Coincidence detection of RAB11A and PI(3)P by WIPI2 directs autophagosome formation

Macroautophagy, hereafter referred to as autophagy, is a process that delivers cytoplasmic material to lysosomes for degradation. When nutrients are scarce, autophagy sustains cellular renovation by recycling cellular constituents (amino acids and fatty acids) for anabolic processes. In nutrient-replete conditions, autophagy maintains cellular homeostasis by selectively degrading disease-related ‘cargoes’ (pathogenic aggregate-prone proteins, damaged organelles, invasive bacteria)

Arf6 promotes autophagosome formation via effects on phosphatidylinositol 4,5-bisphosphate and phospholipase D.

Macroautophagy (in this paper referred to as autophagy) and the ubiquitin-proteasome system are the two major catabolic systems in cells. Autophagy involves sequestration of cytosolic contents in double membrane-bounded vesicles called autophagosomes. The membrane source for autophagosomes has received much attention, and diverse sources, such as the plasma membrane, Golgi, endoplasmic reticulum, and mitochondria, have been implicated. These may not be mutually exclusive, but the exact sources and mechanism involved in the formation of autophagosomes are still unclear. In this paper, we identify a positive role for the small G protein Arf6 in autophagosome formation. The effect of Arf6 on autophagy is mediated by its role in the generation of phosphatidylinositol 4,5-bisphosphate (PIP(2)) and in inducing phospholipase D (PLD) activity. PIP(2) and PLD may themselves promote autophagosome biogenesis by influencing endocytic uptake of plasma membrane into autophagosome precursors. However, Arf6 may also influence autophagy by indirect effects, such as either by regulating membrane flow from other compartments or by modulating PLD activity independently of the mammalian target of rapamycin.We are grateful for funding from a Wellcome Trust Senior and Principal Research Fellowships (to D.C. Rubinsztein) and Cancer Research UK (to C. Puri)

Myosin VI is required for sorting of AP-1B-dependent cargo to the basolateral domain in polarized MDCK cells.

In polarized epithelial cells, newly synthesized membrane proteins are delivered on specific pathways to either the apical or basolateral domains, depending on the sorting motifs present in these proteins. Because myosin VI has been shown to facilitate secretory traffic in nonpolarized cells, we investigated its role in biosynthetic trafficking pathways in polarized MDCK cells. We observed that a specific splice isoform of myosin VI with no insert in the tail domain is required for the polarized transport of tyrosine motif containing basolateral membrane proteins. Sorting of other basolateral or apical cargo, however, does not involve myosin VI. Site-directed mutagenesis indicates that a functional complex consisting of myosin VI, optineurin, and probably the GTPase Rab8 plays a role in the basolateral delivery of membrane proteins, whose sorting is mediated by the clathrin adaptor protein complex (AP) AP-1B. Our results suggest that myosin VI is a crucial component in the AP-1B-dependent biosynthetic sorting pathway to the basolateral surface in polarized epithelial cells

LC3-positive structures are prominent in autophagy-deficient cells

Abstract: Autophagy is an evolutionarily conserved process across eukaryotes that degrades cargoes like aggregate-prone proteins, pathogens, damaged organelles and macromolecules via delivery to lysosomes. The process involves the formation of double-membraned autophagosomes that engulf the cargoes destined for degradation, sometimes with the help of autophagy receptors like p62, which are themselves autophagy substrates. LC3-II, a standard marker for autophagosomes, is generated by the conjugation of cytosolic LC3-I to phosphatidylethanolamine (PE) on the surface of nascent autophagosomes. As LC3-II is relatively specifically associated with autophagosomes and autolysosomes (in the absence of conditions stimulating LC3-associated phagocytosis), quantification of LC3-positive puncta is considered as a gold-standard assay for assessing the numbers of autophagosomes in cells. Here we find that the endogenous LC3-positive puncta become larger in cells where autophagosome formation is abrogated, and are prominent even when LC3-II is not formed. This occurs even with transient and incomplete inhibition of autophagosome biogenesis. This phenomenon is due to LC3-I sequestration to p62 aggregates, which accumulate when autophagy is impaired. This observation questions the reliability of LC3-immunofluorescence assays in cells with compromised autophagy

Design of compensated ferrimagnetic Heusler alloys for giant tunable exchange bias

The discovery of materials with improved functionality can be accelerated by rational material design. Heusler compounds with tunable magnetic sublattices allow to implement this concept to achieve novel magnetic properties. Here, we have designed a family of Heusler alloys with a compensated ferrimagnetic state. In the vicinity of the compensation composition in Mn-Pt-Ga, a giant exchange bias (EB) of more than 3 T and a similarly large coercivity are established. The large exchange anisotropy originates from the exchange interaction between the compensated host and ferrimagnetic clusters that arise from intrinsic anti-site disorder. We demonstrate the applicability of our design concept on a second material, Mn-Fe-Ga, with a magnetic transition above room temperature, exemplifying the universality of the concept and the feasibility of room-temperature applications. Our study points to a new direction for novel magneto-electronic devices. At the same time it suggests a new route for realizing rare-earth free exchange-biased hard magnets, where the second quadrant magnetization can be stabilized by the exchange bias.Comment: Four figure

PI(5)P regulates autophagosome biogenesis.

