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.

A high-resolution morphological and ultrastructural map of anterior sensory cilia and glia in Caenorhabditis elegans.

Many primary sensory cilia exhibit unique architectures that are critical for transduction of specific sensory stimuli. Although basic ciliogenic mechanisms are well described, how complex ciliary structures are generated remains unclear. Seminal work performed several decades ago provided an initial but incomplete description of diverse sensory cilia morphologies in C. elegans. To begin to explore the mechanisms that generate these remarkably complex structures, we have taken advantage of advances in electron microscopy and tomography, and reconstructed three-dimensional structures of fifty of sixty sensory cilia in the C. elegans adult hermaphrodite at high resolution. We characterize novel axonemal microtubule organization patterns, clarify structural features at the ciliary base, describe new aspects of cilia-glia interactions, and identify structures suggesting novel mechanisms of ciliary protein trafficking. This complete ultrastructural description of diverse cilia in C. elegans provides the foundation for investigations into underlying ciliogenic pathways, as well as contributions of defined ciliary structures to specific neuronal functions. DOI: http://dx.doi.org/10.7554/eLife.01948.001.

Primary cilia and dendritic spines: different but similar signaling compartments.

Primary non-motile cilia and dendritic spines are cellular compartments that are specialized to sense and transduce environmental cues and presynaptic signals, respectively. Despite their unique cellular roles, both compartments exhibit remarkable parallels in the general principles, as well as molecular mechanisms, by which their protein composition, membrane domain architecture, cellular interactions, and structural and functional plasticity are regulated. We compare and contrast the pathways required for the generation and function of cilia and dendritic spines, and suggest that insights from the study of one may inform investigations into the other of these critically important signaling structures.

Endocytosis genes facilitate protein and membrane transport in C. elegans sensory cilia.

BACKGROUND: Multiple intracellular transport pathways drive the formation, maintenance, and function of cilia, a compartmentalized organelle associated with motility, chemo-/mechano-/photosensation, and developmental signaling. These pathways include cilium-based intraflagellar transport (IFT) and poorly understood membrane trafficking events. Defects in ciliary transport contribute to the etiology of human ciliary disease such as Bardet-Biedl syndrome (BBS). In this study, we employ the genetically tractable nematode Caenorhabditis elegans to investigate whether endocytosis genes function in cilium formation and/or the transport of ciliary membrane or ciliary proteins. RESULTS: Here we show that localization of the clathrin light chain, AP-2 clathrin adaptor, dynamin, and RAB-5 endocytic proteins overlaps with a morphologically discrete periciliary membrane compartment associated with sensory cilia. In addition, ciliary transmembrane proteins such as G protein-coupled receptors concentrate at periciliary membranes. Disruption of endocytic gene function causes expansion of ciliary and/or periciliary membranes as well as defects in the ciliary targeting and/or transport dynamics of ciliary transmembrane and IFT proteins. Finally, genetic analyses reveal that the ciliary membrane expansions in dynamin and AP-2 mutants require bbs-8 and rab-8 function and that sensory signaling and endocytic genes may function in a common pathway to regulate ciliary membrane volume. CONCLUSIONS: These data implicate C. elegans endocytosis proteins localized at the ciliary base in regulating ciliary and periciliary membrane volume and suggest that membrane retrieval from these compartments is counterbalanced by BBS-8 and RAB-8-mediated membrane delivery.

Split ends antagonizes the Notch and potentiates the EGFR signaling pathways during Drosophila eye development.

The Notch and Epidermal Growth Factor Receptor (EGFR) signaling pathways interact cooperatively and antagonistically to regulate many aspects of Drosophila development, including the eye. How output from these two signaling networks is fine-tuned to achieve the precise balance needed for specific inductive interactions and patterning events remains an open and important question. Previously, we reported that the gene split ends (spen) functions within or parallel to the EGFR pathway during midline glial cell development in the embryonic central nervous system. Here, we report that the cellular defects caused by loss of spen function in the developing eye imaginal disc place spen as both an antagonist of the Notch pathway and a positive contributor to EGFR signaling during retinal cell differentiation. Specifically, loss of spen results in broadened expression of Scabrous, ectopic activation of Notch signaling, and a corresponding reduction in Atonal expression at the morphogenetic furrow. Consistent with Spen's role in antagonizing Notch signaling, reduction of spen levels is sufficient to suppress Notch-dependent phenotypes. At least in part due to loss of Spen-dependent down-regulation of Notch signaling, loss of spen also dampens EGFR signaling as evidenced by reduced activity of MAP kinase (MAPK). This reduced MAPK activity in turn leads to a failure to limit expression of the EGFR pathway antagonist and the ETS-domain transcriptional repressor Yan and to a corresponding loss of cell fate specification in spen mutant ommatidia. We propose that Spen plays a role in modulating output from the Notch and EGFR pathways to ensure appropriate patterning during eye development.

Signal integration during development: mechanisms of EGFR and Notch pathway function and cross-talk.

Metazoan development relies on a highly regulated network of interactions between conserved signal transduction pathways to coordinate all aspects of cell fate specification, differentiation, and growth. In this review, we discuss the intricate interplay between the epidermal growth factor receptor (EGFR; Drosophila EGFR/DER) and the Notch signaling pathways as a paradigm for signal integration during development. First, we describe the current state of understanding of the molecular architecture of the EGFR and Notch signaling pathways that has resulted from synergistic studies in vertebrate, invertebrate, and cultured cell model systems. Then, focusing specifically on the Drosophila eye, we discuss how cooperative, sequential, and antagonistic relationships between these pathways mediate the spatially and temporally regulated processes that generate this sensory organ. The common themes underlying the coordination of the EGFR and Notch pathways appear to be broadly conserved and should, therefore, be directly applicable to elucidating mechanisms of information integration and signaling specificity in vertebrate systems.

Splits ends is a tissue/promoter specific regulator of Wingless signaling.

Wingless directs many developmental processes in Drosophila by regulating expression of specific target genes through a conserved signaling pathway. Although many nuclear factors have been implicated in mediating Wingless-induced transcription, the mechanism of how Wingless regulates different targets in different tissues remains poorly understood. We report here that the split ends gene is required for Wingless signaling in the eye, wing and leg imaginal discs. Expression of a dominant-negative version of split ends resulted in more dramatic reductions in Wingless signaling than split ends-null alleles, suggesting that it may have a redundant partner. However, removal of split ends or expression of the dominant-negative had no effect on several Wingless signaling readouts in the embryo. The expression pattern of Split ends cannot explain this tissue-specific requirement, as the protein is predominantly nuclear and present throughout embryogenesis and larval tissues. Consistent with its nuclear location, the split ends dominant-negative acts downstream of Armadillo stabilization. Our data indicate that Split ends is an important positive regulator of Wingless signaling in larval tissues. However, it has no detectable role in the embryonic Wingless pathway, suggesting that it is a tissue or promoter-specific factor.