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Inflammatory bowel disease (IBD) is a complex multi-factorial disease for which physiologically relevant in vitro models are lacking. Existing models are often a compromise between biological relevance and scalability. Here, we integrated intestinal epithelial cells (IEC) derived from human intestinal organoids with monocyte-derived macrophages, in a gut-on-a-chip platform to model the human intestine and key aspects of IBD. The microfluidic culture of IEC lead to an increased polarization and differentiation state that closely resembled the expression profile of human colon in vivo. Activation of the model resulted in the polarized secretion of CXCL10, IL-8 and CCL-20 by IEC and could efficiently be prevented by TPCA-1 exposure. Importantly, upregulated gene expression by the inflammatory trigger correlated with dysregulated pathways in IBD patients. Finally, integration of activated macrophages offers a first-step towards a multi-factorial amenable IBD platform that could be scaled up to assess compound efficacy at early stages of drug development or in personalized medicine.

Lowe syndrome and Dent II disease are X-linked monogenetic diseases characterised by a renal reabsorption defect in the proximal tubules and caused by mutations in the OCRL gene, which codes for an inositol-5-phosphatase. The life expectancy of patients suffering from Lowe syndrome is largely reduced because of the development of chronic kidney disease and related complications. There is a need for physiological human in vitro models for Lowe syndrome/Dent II disease to study the underpinning disease mechanisms and to identify and characterise potential drugs and drug targets. Here, we describe a proximal tubule organ on chip model combining a 3D tubule architecture with fluid flow shear stress that phenocopies hallmarks of Lowe syndrome/Dent II disease. We demonstrate the high suitability of our in vitro model for drug target validation. Furthermore, using this model, we demonstrate that proximal tubule cells lacking OCRL expression upregulate markers typical for epithelial–mesenchymal transition (EMT), including the transcription factor SNAI2/Slug, and show increased collagen expression and deposition, which potentially contributes to interstitial fibrosis and disease progression as observed in Lowe syndrome and Dent II disease.

In the spinal cord, the central canal forms through a poorly understood process termed dorsal collapse that involves attrition and remodelling of pseudostratified ventricular layer (VL) cells. Here, we use mouse and chick models to show that dorsal ventricular layer (dVL) cells adjacent to dorsal midline Nestin(+) radial glia (dmNes+RG) down-regulate apical polarity proteins, including Crumbs2 (CRB2) and delaminate in a stepwise manner; live imaging shows that as one cell delaminates, the next cell ratchets up, the dmNes+RG endfoot ratchets down, and the process repeats. We show that dmNes+RG secrete a factor that promotes loss of cell polarity and delamination. This activity is mimicked by a secreted variant of Crumbs2 (CRB2S) which is specifically expressed by dmNes+RG. In cultured MDCK cells, CRB2S associates with apical membranes and decreases cell cohesion. Analysis of Crb2F/F/Nestin-Cre+/− mice, and targeted reduction of Crb2/CRB2S in slice cultures reveal essential roles for transmembrane CRB2 (CRB2TM) and CRB2S on VL cells and dmNes+RG, respectively. We propose a model in which a CRB2S–CRB2TM interaction promotes the progressive attrition of the dVL without loss of overall VL integrity. This novel mechanism may operate more widely to promote orderly progenitor delamination., During prenatal development, the lumen of the vertebrate spinal cord is remodelled into the central canal when progenitor cells progressively delaminate. Imaging studies in the mouse provide novel insights into how roof plate–derived dorsal midline glia secrete a factor that regulates local delamination and ratcheting. Gain- and loss-of-function approaches show that the apical polarity protein, Crumbs2, mediates these events, and highlight the role of a secreted variant of Crumbs2.

Protein tyrosine phosphatase PTPN13, also known as PTP-BL in mice, represents a large multi-domain non-transmembrane scaffolding protein that contains five consecutive PDZ domains. Here, we report the solution structures of the extended murine PTPN13 PDZ3 domain in its apo form and in complex with its physiological ligand, the carboxy-terminus of protein kinase C-related kinase-2 (PRK2), determined by multidimensional NMR spectroscopy. Both in its ligand-free state and when complexed to PRK2, PDZ3 of PTPN13 adopts the classical compact, globular D/E fold. PDZ3 of PTPN13 binds five carboxy-terminal amino acids of PRK2 via a groove located between the EB-strand and the DB-helix. The PRK2 peptide resides in the canonical PDZ3 binding cleft in an elongated manner and the amino acid side chains in position P0 and P-2, cysteine and aspartate, of the ligand face the groove between EB-strand and DB-helix, whereas the PRK2 side chains of tryptophan and alanine located in position P-1 and P-3 point away from the binding cleft. These structures are rare examples of selective class III ligand recognition by a PDZ domain and now provide a basis for the detailed structural investigation of the promiscuous interaction between the PDZ domains of PTPN13 and their ligands. They will also lead to a better understanding of the proposed scaffolding function of these domains in multi-protein complexes assembled by PTPN13 and could ultimately contribute to low molecular weight antagonists that might even act on the PRK2 signaling pathway to modulate rearrangements of the actin cytoskeleton.

