A 3D Renal Proximal Tubule on Chip Model Phenocopies Lowe Syndrome and Dent II Disease Tubulopathy.

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.

Development of a human primary gut-on-a-chip to model inflammatory processes.

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.

Direct On-Chip Differentiation of Intestinal Tubules from Induced Pluripotent Stem Cells.

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.

Crumbs2 mediates ventricular layer remodelling to form the spinal cord central canal.

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.

Development of a Gut-On-A-Chip Model for High Throughput Disease Modeling and Drug Discovery.

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.

The binding affinity of PTPN13's tandem PDZ2/3 domain is allosterically modulated.

BACKGROUND: Protein tyrosine phosphatase PTPN13, also known as PTP-BL in mice, is a large multi-domain non-transmembrane scaffolding protein with a molecular mass of 270 kDa. It is involved in the regulation of several cellular processes such as cytokinesis and actin-cytoskeletal rearrangement. The modular structure of PTPN13 consists of an N-terminal KIND domain, a FERM domain, and five PDZ domains, followed by a C-terminal protein tyrosine phosphatase domain. PDZ domains are among the most abundant protein modules and they play a crucial role in signal transduction of protein networks. RESULTS: Here, we have analysed the binding characteristics of the isolated PDZ domains 2 and 3 from PTPN13 and compared them to the tandem domain PDZ2/3, which interacts with 12 C-terminal residues of the tumour suppressor protein of APC, using heteronuclear multidimensional NMR spectroscopy. Furthermore, we could show for the first time that PRK2 is a weak binding partner of PDZ2 and we demonstrate that the presence of PDZ3 alters the binding affinity of PDZ2 for APC, suggesting an allosteric effect and thereby modulating the binding characteristics of PDZ2. A HADDOCK-based molecular model of the PDZ2/3 tandem domain from PTPN13 supports these results. CONCLUSIONS: Our study of tandem PDZ2/3 in complex with APC suggests that the interaction of PDZ3 with PDZ2 induces an allosteric modulation within PDZ2 emanating from the back of the domain to the ligand binding site. Thus, the modified binding preference of PDZ2 for APC could be explained by an allosteric effect and provides further evidence for the pivotal function of PDZ2 in the PDZ123 domain triplet within PTPN13.

Apoptotic signalling targets the post-endocytic sorting machinery of the death receptor Fas/CD95.

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.

Molecular Basis of Class III Ligand Recognition by PDZ3 in Murine Protein Tyrosine Phosphatase PTPN13.

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.

A novel interaction between ATOH8 and PPP3CB.

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.

Crystal structure of the Rab binding domain of OCRL1 in complex with Rab8 and functional implications of the OCRL1/Rab8 module for Lowe syndrome.

Mutations of the inositol-5-phosphatase OCRL1 cause Lowe syndrome. Lowe syndrome is an inherited disease characterized by renal dysfunction and impaired development of the eye and the nervous system. OCRL1 is a Rab effector protein that can bind to a large number of different Rab proteins. We have recently determined the X-ray structure of the Rab-binding domain of OCRL1 in complex with Rab8. Furthermore, we have characterized point mutations that abolish binding to Rab proteins and cause Lowe syndrome. Here we shortly review our recent biophysical and structural work and discuss possible functional implications of our finding that Rab8 binds with the highest affinity to OCRL1 among the Rab proteins tested. This could direct further work on OCRL1 leading to a better understanding of the complex disease mechanism of Lowe syndrome.

A structural basis for Lowe syndrome caused by mutations in the Rab-binding domain of OCRL1.

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 ß-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.

Sequence-specific 1H, 13C, and 15N assignment of the extended PDZ3 domain of the protein tyrosine phosphatase basophil-like PTP-BL.

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.

PDZ-domain-directed basolateral targeting of the peripheral membrane protein FRMPD2 in epithelial cells.

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.

A PH domain within OCRL bridges clathrin-mediated membrane trafficking to phosphoinositide metabolism.

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.

The protein tyrosine phosphatase-BL, modulates pancreatic beta-cell proliferation by interaction with the Wnt signalling pathway.

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.

A role of the Lowe syndrome protein OCRL in early steps of the endocytic pathway.

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.

Sequence-specific (1)H, (13)C, and (15)N backbone assignment of the 28 kDa PDZ2/PDZ3 tandem domain of the protein tyrosine phosphatase PTP-BL.

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.

Dynamin and the actin cytoskeleton cooperatively regulate plasma membrane invagination by BAR and F-BAR proteins.

Cell 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.

Truncated TrkB receptor-induced outgrowth of dendritic filopodia involves the p75 neurotrophin receptor.

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.

The protein tyrosine phosphatase PTP-Basophil/Basophil-like. Interacting proteins and molecular functions.

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.