The Itinerary of Autophagosomes: From Peripheral Formation to Kiss-and-Run Fusion with Lysosomes

Macroautophagy, a constitutive process in higher eukaryotic cells, mediates degradation of many long-lived proteins and organelles. The actual events occurring during the process in the dynamic system of a living cell have never been thoroughly investigated. We aimed to develop a live-cell assay in which to follow the complete itinerary of an autophagosome. Our experiments show that autophagosomes are formed randomly in peripheral regions of the cell. They then move bidirectionally along microtubules, accumulating at the microtubule-organizing centre, in a similar way to lysosomes. Their centripetal movement is dependent on the motor protein dynein and is important for their fusion with lysosomes. Initially, autophagosomes dock on to lysosomes, independent of lysosomal acidification. Two kinds of fusion then occur: complete fusions, creating a hybrid organelle, or more often kiss-and-run fusions, i.e. transfer of some content while still maintaining two separate vesicles. Surprisingly, the autophagolysosomal compartment seems to be more long lived than expected. Our study documents many aspects of autophagosome behaviour, adding to our understanding of the mechanism and control of autophagy. Indeed, although the formation of autophagosomes is completely different from any other vesicular structures, their later itinerary appears to be very similar to those of other trafficking pathways

Kondo Effects and Multipolar Order in the cubic PrTr2Al20 (Tr=Ti, V)

Our single crystal study reveals that PrTr2Al20 (Tr = Ti and V) provides the first examples of a cubic {\Gamma}3 nonmagnetic ground doublet system that shows the Kondo effect including a -ln T dependent resistivity. The {\Gamma}3 quadrupolar moments in PrV2Al20 induce anomalous metallic behavior through hybridization with conduction electrons, such as T^{1/2} dependent resistivity and susceptibility below ~ 20 K down to its ordering temperature T_O = 0.6 K. In PrTi2Al20, however, quadrupoles are well-localized and exhibit an order at T_O = 2.0 K. Stronger Kondo coupling in PrV2Al20 than in PrTi2Al20 suppresses quadrupolar ordering, and instead promotes hybridization between the {\Gamma}3 doublet and conduction electrons, leading to most likely the quadrupolar Kondo effect.Comment: 12 pages, 4 figure

Magnetic and structural characterization of thiol capped ferromagnetic Ag nanoparticles

Dodecanethiol capped Ag nanoparticles (NPs) have been independently synthesized by the well-known Brust method under the same physical-chemical conditions. The obtained NP present similar sizes ( ∼ 2 nm) but different magnetic behaviors. The extended x-ray absorption fine structure analyses at the K-edge of Ag did not reveal any noticeable structural nor topological differences among the samples. In clear contrast with the structure provided for thiol capped ferromagnetic Au NPs, the analysis also brings out the existence of Ag–S bonds in a diffuse region surrounding a reduced Ag core where the magnetism of the Ag NPs would be located. This record was migrated from the OpenDepot repository service in June, 2017 before shutting down

Exclusive Measurement of the Nonmesonic Weak Decay of ^{5}_{\Lambda}He Hypernucleus

We performed a coincidence measurement of two nucleons emitted from the nonmesonic weak decay (NMWD) of ^{5}_{\Lambda}He formed via the ^{6}Li(\pi^+,K^+) reaction. The energies of two nucleons and the pair number distributions in the opening angle between them were measured. In both np and nn pairs, we observed a clean back-to-back correlation coming from the two-body decay of \Lambda p --> n p and \Lambda n --> n n, respectively. The ratio of the nucleon pair numbers was N_{nn}/N_{np}=0.45 \pm 0.11(stat)\pm 0.03(syst) in the kinematic region of cos(theta_{NN}) < -0.8. Since each decay mode was exclusively detected, the measured ratio should be close to the ratio of \Gamma(\Lambda p --> np)/\Gamma(\Lambda n --> nn). The ratio is consistent with recent theoretical calculations based on the heavy meson/direct quark exchange picture.Comment: Submitted to Phys. Rev. lett., 4 pages, 3 figure

A reversible phospho-switch mediated by ULK1 regulates the activity of autophagy protease ATG4B

Upon induction of autophagy, the ubiquitin-like protein LC3 is conjugated to phosphatidylethanolamine (PE) on the inner and outer membrane of autophagosomes to allow cargo selection and autophagosome formation. LC3 undergoes two processing steps, the proteolytic cleavage of pro-LC3 and the de-lipidation of LC3-PE from autophagosomes, both executed by the same cysteine protease ATG4. How ATG4 activity is regulated to co-ordinate these events is currently unknown. Here we find that ULK1, a protein kinase activated at the autophagosome formation site, phosphorylates human ATG4B on serine 316. Phosphorylation at this residue results in inhibition of its catalytic activity in vitro and in vivo. On the other hand, phosphatase PP2A-PP2R3B can remove this inhibitory phosphorylation. We propose that the opposing activities of ULK1-mediated phosphorylation and PP2A-mediated dephosphorylation provide a phospho-switch that regulates the cellular activity of ATG4B to control LC3 processing