Intestinal organoids have emerged as the new paradigm for modelling the healthy and diseased intestine with patient-relevant properties. In this study, we show directed differentiation of induced pluripotent stem cells towards intestinal-like phenotype within a microfluidic device. iPSCs are cultured against a gel in microfluidic chips of the OrganoPlate, in which they undergo stepwise differentiation. Cells form a tubular structure, lose their stem cell markers and start expressing mature intestinal markers, including markers for Paneth cells, enterocytes and neuroendocrine cells. Tubes develop barrier properties as confirmed by transepithelial electrical resistance (TEER). Lastly, we show that tubules respond to pro-inflammatory cytokine triggers. The whole procedure for differentiation lasts 14 days, making it an efficient process to make patient-specific organoid tubules. We anticipate the usage of the platform for disease modelling and drug candidate screening.

Fas plays a major role in regulating ligand-induced apoptosis in many cell types. It is well known that several cancers demonstrate reduced cell surface levels of Fas and thus escape a potential control system via ligand-induced apoptosis, although underlying mechanisms are unclear. Here we report that the endosome associated trafficking regulator 1 (ENTR1), controls cell surface levels of Fas and Fas-mediated apoptotic signalling. ENTR1 regulates, via binding to the coiled coil domain protein Dysbindin, the delivery of Fas from endosomes to lysosomes thereby controlling termination of Fas signal transduction. We demonstrate that ENTR1 is cleaved during Fas-induced apoptosis in a caspase-dependent manner revealing an unexpected interplay of apoptotic signalling and regulation of endolysosomal trafficking resulting in a positive feedback signalling-loop. Our data provide insights into the molecular mechanism of Fas post-endocytic trafficking and signalling, opening possible explanations on how cancer cells regulate cell surface levels of death receptors., Fas is a death receptor that regulates apoptosis in many cell types and is downregulated on the cell surface in many cancers. Here, Sharma et al. show that endosome associated trafficking regulator ENTR1 regulates delivery of Fas to lysosomes, thereby controlling its degradation and signalling.

A primary cilium is present on most eukaryotic cells and represents a specialized organelle dedicated to signal transduction and mechanosensing. Defects in cilia function are the cause for several human diseases called ciliopathies. The serologically defined colon cancer antigen-3 (SDCCAG3) is a recently described novel endosomal protein mainly localized at early and recycling endosomes and interacting with several components of membrane trafficking pathways. Here we describe localization of SDCCAG3 to the basal body of primary cilia. Furthermore, we demonstrate that decreased expression levels of SDCCAG3 correlate with decreased ciliary length and a reduced percentage of ciliated cells. We show that SDCCAG3 interacts with the intraflagellar transport protein 88 (IFT88), a crucial component of ciliogenesis and intraciliary transport. Mapping experiments revealed that the N-terminus of SDCCAG3 mediates this interaction by binding to a region within IFT88 comprising several tetratricopeptide (TRP) repeats. Finally, we demonstrate that SDCCAG3 is important for ciliary localization of the membrane protein Polycystin-2, a protein playing an important role in the formation of polycystic kidney disease, but not for Rab8 another ciliary protein. Together these data suggest a novel role for SDCCAG3 in ciliogenesis and in localization of cargo to primary cilia.

The intracellular nucleotide-binding oligomerization domain-2 (NOD2) receptor detects bacteria-derived muramyl dipeptide (MDP) and activates the transcription factor NF-κB. Here we describe the regulatome of NOD2 signaling using a systematic RNAi screen. Using three consecutive screens, we identified a set of 20 positive NF-κB regulators including the known pathway members RIPK2, RELA, and BIRC4 (XIAP) as well as FRMPD2 (FERM and PDZ domain-containing 2). FRMPD2 interacts with NOD2 via leucine-rich repeats and forms a complex with the membrane-associated protein ERBB2IP. We demonstrate that FRMPD2 spatially assembles the NOD2-signaling complex, hereby restricting NOD2-mediated immune responses to the basolateral compartment of polarized intestinal epithelial cells. We show that genetic truncation of the NOD2 leucine-rich repeat domain, which is associated with Crohn disease, impairs the interaction with FRMPD2, and that intestinal inflammation leads to down-regulation of FRMPD2 . These results suggest a structural mechanism for how polarity of epithelial cells acts on intestinal NOD-like receptor signaling to mediate spatial specificity of bacterial recognition and control of immune responses.

