polarization and dendritic Golgi deployment.

Cell 2010 Nov;143(5):826-36

Department of Neuroscience and Physiology, SUNY Upstate Medical University, Syracuse, NY 13210, USA.

The Reelin ligand regulates a Dab1-dependent signaling pathway required for brain lamination and normal dendritogenesis, but the specific mechanisms underlying these actions remain unclear. We find that Stk25, a modifier of Reelin-Dab1 signaling, regulates Golgi morphology and neuronal polarization as part of an LKB1-Stk25-Golgi matrix protein 130 (GM130) signaling pathway. Overexpression of Stk25 induces Golgi condensation and multiple axons, both of which are rescued by Reelin treatment. Reelin stimulation of cultured neurons induces the extension of the Golgi into dendrites, which is suppressed by Stk25 overexpression. In vivo, Reelin and Dab1 are required for the normal extension of the Golgi apparatus into the apical dendrites of hippocampal and neocortical pyramidal neurons. This demonstrates that the balance between Reelin-Dab1 signaling and LKB1-Stk25-GM130 regulates Golgi dispersion, axon specification, and dendrite growth and provides insights into the importance of the Golgi apparatus for cell polarization.
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http://dx.doi.org/10.1016/j.cell.2010.10.029DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3033572PMC
November 2010

Epigenetic downregulation of human disabled homolog 2 switches TGF-beta from a tumor suppressor to a tumor promoter.

J Clin Invest 2010 Aug 1;120(8):2842-57. Epub 2010 Jul 1.

The Beatson Institute for Cancer Research, Garscube Estate, Glasgow, United Kingdom.

The cytokine TGF-beta acts as a tumor suppressor in normal epithelial cells and during the early stages of tumorigenesis. During malignant progression, cancer cells can switch their response to TGF-beta and use this cytokine as a potent oncogenic factor; however, the mechanistic basis for this is poorly understood. Here we demonstrate that downregulation of disabled homolog 2 (DAB2) gene expression via promoter methylation frequently occurs in human squamous cell carcinomas (SCCs) and acts as an independent predictor of metastasis and poor prognosis. Retrospective microarray analysis in an independent data set indicated that low levels of DAB2 and high levels of TGFB2 expression correlate with poor prognosis. Immunohistochemistry, reexpression, genetic knockout, and RNAi silencing studies demonstrated that downregulation of DAB2 expression modulated the TGF-beta/Smad pathway. Simultaneously, DAB2 downregulation abrogated TGF-beta tumor suppressor function, while enabling TGF-beta tumor-promoting activities. Downregulation of DAB2 blocked TGF-beta-mediated inhibition of cell proliferation and migration and enabled TGF-beta to promote cell motility, anchorage-independent growth, and tumor growth in vivo. Our data indicate that DAB2 acts as a tumor suppressor by dictating tumor cell TGF-beta responses, identify a biomarker for SCC progression, and suggest a means to stratify patients with advanced SCC who may benefit clinically from anti-TGF-beta therapies.
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http://dx.doi.org/10.1172/JCI36125DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2912175PMC
August 2010

Cullin 5 regulates cortical layering by modulating the speed and duration of Dab1-dependent neuronal migration.

J Neurosci 2010 Apr;30(16):5668-76

Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington 98109, USA.

The multilayered mammalian neocortex develops by the coordinated immigration and differentiation of cells that are produced at distant sites. Correct layering requires an extracellular protein, Reelin (Reln), an intracellular signaling molecule, Disabled-1 (Dab1), and an E3 ubiquitin ligase, Cullin-5 (Cul5). Reln activates Dab1, which is then degraded by Cul5. Here we test whether Cul5 regulates neuron layering by affecting Dab1 stability or other mechanisms. We find that a stabilized mutant Dab1, which resists Cul5-dependent degradation, causes a similar phenotype to Cul5 deficiency. Moreover, Cul5 has no effect when Dab1 is absent. The effects of Cul5 and Dab1 are cell autonomous, and Cul5 regulates movement of early as well as late cortical neurons. Removing Cul5 increases the speed at which neurons migrate through the cortical plate by reducing the time spent stationary and increasing the speed of individual steps. These results show that Cul5 regulates neuron layering by stimulating Dab1 degradation and that Cul5 controls migration speed and stopping point, and they demonstrate the importance of negative feedback in signaling during cortical development.
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http://dx.doi.org/10.1523/JNEUROSCI.0035-10.2010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2866641PMC
April 2010

Cutting edge: Dab2 is a FOXP3 target gene required for regulatory T cell function.

