Analysis of Endocytic Pathways in Drosophila Cells Reveals a Conserved Role for GBF1 in Internalization via GEECs

In mammalian cells, endocytosis of the fluid phase and glycosylphosphatidylinositol-anchored proteins (GPI-APs) forms GEECs (GPI-AP enriched early endosomal compartments) via an Arf1- and Cdc42-mediated, dynamin independent mechanism. Here we use four different fluorescently labeled probes and several markers in combination with quantitative kinetic assays, RNA interference and high resolution imaging to delineate major endocytic routes in Drosophila cultured cells. We find that the hallmarks of the pinocytic GEEC pathway are conserved in Drosophila and identify garz, the fly ortholog of the GTP exchange factor GBF1, as a novel component of this pathway. Live confocal and TIRF imaging reveals that a fraction of GBF1 GFP dynamically associates with ABD RFP (a sensor for activated Arf1 present on nascent pinosomes). Correspondingly, a GTP exchange mutant of GBF1 has altered ABD RFP localization in the evanescent field and is impaired in fluid phase uptake. Furthermore, GBF1 activation is required for the GEEC pathway even in the presence of Brefeldin A, implying that, like Arf1, it has a role in endocytosis that is separable from its role in secretion

β-Catenin asymmetry is regulated by PLA1 and retrograde traffic in C. elegans stem cell divisions

Asymmetric division is an important property of stem cells. In Caenorhabditis elegans, the Wnt/β-catenin asymmetry pathway determines the polarity of most asymmetric divisions. The Wnt signalling components such as β-catenin localize asymmetrically to the cortex of mother cells to produce two distinct daughter cells. However, the molecular mechanism to polarize them remains to be elucidated. Here, we demonstrate that intracellular phospholipase A1 (PLA1), a poorly characterized lipid-metabolizing enzyme, controls the subcellular localizations of β-catenin in the terminal asymmetric divisions of epithelial stem cells (seam cells). In mutants of ipla-1, a single C. elegans PLA1 gene, cortical β-catenin is delocalized and the asymmetry of cell-fate specification is disrupted in the asymmetric divisions. ipla-1 mutant phenotypes are rescued by expression of ipla-1 in seam cells in a catalytic activity-dependent manner. Furthermore, our genetic screen utilizing ipla-1 mutants reveals that reduction of endosome-to-Golgi retrograde transport in seam cells restores normal subcellular localization of β-catenin to ipla-1 mutants. We propose that membrane trafficking regulated by ipla-1 provides a mechanism to control the cortical asymmetry of β-catenin

In-Cell RNA Hydrolysis Assay: A Method for the Determination of the RNase Activity of Potential RNases

Conventional procedures to assay RNA degradation by a protein with ribonuclease (RNase) activity require a step to isolate intact RNA molecules, which are used as a substrate. Here, we established a novel “In-cell RNA hydrolysis assay” in which RNAs within cells are used as a substrate for the RNA-hydrolyzing protein, thereby avoiding the need to prepare intact RNA molecules. In this method, the degree of RNA degradation is indicated by the fluorescence intensity of RNA molecules released from fixed and permeabilized cells following treatment with the potential RNase. A catalytic 3D8 antibody capable of degrading RNAs and pancreatic RNase A were used as model RNases. Our data demonstrate that the novel In-cell RNA hydrolysis assay is a reliable and sensitive method to analyze the activities of potential RNA-hydrolyzing proteins such as catalytic antibodies

Amino acid analysis by precolumn derivatization with 1-fluoro-2,4-dinitrophenyl-5-L-alanine amide (Marfey agent)

For the determination of the chirality of amino acids and other compounds containing primary amino groups, derivatization with the chiral Marfey’s reagent has kept its place as a complementary method beside direct chromatographic analysis with chiral stationary phases. Here, we describe the use of Marfey’s reagent for common amino acid analysis. The simplicity of pre-column derivatization of amino acids with this reagent, the ease of separation of the derivatized amino acids with non-dedicated reverse-phase highperformance liquid chromatography (RP-HPLC) equipment and the high sensitivity of their chromatographic detection at 340 nm make the described method an attractive alternative to the dedicated amino acid analyzer

Remodeling a DNA-binding protein as a specific in vivo inhibitor of bacterial secretin PulD.

International audienceWe engineered a class of proteins that binds selected polypeptides with high specificity and affinity. Use of the protein scaffold of Sac7d, belonging to a protein family that binds various ligands, overcomes limitations inherent in the use of antibodies as intracellular inhibitors: it lacks disulfide bridges, is small and stable, and can be produced in large amounts. An in vitro combinatorial/selection approach generated specific, high-affinity (up to 140 pM) binders against bacterial outer membrane secretin PulD. When exported to the Escherichia coli periplasm, they inhibited PulD oligomerization, thereby blocking the type II secretion pathway of which PulD is part. Thus, high-affinity inhibitors of protein function can be derived from Sac7d and can be exported to, and function in, a cell compartment other than that in which they are produced