ATOH8 is a bHLH transcription factor playing roles in a variety of developmental processes such as neurogenesis, differentiation of pancreatic precursor cells, development of kidney and muscle, and differentiation of endothelial cells. PPP3CB belongs to the catalytic subunit of the serine/threonine phosphatase, calcineurin, which can dephosphorylate its substrate proteins to regulate their physiological activities. In our study, we demonstrated that ATOH8 interacts with PPP3CB in vitro with different approaches. We show that the conserved catalytic domain of PPP3CB interacts with both the N-terminus and the bHLH domain of ATOH8. Although the interaction domain of PPP3CB is conserved among all isoforms of calcineurin A, ATOH8 selectively interacts with PPP3CB instead of PPP3CA, probably due to the unique proline-rich region present in the N-terminus of PPP3CB, which controls the specificity of its interaction partners. Furthermore, we show that inhibition of the interaction with calcineurin inhibitor, cyclosporin A (CsA), leads to the retention of ATOH8 to the cytoplasm, suggesting that the interaction renders nuclear localization of ATOH8 which may be critical to control its activity as transcription factor. Electronic supplementary material The online version of this article (doi:10.1007/s00418-015-1368-5) contains supplementary material, which is available to authorized users.

The oculocerebrorenal syndrome of Lowe (OCRL), also called Lowe syndrome, is characterized by defects of the nervous system, the eye and the kidney. Lowe syndrome is a monogenetic X-linked disease caused by mutations of the inositol-5-phosphatase OCRL1. OCRL1 is a membrane-bound protein recruited to membranes via interaction with a variety of Rab proteins. The structural and kinetic basis of OCRL1 for the recognition of several Rab proteins is unknown. In this study, we report the crystal structure of the Rab-binding domain (RBD) of OCRL1 in complex with Rab8a and the kinetic binding analysis of OCRL1 with several Rab GTPases (Rab1b, Rab5a, Rab6a and Rab8a). In contrast to other effectors that bind their respective Rab predominantly via α-helical structure elements, the Rab-binding interface of OCRL1 consists mainly of the IgG-like β-strand structure of the ASPM-SPD-2-Hydin domain as well as one α-helix. Our results give a deeper structural understanding of disease-causing mutations of OCRL1 affecting Rab binding.

Protein tyrosine phosphatase basophil-like (PTP-BL), also known as PTPN13, represents a large multi domain non-transmembrane scaffolding protein that contains five PDZ domains. Here we report the complete resonance assignments of the extended PDZ3 domain of PTP-BL. These assignments provide a basis for the detailed structural investigation of the interaction between the PDZ domains of PTP-BL as well as of their interaction with ligands. It will also lead to a better understanding of the proposed scaffolding function of these domains in multi-protein complexes assembled by PTB-BL.

Multi-PDZ (PSD-95/Discs large/Zonula-occludens-1) domain proteins play a crucial role in the establishment and maintenance of cell polarization. The novel multi-PDZ domain protein FRMPD2 is a potential scaffolding protein consisting of an N-terminal KIND domain, a FERM domain and three PDZ domains. Here we show that FRMPD2 is localized in a polarized fashion in epithelial cells at the basolateral membrane and partially colocalizes with the tight-junction marker protein Zonula-occludens-1. Downregulation of FRMPD2 protein in Caco-2 cells is associated with an impairment of tight junction formation. We find that the FERM domain of FRMPD2 binds phosphatidylinositols and is sufficient for membrane localization. Moreover, we demonstrate that recruitment of FRMPD2 to cell-cell junctions is strictly E-cadherin-dependent, which is in line with our identification of catenin family proteins as binding partners for FRMPD2. We demonstrate that the FERM domain and binding of the PDZ2 domain to the armadillo protein p0071 are required for basolateral restriction of FRMPD2. Moreover, the PDZ2 domain of FRMPD2 is sufficient to partially redirect an apically localized protein to the basolateral membrane. Our results provide novel insights into the molecular function of FRMPD2 and into the targeting mechanism of peripheral membrane proteins in polarized epithelial cells.

