Developmental patterning requires the precise interplay of numerous intercellular signaling pathways to ensure that cells are properly specified during tissue formation and organogenesis. The spatiotemporal function of the Notch signaling pathway is strongly influenced by the biosynthesis and intracellular trafficking of signaling components. Receptors and ligands must be trafficked to the cell surface where they interact, and their subsequent endocytic internalization and endosomal trafficking is crucial for both signal propagation and its down-modulation. In a forward genetic screen for mutations that alter intracellular Notch receptor trafficking in Drosophila epithelial tissues, we recovered mutations that disrupt the Catsup gene, which encodes the Drosophila ortholog of the mammalian ZIP7 zinc transporter. Loss of Catsup function causes Notch to accumulate abnormally in the endoplasmic reticulum (ER) and Golgi compartments, resulting in impaired Notch signaling. In addition, Catsup mutant cells exhibit elevated ER stress, suggesting that impaired zinc homeostasis causes increased levels of misfolded proteins within the secretory compartment.

A group of neurodegenerative diseases referred to as tauopathies are characterized by the presence of brain cells harboring inclusions of pathological species of the tau protein. These disorders include Alzheimer’s disease (AD) and frontotemporal lobar degeneration (FTLD) due to tau pathology, including progressive supranuclear palsy, corticobasal degeneration and Pick’s disease. Tau is normally a microtubule-associated protein that appears to play an important role in ensuring proper axonal transport, but in tauopathies tau becomes hyperphosphorylated and disengages from microtubules, with consequent misfolding and deposition into inclusions that mainly affect neurons, but also glia. A body of experimental evidence suggests that the development of tau inclusions leads to the neurodegeneration observed in tauopathies, and there is a growing interest in developing tau-directed therapeutic agents. The following review provides a summary of strategies under investigation for the potential treatment of tauopathies, highlighting both the promises and challenges associated with these various therapeutic approaches.

SummaryThe role of the cytoskeleton in protein trafficking is still being defined. Here, we describe a relationship between the small Ca 2+ -dependent membrane-binding protein Annexin B9 (AnxB9), apical b Heavy -spectrin (b H ) and the multivesicular body (MVB) in Drosophila. AnxB9 binds to a subset of b H spliceoforms, and loss of AnxB9 results in an increase in basolateral b H and its appearance on cytoplasmic vesicles that overlap with the MVB markers Hrs, Vps16 and EPS15. Similar colocalizations are seen when b H -positive endosomes are generated either by upregulation of b H in pak mutants or through the expression of the dominant-negative version of b H . In common with other mutations disrupting the MVB, we also show that there is an accumulation of ubiquitylated proteins and elevated EGFR signaling in the absence of AnxB9 or b H . Loss of AnxB9 or b H function also causes the redistribution of the DE-Cadherin (encoded by shotgun) to endosomal vesicles, suggesting a rationale for the previously documented destabilization of the zonula adherens in karst (which encodes b H ) mutants. Reduction of AnxB9 results in degradation of the apical-lateral boundary and the appearance of the basolateral proteins Coracle and Dlg on internal vesicles adjacent to b H . These results indicate that AnxB9 and b H are intimately involved in endosomal trafficking to the MVB and play a role in maintaining high-fidelity segregation of the apical and lateral domains.

BackgroundThe plasma membrane (PM) is a compartment of significant interest because cell surface proteins influence the way in which a cell interacts with its neighbours and its extracellular environment. However, PM is hard to isolate because of its low abundance. Aqueous two-phase affinity purification (2PAP), based on PEG/Dextran two-phase fractionation and lectin affinity for PM-derived microsomes, is an emerging method for the isolation of high purity plasma membranes from several vertebrate sources. In contrast, PM isolation techniques in important invertebrate genetic model systems, such as Drosophila melanogaster, have relied upon enrichment by density gradient centrifugation. To facilitate genetic investigation of activities contributing to the content of the PM sub-proteome, we sought to adapt 2PAP to this invertebrate model to provide a robust PM isolation technique for Drosophila.ResultsWe show that 2PAP alone does not completely remove contaminating endoplasmic reticulum and mitochondrial membrane. However, a novel combination of density gradient centrifugation plus 2PAP results in a robust PM preparation. To demonstrate the utility of this technique we isolated PM from fly heads and successfully identified 432 proteins using MudPIT, of which 37% are integral membrane proteins from all compartments. Of the 432 proteins, 22% have been previously assigned to the PM compartment, and a further 34% are currently unassigned to any compartment and represent candidates for assignment to the PM. The remainder have previous assignments to other compartments.ConclusionA combination of density gradient centrifugation and 2PAP results in a robust, high purity PM preparation from Drosophila, something neither technique can achieve on its own. This novel preparation should lay the groundwork for the proteomic investigation of the PM in different genetic backgrounds in Drosophila. Our results also identify two key steps in this procedure: The optimization of membrane partitioning in the PEG/Dextran mixture, and careful choice of the correct lectin for the affinity purification step in light of variations in bulk membrane lipid composition and glycosylation patterns respectively. This points the way for further adaptations into other systems.