J Immunol 2009 Oct;183(7):4192-6

Department of Pathology, Graduate Program in Immunology and Virology, University of Massachusetts Medical School, Worcester, MA 01655, USA.

FOXP3-expressing regulatory T (Treg) cells are vital for maintaining peripheral T cell tolerance and homeostasis. The mechanisms by which FOXP3 target genes orchestrate context-dependent Treg cell function are largely unknown. In this study we show that in mouse peripheral lymphocytes the Drosophila Disabled-2 (Dab2) homolog, a gene that is involved in enhancing TGFbeta responses, is exclusively expressed in FOXP3+ regulatory T cells. Dab2 is a direct target of FOXP3, and regulatory T cells lacking DAB2 are functionally impaired in vitro and in vivo. However, not all aspects of Treg cell function are perturbed, and DAB2 appears to be dispensable for Treg cell function in maintaining naive T cell homeostasis.
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http://dx.doi.org/10.4049/jimmunol.0902041DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2852251PMC
October 2009

Quantitative proteomics identifies a Dab2/integrin module regulating cell migration.

J Cell Biol 2009 Jul 6;186(1):99-111. Epub 2009 Jul 6.

Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA.

Clathrin-associated endocytic adapters recruit cargoes to coated pits as a first step in endocytosis. We developed an unbiased quantitative proteomics approach to identify and quantify glycoprotein cargoes for an endocytic adapter, Dab2. Surface levels of integrins beta1, alpha1, alpha2, and alpha3 but not alpha5 or alphav chains were specifically increased on Dab2-deficient HeLa cells. Dab2 colocalizes with integrin beta1 in coated pits that are dispersed over the cell surface, suggesting that it regulates bulk endocytosis of inactive integrins. Depletion of Dab2 inhibits cell migration and polarized movement of integrin beta1 and vinculin to the leading edge. By manipulating intracellular and surface integrin beta1 levels, we show that migration speed correlates with the intracellular integrin pool but not the surface level. Together, these results suggest that Dab2 internalizes integrins freely diffusing on the cell surface and that Dab2 regulates migration, perhaps by maintaining an internal pool of integrins that can be recycled to create new adhesions at the leading edge.
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http://dx.doi.org/10.1083/jcb.200812160DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2712992PMC
July 2009

Restriction of Src activity by Cullin-5.

Curr Biol 2009 Jan 15;19(2):157-62. Epub 2009 Jan 15.

Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA.

Src is a nonreceptor tyrosine kinase that coordinates responses to diverse soluble and adhesive signaling molecules and regulates cell proliferation, survival, differentiation and migration. Normally, Src activity is tightly regulated, and Src-catalyzed phosphorylation is counterbalanced by phosphotyrosine phosphatases. However, deregulated mutant Src causes malignant transformation when highly expressed. Src transformation is dose dependent, but it has been unclear how much mutant Src, compared with endogenous Src, is required for transformation. Here, we show that transformation requires high-level overexpression of mutant src mRNA, in part because active Src protein is degraded by ubiquitin-mediated proteolysis. We show that active but not inactive Src protein is downregulated depending on the putative tumor suppressor and E3 ubiquitin ligase component, Cullin-5 (Cul5). Cul5 removal synergizes with physiological levels of mutant src mRNA to increase protein tyrosine phosphorylation, induce morphological transformation, and deregulate growth. Cul5 also represses Src-induced tumorigenesis and regulates Src signaling in normal cells. These results suggest that, when Src is activated by mutation or physiological mechanisms, its effects are limited by Cul5, which downregulates active Src and its phosphorylated substrates. These findings demonstrate the importance of a new mechanism that downregulates Src signaling in cells.
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http://dx.doi.org/10.1016/j.cub.2008.12.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2730496PMC
January 2009