In pancreatic beta-cells, increased expression of the MODY5 gene product, HNF1 beta, leads to enhanced rates of apoptosis and altered regulation of the cell cycle, suggesting that control of HNF1 beta expression may be important for the control of beta-cell proliferation and viability. It is unclear how these effects of HNF1 beta are mediated, but previously we have identified a protein tyrosine phosphatase, (PTP)-BL, as an HNF1 beta-regulated protein in beta-cells and have now studied the role of this protein in INS-1 beta-cells. Stably transfected cells were generated, which express either wild-type (WT) or a phosphatase-deficient mutant (PTP-BL-CS) of PTP-BL conditionally under the control of a tetracycline-regulated promoter. Enhanced expression of WT PTP-BL inhibited INS-1 cell growth dose dependently, but this effect was not observed when PTP-BL-CS was expressed. Neither construct altered the rate of apoptosis. PTP-BL has been reported to interact with components of the Wnt signalling pathway, and we observed that addition of exogenous Wnt3a resulted in an increase in cell proliferation and a rise in beta-catenin levels, consistent with the operation of this pathway in INS-1 cells. Up-regulation of WT PTP-BL antagonised these responses but PTP-BL-CS failed to inhibit Wnt3a-induced proliferation. The rise in beta-catenin caused by Wnt3a was also suppressed by over-expression of HNF1 beta, suggesting that HNF1 beta may interact with the Wnt signalling pathway via an increase in PTP-BL levels. We conclude that PTP-BL plays an important role in the regulation of cell cycle progression in pancreatic beta-cells, and that it interacts functionally with components of the Wnt signalling pathway.

Mutations of the inositol-5-phosphatase OCRL cause Lowe syndrome and Dent-II disease. Both are rare genetic disorders characterized by renal defects. Lowe syndrome is furthermore characterized by defects of the eye (congenital cataracts) and nervous system (mental disabilities, hypotonia). OCRL has been localised to various endocytic compartments suggesting impairments in the endocytic pathway as possible disease mechanism. Recent evidence strongly supports this view and shows essential roles of OCRL at clathrin coated pits, transport of cargo from endosomes to the trans-Golgi network as well as recycling of receptors from endosomes to the plasma membrane. In particularin vitroandin vivoevidence demonstrates an important role of OCRL in recycling of megalin, a multi-ligand receptor crucial for reabsorption of nutrients in the proximal tubulus, a process severely impaired in Lowe syndrome patients. Thus defects in the endocytic pathway are likely to significantly contribute to the kidney phenotype in Lowe syndrome and Dent-II disease.

The protein tyrosine phosphatase PTP-Basophil (PTP-Bas) and its mouse homologue, PTP-Basophil-like (PTP-BL), are high molecular mass protein phosphatases consisting of a number of diverse protein-protein interaction modules. Several splicing variants of these phosphatases are known to exist thus demonstrating the complexity of these molecules. PTP-Bas/BL serves as a central scaffolding protein facilitating the assembly of a multiplicity of different proteins mainly via five different PDZ domains. Many of these interacting proteins are implicated in the regulation of the actin cytoskeleton. However, some proteins demonstrate a nuclear function of this protein tyrosine phosphatase. PTP-Bas is involved in the regulation of cell surface expression of the cell death receptor, Fas. Moreover, it is a negative regulator of ephrinB phosphorylation, a receptor playing an important role during development. The phosphorylation status of other proteins such as RIL, IkappaBalpha and beta-catenin can also be regulated by this phosphatase. Finally, PTP-BL has been shown to be involved in the regulation of cytokinesis, the last step in cell division. Although the precise molecular function of PTP-Bas/BL is still elusive, current data suggest clearly that PTP-Bas/BL belongs to the family of PDZ domain containing proteins involved in the regulation of the cytoskeleton and of intracellular vesicular transport processes.

PTP-BL is a highly modular protein tyrosine phosphatase of unknown function. It consists of an N-terminal FERM domain, five PDZ domains, and a C-terminally located tyrosine phosphatase domain. Here we show that PTP-BL is involved in the regulation of cytokinesis. We demonstrate localization of endogenous PTP-BL at the centrosomes during inter- and metaphase and at the spindle midzone during anaphase. Finally PTP-BL is concentrated at the midbody in cytokinesis. We show that PTP-BL is targeted to the midbody and centrosome by a specific splicing variant of the N-terminus characterized by an insertion of 182 amino acids. Moreover, we demonstrate that the FERM domain of PTP-BL is associated with the contractile ring and can be cosedimented with filamentous actin, whereas the N-terminus can be cosedimented with microtubules. We demonstrate that elevating the expression level of wild-type PTP-BL or expression of PTP-BL with an inactive tyrosine phosphatase domain leads to defects in cytokinesis and to the generation of multinucleate cells. We suggest that PTP-BL plays a role in the regulation of cytokinesis.

