Congenital chloride-losing diarrhea in a Mexican child with the novel homozygous SLC26A3 mutation G393W

Congenital chloride diarrhea is an autosomal recessive disease caused by mutations in the intestinal lumenal membrane Cl−/HCO−3 exchanger, SLC26A3. We report here the novel SLC26A3 mutation G393W in a Mexican child, the first such report in a patient from Central America. SLC26A3 G393W expression in Xenopus oocytes exhibits a mild hypomorphic phenotype, with normal surface expression and moderately reduced anion transport function. However, expression of HA-SLC26A3 in HEK-293 cells reveals intracellular retention and greatly decreased steady-state levels of the mutant polypeptide, in contrast to peripheral membrane expression of the wildtype protein. Whereas wildtype HA-SLC26A3 is apically localized in polarized monolayers of filter-grown MDCK cells and Caco2 cells, mutant HA-SLC26A3 G393W exhibits decreased total polypeptide abundance, with reduced or absent surface expression and sparse punctate (or absent) intracellular distribution. The WT protein is similarly localized in LLC-PK1 cells, but the mutant fails to accumulate to detectable levels. We conclude that the chloride-losing diarrhea phenotype associated with homozygous expression of SLC26A3 G393W likely reflects lack of apical surface expression in enterocytes, secondary to combined abnormalities in polypeptide trafficking and stability. Future progress in development of general or target-specific folding chaperonins and correctors may hold promise for pharmacological rescue of this and similar genetic defects in membrane protein targeting

Combined Inactivation of pRB and Hippo Pathways Induces Dedifferentiation in the Drosophila Retina

Functional inactivation of the Retinoblastoma (pRB) pathway is an early and obligatory event in tumorigenesis. The importance of pRB is usually explained by its ability to promote cell cycle exit. Here, we demonstrate that, independently of cell cycle exit control, in cooperation with the Hippo tumor suppressor pathway, pRB functions to maintain the terminally differentiated state. We show that mutations in the Hippo signaling pathway, wts or hpo, trigger widespread dedifferentiation of rbf mutant cells in the Drosophila eye. Initially, rbf wts or rbf hpo double mutant cells are morphologically indistinguishable from their wild-type counterparts as they properly differentiate into photoreceptors, form axonal projections, and express late neuronal markers. However, the double mutant cells cannot maintain their neuronal identity, dedifferentiate, and thus become uncommitted eye specific cells. Surprisingly, this dedifferentiation is fully independent of cell cycle exit defects and occurs even when inappropriate proliferation is fully blocked by a de2f1 mutation. Thus, our results reveal the novel involvement of the pRB pathway during the maintenance of a differentiated state and suggest that terminally differentiated Rb mutant cells are intrinsically prone to dedifferentiation, can be converted to progenitor cells, and thus contribute to cancer advancement

Polarized Secretion of Drosophila EGFR Ligand from Photoreceptor Neurons Is Controlled by ER Localization of the Ligand-Processing Machinery

Trafficking within the endoplasmic reticulum and specialized localization of the intra-membrane protease Rhomboid regulate EGF ligand-dependent signaling in Drosophila photoreceptor axon termini

Multiple Signals Converge on a Differentiation MAPK Pathway

An important emerging question in the area of signal transduction is how information from different pathways becomes integrated into a highly coordinated response. In budding yeast, multiple pathways regulate filamentous growth, a complex differentiation response that occurs under specific environmental conditions. To identify new aspects of filamentous growth regulation, we used a novel screening approach (called secretion profiling) that measures release of the extracellular domain of Msb2p, the signaling mucin which functions at the head of the filamentous growth (FG) MAPK pathway. Secretion profiling of complementary genomic collections showed that many of the pathways that regulate filamentous growth (RAS, RIM101, OPI1, and RTG) were also required for FG pathway activation. This regulation sensitized the FG pathway to multiple stimuli and synchronized it to the global signaling network. Several of the regulators were required for MSB2 expression, which identifies the MSB2 promoter as a target “hub” where multiple signals converge. Accessibility to the MSB2 promoter was further regulated by the histone deacetylase (HDAC) Rpd3p(L), which positively regulated FG pathway activity and filamentous growth. Our findings provide the first glimpse of a global regulatory hierarchy among the pathways that control filamentous growth. Systems-level integration of signaling circuitry is likely to coordinate other regulatory networks that control complex behaviors

Characterization of Spen function during Drosophila eye development

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2007.This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.Pages 327-328 blank.Includes bibliographical references.Conserved signal transduction pathways coordinate all aspects of metazoan development, including cell fate specification, differentiation, and growth. Rather than functioning as completely independent modules, signaling pathways interface to create a web of specific interactions that a cell integrates in a spatial and temporal manner. The Epidermal Growth Factor Receptor (EGFR) and Notch pathways are evolutionarily conserved signal transduction mechanisms that interact intimately to regulate a broad spectrum of developmental processes in metazoa. The molecular bases underlying cross-talk and signal integration between these two pathways are just beginning to be elucidated. In this thesis, we have focused on the role of Split ends (Spen), the founding member of a family of transcriptional co-repressors, as a node of cross-talk between the EGFR and Notch signaling pathways during Drosophila eye development. At the morphogenetic furrow (MF), which marks the wave of differentiation that passes through the imaginal disc eye primordium, Notch signaling is required for establishing and refining the expression of proneural Atonal (Ato). Ato promotes the EGFR pathway's reiterative signaling for progressive and sequential recruitment of cells within each ommatidial facet of the eye.(cont.) Previous studies found that spen functioned within or in parallel to the EGFR pathway during midline glial cell development in the embryonic central nervous system. In vertebrates, Spen orthologs function in repressor complexes that antagonize the transcription of Notch pathway targets. The involvement of Spen proteins in EGFR and Notch signaling in these systems thus motivated us to explore the consequences of loss of spen function with respect to each pathway during eye development. Here we report that Spen acts as both a positive regulator of EGFR signaling and as an antagonist of Notch signaling in the eye. We find that loss of spen results in hyper-activation of the Notch pathway via upregulation of the Notch activator Scabrous. This results in loss of Ato and activated MAP kinase at the MF, therefore antagonizing output from the EGFR signaling pathway. As a consequence, there is a failure of cell fate specification in spen mutant ommatidia. These observations suggest that Spen modulates output from the Notch and EGFR pathways to ensure appropriate patterning during eye development. Additionally, we have characterized a transgene encoding nuclear localization sequence-tagged Spen C-terminus that functions as a dominant-negative (SpenDN).(cont.) The Spen C-terminus contains the evolutionarily conserved SPOC domain that is required for transcriptional repression. In order to identify components related to Spen function and to understand the processes in which Spen may be involved, we performed a genetic screen to identify dominant modifiers of the rough-eye associated with eye-expressed SpenDN. Our results confirm interactions with the EGFR and Notch pathways, but also suggest functions for Spen in chromatin regulation and programmed cell death. As Spen-like proteins are involved in human development and disease, it will be important to elucidate the underlying molecular mechanism behind Spen function.by David Bagon Doroquez.Ph.D