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.

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.

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.

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.

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).

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.

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.

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.

The serologically defined colon cancer antigen-3 (SDCCAG3) is involved in the regulation of ciliogenesis.

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.

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.

RNAi screening identifies mediators of NOD2 signaling: implications for spatial specificity of MDP recognition.

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).

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Structure determination and ligand interactions of the PDZ2b domain of PTP-Bas (hPTP1E): splicing-induced modulation of ligand specificity.

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.

The protein tyrosine phosphatase PTP-BL associates with the midbody and is involved in the regulation of cytokinesis.

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.