Ephrins are cell surface-associated ligands for Eph receptors and are important regulators of morphogenic processes such as axon guidance and angiogenesis. Transmembrane ephrinB ligands act as "receptor-like" signaling molecules, in part mediated by tyrosine phosphorylation and by engagement with PDZ domain proteins. However, the underlying cell biology and signaling mechanisms are poorly understood. Here we show that Src family kinases (SFKs) are positive regulators of ephrinB phosphorylation and phosphotyrosine-mediated reverse signaling. EphB receptor engagement of ephrinB causes rapid recruitment of SFKs to ephrinB expression domains and transient SFK activation. With delayed kinetics, ephrinB ligands recruit the cytoplasmic PDZ domain containing protein tyrosine phosphatase PTP-BL and are dephosphorylated. Our data suggest the presence of a switch mechanism that allows a shift from phosphotyrosine/SFK-dependent signaling to PDZ-dependent signaling.

Semaphorins are a family of secreted and membraneassociated proteins involved in growth cone guidance during development. Here, we describe the interaction of Semaphorin4F (Sema4F) with the post-synaptic density protein SAP90/ PSD-95. Using the yeast two-hybrid system and coprecipitation assays we were able to show an interaction between the extreme C-terminus of Sema4F and the PDZ domains of SAP90/PSD-95. Heterologous coexpression of a chimeric EphrinB1/Semaphorin4F protein with SAP90/PSD-95 in COS cells leads to translocation of SAP90/PSD-95 from the cytosol to the membrane. Deletion analysis shows that this translocation activity of Sema4F is completely dependent on the presence of the last three C-terminal amino acids. In addition, Sema4F immunoreactivity is present in synaptosome fractions and enriched in post-synaptic density fractions. Consistently, in cultured hippocampal neurons, we demonstrate punctate colocalization of Sema4F and SAP90/PSD-95 in dendrites, furthermore we found colocalization of Sema4F with synapsin1 suggesting a synaptic localization. Our data implicate a new functional context for semaphorins at glutamatergic synapses.

Protein tyrosine phosphatase-basophil like (PTP-BL) is a large non-transmembrane protein tyrosine phosphatase implicated in the modulation of the cytoskeleton. Here we describe a novel interaction of PTP-BL with the protein kinase C-related kinase 2 (PRK2), a serine/threonine kinase regulated by the G-protein rho. This interaction is mediated by the PSD-95, Drosophila discs large, zonula occludens (PDZ)3 domain of PTP-BL and the extreme C-terminus of PRK2 as shown by yeast two-hybrid assays and coimmunoprecipitation experiments from transfected HeLa cells. In particular, we demonstrate that a conserved C-terminal cysteine of PRK2 is indispensable for the interaction with PTP-BL. In HeLa cells we demonstrate colocalization of both proteins in lamellipodia like structures. Interaction of PTP-BL with the rho effector kinase PRK2 gives further evidence for a possible function of PTP-BL in the regulation of the actin cytoskeleton.

Pex14p is a central component of the peroxisomal protein import machinery, which has been suggested to provide the point of convergence for PTS1- and PTS2-dependent protein import in yeast cells. Here we describe the identification of a human peroxisome-associated protein (HsPex14p) which shows significant similarity to the yeast Pex14p. HsPex14p is a carbonate-resistant peroxisomal membrane protein with its C terminus exposed to the cytosol. The N terminus of the protein is not accessible to exogenously added antibodies or protease and thus might protrude into the peroxisomal lumen. HsPex14p overexpression leads to the decoration of tubular structures and mislocalization of peroxisomal catalase to the cytosol. HsPex14p binds the cytosolic receptor for the peroxisomal targeting signal 1 (PTS1), a result consistent with a function as a membrane receptor in peroxisomal protein import. Homo-oligomerization of HsPex14p or interaction of the protein with the PTS2-receptor or HsPex13p was not observed. This distinguishes the human Pex14p from its counterpart in yeast cells and thus supports recent data suggesting that not all aspects of peroxisomal protein import are conserved between yeasts and humans. The role of HsPex14p in mammalian peroxisome biogenesis makes HsPEX14 a candidate PBD gene for being responsible for an unrecognized complementation group of human peroxisome biogenesis disorders.