Phosphatidylinositol 3-phosphate (PI(3)P), the product of class III PI3K VPS34, recruits specific autophagic effectors, like WIPI2, during the initial steps of autophagosome biogenesis and thereby regulates canonical autophagy. However, mammalian cells can produce autophagosomes through enigmatic noncanonical VPS34-independent pathways. Here we show that PI(5)P can regulate autophagy via PI(3)P effectors and thereby identify a mechanistic explanation for forms of noncanonical autophagy. PI(5)P synthesis by the phosphatidylinositol 5-kinase PIKfyve was required for autophagosome biogenesis, and it increased levels of PI(5)P, stimulated autophagy, and reduced the levels of autophagic substrates. Inactivation of VPS34 impaired recruitment of WIPI2 and DFCP1 to autophagic precursors, reduced ATG5-ATG12 conjugation, and compromised autophagosome formation. However, these phenotypes were rescued by PI(5)P in VPS34-inactivated cells. These findings provide a mechanistic framework for alternative VPS34-independent autophagy-initiating pathways, like glucose starvation, and unravel a cytoplasmic function for PI(5)P, which previously has been linked predominantly to nuclear roles.We are grateful for funding from a Wellcome Trust Principal Research Fellowship (095317/Z/11/Z to D.C.R.), a Wellcome Trust Strategic Award (100140/Z/ 12/Z), the NIHR Biomedical Research Centre in Dementia at Addenbrooke’s Hospital, an MRC Confidence in Concepts grant (D.C.R.), and a FEBS Long- Term Fellowship (A.A.).This article was originally published in Molecular Cell (M Vicinanza, VI Korolchuk, A Ashkenazi, C Puri, FM Menzies, JH Clarke, DC Rubinsztein, Molecular Cell 2015, 57, 219-234

Numb Is an Endocytic Protein

Numb is a protein that in Drosophila determines cell fate as a result of its asymmetric partitioning at mitosis. The function of Numb has been linked to its ability to bind and to biologically antagonize Notch, a membrane receptor that also specifies cell fate. The biochemical mechanisms underlying the action of Numb, however, are still largely unknown. The wide pattern of expression of Numb suggests a general function in cellular homeostasis that could be additional to, or part of, its action in fate determination. Such a function could be endocytosis, as suggested by the interaction of Numb with Eps15, a component of the endocytic machinery. Here, we demonstrate that Numb is an endocytic protein. We found that Numb localizes to endocytic organelles and is cotrafficked with internalizing receptors. Moreover, it associates with the appendage domain of α adaptin, a subunit of AP2, a major component of clathrin-coated pits. Finally, fragments of Numb act as dominant negatives on both constitutive and ligand-regulated receptor-mediated internalization, suggesting a general role for Numb in the endocytic process

Use of a Zwitterionic Surfactant to Improve the Biofunctional Properties of Wool Dyed with an Onion (Allium cepa L.) Skin Extract

To improve the loadability and antioxidant properties of wool impregnated with onion skin extract, the introduction of SB3-14 surfactant in the dyeing process was evaluated. A preliminary investigation on the surfactant–quercetin interaction indicated that the optimal conditions for dye solubility, stability, and surfactant affinity require double-distilled water (pH = 5.5) as a medium and SB3-14 in a concentration above the c.m.c. (2.5 × 10−3 M). The absorption profile of textiles showed the flavonoid absorption band (390 nm) and a bathochromic feature (510 nm), suggesting flavonoid aggregates. The higher absorbance for the sample dyed with SB3-14 indicated greater dye uptake, which was further confirmed by HPLC analysis. The Folin–Ciocalteu method was applied to evaluate the total phenol content (TPC) released from the treated wool, while the assays FRAP, DPPH, ABTS, and ORAC were applied to evaluate the corresponding total antioxidant activity (TAC). Higher TPCs (about 20%) and TACs (5–55%) were measured with SB3-14, highlighting textiles with improved biofunctional properties. Spectrophotometric analyses were also performed with an artificial sweat. The potential cytotoxic effect of SB3-14 in both monomeric and aggregated forms, cell viability, and induction of apoptosis were evaluated in RAW 264.7 cells. These analyses revealed that SB3-14 is safe at concentrations below the c.m.c

Lipid Rafts and Clathrin Cooperate in the Internalization of PrPC in Epithelial FRT Cells

The cellular prion protein (PrP(C)) plays a key role in the pathogenesis of Transmissible Spongiform Encephalopathies in which the protein undergoes post-translational conversion to the infectious form (PrP(Sc)). Although endocytosis appears to be required for this conversion, the mechanism of PrP(C) internalization is still debated, as caveolae/raft- and clathrin-dependent processes have all been reported to be involved. METHODOLOGY/PRINCIPAL FINDINGS: We have investigated the mechanism of PrP(C) endocytosis in Fischer Rat Thyroid (FRT) cells, which lack caveolin-1 (cav-1) and caveolae, and in FRT/cav-1 cells which form functional caveolae. We show that PrP(C) internalization requires activated Cdc-42 and is sensitive to cholesterol depletion but not to cav-1 expression suggesting a role for rafts but not for caveolae in PrP(C) endocytosis. PrP(C) internalization is also affected by knock down of clathrin and by the expression of dominant negative Eps15 and Dynamin 2 mutants, indicating the involvement of a clathrin-dependent pathway. Notably, PrP(C) co-immunoprecipitates with clathrin and remains associated with detergent-insoluble microdomains during internalization thus indicating that PrP(C) can enter the cell via multiple pathways and that rafts and clathrin cooperate in its internalization. CONCLUSIONS/SIGNIFICANCE: These findings are of particular interest if we consider that the internalization route/s undertaken by PrP(C) can be crucial for the ability of different prion strains to infect and to replicate in different cell lines