Protein tyrosine phosphatase-basophil like (PTP-BL) represents a large multi domain non-transmembrane scaffolding protein that contains five PDZ domains. Here we report the backbone assignments of the PDZ2/PDZ3 tandem domain of PTP-BL. These assignments now provide a basis for the detailed structural investigation of the interaction between the PDZ domains 2 and 3 of PTP-BL. It will lead to a better understanding of the proposed scaffolding function of this tandem domain in multi-protein complexes assembled by PTB-BL.

Cytokinesis is the final step of cell division. Increasing evidence suggests failure of cytokinesis might contribute to the development of cancer. Here, we demonstrate that the serologically defined colon cancer antigen-3 (SDCCAG3) forms a complex with PTPN13, a protein tyrosine phosphatase known to be involved in the regulation of cytokinesis, carcinogenesis and tumor aggressiveness. We show that SDCCAG3 is a novel endosomal protein, primarily localized at the early/recycling endosomal compartment. SDCCAG3 undergoes dynamic localization during cell division with strong accumulation at the midbody during cytokinesis. Overexpression as well as downregulation correlates with the generation of multinucleate cells. Furthermore, we show interaction of SDCCAG3 with the Arf GTPase activating protein GIT1 (G protein-coupled receptor kinase interactor-1). Overexpression of an ArfGAP-negative version of GIT1 also results in an increased number of multinucleate cells suggesting regulation of Arf-mediated vesicular trafficking or signaling via SDCCAG3. Finally, we demonstrate that SDCCAG3 expression levels are elevated in colon cancers. In summary, we have established SDCCAG3 as a novel endosomal protein, which is involved in the regulation of cytokinesis.

OCRL, whose mutations are responsible for Lowe syndrome and Dent disease, and INPP5B are two similar proteins comprising a central inositol 5-phosphatase domain followed by an ASH and a RhoGAP-like domain. Their divergent NH2-terminal portions remain uncharacterized. We show that the NH2-terminal region of OCRL, but not of INPP5B, binds clathrin heavy chain. OCRL, which in contrast to INPP5B visits late stage endocytic clathrin-coated pits, was earlier shown to contain another binding site for clathrin in its COOH-terminal region. NMR structure determination further reveals that despite their primary sequence dissimilarity, the NH2-terminal portions of both OCRL and INPP5B contain a PH domain. The novel clathrin-binding site in OCRL maps to an unusual clathrin-box motif located in a loop of the PH domain, whose mutations reduce recruitment efficiency of OCRL to coated pits. These findings suggest an evolutionary pressure for a specialized function of OCRL in bridging phosphoinositide metabolism to clathrin-dependent membrane trafficking.

SummaryCell membranes undergo continuous curvature changes as a result of membrane trafficking and cell motility. Deformations are achieved both by forces extrinsic to the membrane as well as by structural modifications in the bilayer or at the bilayer surface that favor the acquisition of curvature. We report here that a family of proteins previously implicated in the regulation of the actin cytoskeleton also have powerful lipid bilayer-deforming properties via an N-terminal module (F-BAR) similar to the BAR domain. Several such proteins, like a subset of BAR domain proteins, bind to dynamin, a GTPase implicated in endocytosis and actin dynamics, via SH3 domains. The ability of BAR and F-BAR domain proteins to induce tubular invaginations of the plasma membrane is enhanced by disruption of the actin cytoskeleton and is antagonized by dynamin. These results suggest a close interplay between the mechanisms that control actin dynamics and those that mediate plasma membrane invagination and fission.

The Trk family of receptor tyrosine kinases and the p75 receptor (p75NTR) mediate the effects of neurotrophins on neuronal survival, differentiation and synaptic plasticity. The neurotrophin BDNF and its cognate receptor tyrosine kinase, TrkB.FL, are highly expressed in neurons of the central nervous system. At later stages in postnatal development the truncated TrkB splice variants (TrkB.T1, TrkB.T2) become abundant. However, the signalling and function of these truncated receptors remained largely elusive.We show that overexpression of TrkB.T1 in hippocampal neurons induces the formation of dendritic filopodia, which are known precursors of synaptic spines. The induction of filopodia by TrkB.T1 occurs independently of neurotrophin binding and of kinase activity of endogenous TrkB.FL. Coexpression of a p75NTR lacking an intracellular domain inhibits the TrkB.T1-induced effect in a dominant negative manner. Steric hindrance of extracellular p75NTR interactions with a specific antibody, or absence of p75NTR with an intact extracellular domain also inhibit this TrkB.T1-induced effect.We thus propose a novel signalling pathway initiated by neurotrophin-independent extracellular or intramembrane interaction of TrkB.T1 with the p75NTR receptor, which modulates dendritic growth via p75NTR signalling cascades.

Two versions of the PDZ2 domain of the protein tyrosine phosphatase PTP-Bas/human PTP-BL are generated by alternative splicing. The domains differ by the insertion of five amino acid residues and their affinity to the tumour suppressor protein APC. Whereas PDZ2a is able to bind APC in the nanomolar range, PDZ2b shows no apparent interaction with APC. Here the solution structure of the splicing variant of PDZ2 with the insertion has been determined using 2D and 3D heteronuclear NMR experiments. The structural reason for the changed binding specificity is the reorientation of the loop with extra five amino acid residues, which folds back onto beta-strands two and three. In addition the side-chain of Lys32 closes the binding site of the APC binding protein and the two helices, especially alpha-helix 2, change their relative position to the protein core. Consecutively, the binding site is sterically no longer fully accessible. From the NMR-titration studies with a C-terminal APC-peptide the affinity of the peptide with the protein can be estimated as 540(+/-40)microM. The binding site encompasses part of the analogous binding site of PDZ2a as already described previously, yet specific interaction sites are abolished by the insertion of amino acids in PDZ2b. As shown by high-affinity chromatography, GST-PDZ2b and GST-PDZ2a bind to phosphatidylinositol 4,5-bisphosphate (PIP(2)) micelles with a dissociation constant K(D) of 21 microM and 55 microM, respectively. In line with these data PDZ2b binds isolated, dissolved PIP(2) and PIP(3) (phosphatidylinositol 3,4,5-trisphosphate) molecules specifically with a lower K(D) of 230(+/-20)microM as detected by NMR spectroscopy. The binding site could be located by our studies and involves the residues Ile24, Val26, Val70, Asn71, Gly77, Ala78, Glu85, Arg88, Gly91 and Gln92. PIP(2) and PIP(3) binding takes place in the groove of the PDZ domain that is normally part of the APC binding site.

Ephrins are cell surface-associated ligands for Eph receptors and are important regulators of morphogenic processes such as axon guidance and angiogenesis. Transmembrane ephrinB ligands act as "receptor-like" signaling molecules, in part mediated by tyrosine phosphorylation and by engagement with PDZ domain proteins. However, the underlying cell biology and signaling mechanisms are poorly understood. Here we show that Src family kinases (SFKs) are positive regulators of ephrinB phosphorylation and phosphotyrosine-mediated reverse signaling. EphB receptor engagement of ephrinB causes rapid recruitment of SFKs to ephrinB expression domains and transient SFK activation. With delayed kinetics, ephrinB ligands recruit the cytoplasmic PDZ domain containing protein tyrosine phosphatase PTP-BL and are dephosphorylated. Our data suggest the presence of a switch mechanism that allows a shift from phosphotyrosine/SFK-dependent signaling to PDZ-dependent signaling.

Brain-derived neurotrophic factor (BDNF) acutely modulates the efficacy of central glutamatergic synapses via activation of the receptor tyrosine kinase TrkB. On a longer time scale, recent evidence suggests an additional role of TrkB signaling in the formation of excitatory synaptic connections. Here, we have overexpressed full-length TrkB receptors (fl-TrkB) in hippocampal neurons, to investigate the contribution of BDNF signaling to the maturation of glutamatergic synapses. Using patch clamp recordings, we show a three-fold reduction in glutamatergic excitatory autaptic and synaptic current amplitudes in neurons overexpressing fl-TrkB, and application of saturating concentrations of BDNF and NT-4/5 completely reverses this effect. Compatible with these overexpression data, in untransfected neurons, scavenging of endogenous BDNF and NT-4/5 by TrkB-IgGs reduces excitatory autaptic current (EAC) amplitudes. By overexpression of truncated TrkB receptors (TrkB.T1, TrkB.T2) and a chimeric receptor containing only the intracellular domain of fl-TrkB, we show that intra- and extracellular domains of fl-TrkB are necessary to observe the EAC reduction. Labeling of presynaptic terminals with FM 4-64 revealed, that the reduced EAC amplitudes in fl-TrkB overexpressing neurons are accompanied by a two-fold reduction in synapse number. These results suggest, that ligand-independent signaling through fl-TrkB receptors can decrease glutamatergic synaptic strength, if sufficient amounts of BDNF or NT-4/5 are not available.

Mutations of the tumor suppressor protein APC (Adenomatous Polyposis Coli) are linked to familiar and sporadic human colon cancer. Here we describe a novel interaction between the APC protein and the protein tyrosine phosphatase PTP-BL carrying five PDZ protein-protein interaction domains. Exclusively, the second PDZ domain (PDZ2) of PTP-BL is binding to the extreme C-terminus of the APC protein, as determined by yeast two-hybrid studies. Using surface plasmon resonance analysis we established a dissociation constant (K(D)) of 8.1 x 10(-9) M. We find that a naturally occurring splice insertion of five amino acids (PDZ2b) abolishes its binding affinity to the APC protein. The in vivo interaction between PTP-BL and the APC protein was shown by coprecipitation experiments in transfected COS cells. Furthermore, in cultured epithelial Madine Carnine Kidney cells the subcellular colocalization was demonstrated for the nucleus and also for the tips of cellular extensions. The interaction of the APC protein with a protein tyrosine phosphatase may indirectly modulate the steady state levels of tyrosine phosphorylations of associated proteins, such as beta-catenin playing a major role in the regulation of cell division, migration and cell adhesion.

The regulation of the density of innervation and the promotion of survival of neurons are the original effects depending on neurotrophins. Here we analyse such effects evoked by trkB tyrosine kinase in transfected PC12 cells and transfected sympathetic neurons. In order to exclude the previously described modulation of trk kinase activity by the extracellular activation of the low-affinity p75 neurotrophin receptor, we applied a chimeric receptor approach: The extracellular domain of colony-stimulating factor-1 (CSF-1) receptor was fused to the transmembrane and cytoplasmic domain of the trkB tyrosine kinase receptor, allowing its selective activation by the heterologous ligand. Protein expression and CSF-1-induced tyrosine phosphorylation of the chimeric receptor protein was demonstrated in transfected COS cells. After stable transfection into nerve growth factor (NGF)-responsive PC12 cells, CSF-1 mediated the K252a-sensitive induction of fiber outgrowth. Furthermore, we were able to show by heterologous expression of the chimeric receptor, that activation of trkB tyrosine kinase activity is sufficient to promote survival of neurotrophin deprived sympathetic neurons.

A cDNA clone encoding a novel truncated form of the chicken TrkB receptor has been isolated and sequenced. Compared to two previously reported forms from mouse and rat, this clone contains an 141-bp insert (47 amino acids) in the cytoplasmic region.

Summary Mutations in the inositol 5-phosphatase OCRL are responsible for Lowe syndrome, whose manifestations include mental retardation and renal Fanconi syndrome. OCRL has been implicated in membrane trafficking, but disease mechanisms remain unclear. We show that OCRL visits late-stage, endocytic clathrin-coated pits and binds the Rab5 effector APPL1 on peripheral early endosomes. The interaction with APPL1, which is mediated by the ASH-RhoGAP-like domains of OCRL and is abolished by disease mutations, provides a link to protein networks implicated in the reabsorptive function of the kidney and in the trafficking and signaling of growth factor receptors in the brain. Crystallographic studies reveal a role of the ASH-RhoGAP-like domains in positioning the phosphatase domain at the membrane interface and a clathrin box protruding from the RhoGAP-like domain. Our results support a role of OCRL in the early endocytic pathway, consistent with the predominant localization of its preferred substrates, PI(4,5)P 2 and PI(3,4,5)P 3 , at the cell surface.

A common bottleneck in any drug development process is finding sufficiently accurate models that capture key aspects of disease development and progression. Conventional drug screening models often rely on simple 2D culture systems that fail to recapitulate the complexity of the organ situation. In this study, we show the application of a robust high throughput 3D gut-on-a-chip model for investigating hallmarks of inflammatory bowel disease (IBD). Using the OrganoPlate platform, we subjected enterocyte-like cells to an immune-relevant inflammatory trigger in order to recapitulate key events of IBD and to further investigate the suitability of this model for compound discovery and target validation activities. The induction of inflammatory conditions caused a loss of barrier function of the intestinal epithelium and its activation by increased cytokine production, two events observed in IBD physiopathology. More importantly, anti-inflammatory compound exposure prevented the loss of barrier function and the increased cytokine release. Furthermore, knockdown of key inflammatory regulators RELA and MYD88 through on-chip adenoviral shRNA transduction alleviated IBD phenotype by decreasing cytokine production. In summary, we demonstrate the routine use of a gut-on-a-chip platform for disease-specific aspects modeling. The approach can be used for larger scale disease modeling, target validation and drug discovery purposes.