MIR376A is a regulator of starvation-induced autophagy

Background: Autophagy is a vesicular trafficking process responsible for the degradation of long-lived, misfolded or abnormal proteins, as well as damaged or surplus organelles. Abnormalities of the autophagic activity may result in the accumulation of protein aggregates, organelle dysfunction, and autophagy disorders were associated with various diseases. Hence, mechanisms of autophagy regulation are under exploration. Methods: Over-expression of hsa-miR-376a1 (shortly MIR376A) was performed to evaluate its effects on autophagy. Autophagy-related targets of the miRNA were predicted using Microcosm Targets and MIRanda bioinformatics tools and experimentally validated. Endogenous miRNA was blocked using antagomirs and the effects on target expression and autophagy were analyzed. Luciferase tests were performed to confirm that 3’ UTR sequences in target genes were functional. Differential expression of MIR376A and the related MIR376B was compared using TaqMan quantitative PCR. Results: Here, we demonstrated that, a microRNA (miRNA) from the DlkI/Gtl2 gene cluster, MIR376A, played an important role in autophagy regulation. We showed that, amino acid and serum starvation-induced autophagy was blocked by MIR376A overexpression in MCF-7 and Huh-7 cells. MIR376A shared the same seed sequence and had overlapping targets with MIR376B, and similarly blocked the expression of key autophagy proteins ATG4C and BECN1 (Beclin 1). Indeed, 3’ UTR sequences in the mRNA of these autophagy proteins were responsive to MIR376A in luciferase assays. Antagomir tests showed that, endogenous MIR376A was participating to the control of ATG4C and BECN1 transcript and protein levels. Moreover, blockage of endogenous MIR376A accelerated starvation-induced autophagic activity. Interestingly, MIR376A and MIR376B levels were increased with different kinetics in response to starvation stress and tissue-specific level differences were also observed, pointing out to an overlapping but miRNA-specific biological role. Conclusions: Our findings underline the importance of miRNAs encoded by the DlkI/Gtl2 gene cluster in stress-response control mechanisms, and introduce MIR376A as a new regulator of autophagy

CD4 T lymphocyte autophagy is upregulated in the salivary glands of primary Sjögren’s syndrome patients and correlates with focus score and disease activity

Background: Primary Sjögren’s syndrome (pSS) is a common chronic autoimmune disease characterized by lymphocytic infiltration of exocrine glands and peripheral lymphocyte perturbation. In the current study, we aimed to investigate the possible pathogenic implication of autophagy in T lymphocytes in patients with pSS. Methods: Thirty consecutive pSS patients were recruited together with 20 patients affected by sicca syndrome a nd/or chronic sialoadenitis and 30 healthy controls. Disease activity and damage were evaluated according to SS disease activity index, EULAR SS disease activity index, and SS disease damage index. T lymphocytes were analyzed for the expression of autophagy-specific markers by biochemical, molecular, and histological assays in peripheral blood and labial gland biopsies. Serum interleukin (IL)-23 and IL-21 levels were quantified by enzyme-linked immunosorbent assay. Results: Our study provides evidence for the first time that autophagy is upregulated in CD4+ T lymphocyte salivary glands from pSS patients. Furthermore, a statistically significant correlation was detected between lymphocyte autophagy levels, disease activity, and damage indexes. We also found a positive correlation between autophagy enhancement and the increased salivary gland expression of IL-21 and IL-23, providing a further link between innate and adaptive immune responses in pSS. Conclusions: These findings suggest that CD4+ T lymphocyte autophagy could play a key role in pSS pathogenesis. Additionally, our data highlight the potential exploitation of T cell autophagy as a biomarker of disease activity and provide new ground to verify the therapeutic implications of autophagy as an innovative drug target in pSS

Developing a High Resolution ZDC for the EIC

The Electron Ion Collider offers the opportunity to make un-paralleled multidimen- sional measurements of the spin structure of the proton and nuclei, as well as a study of the onset of partonic saturation at small Bjorken-x [1]. An important requirement of the physics program is the tagging of spectator neutrons and the identification of forward photons. We propose to design and build a Zero Degree Calorimeter, or ZDC, to measure photons and neutrons with excellent energy & position resolution

Measurement of the strong interaction induced shift and width of the 1s state of kaonic deuterium at J-PARC

The antikaon-nucleon interaction close to threshold provides crucial information on the interplay between spontaneous and explicit chiral symmetry breaking in low-energy QCD. In this context the importance of kaonic deuterium X-ray spectroscopy has been well recognized, but no experimental results have yet been obtained due to the difficulty of the measurement. We propose to measure the shift and width of the kaonic deuterium 1s state with an accuracy of 60 eV and 140 eV respectively at J-PARC. These results together with the kaonic hydrogen data (KpX at KEK, DEAR and SIDDHARTA at DAFNE) will then permit the determination of values of both the isospin I=0 and I=1 antikaon-nucleon scattering lengths and will provide the most stringent constraints on the antikaon-nucleon interaction, promising a breakthrough. Refined Monte Carlo studies were performed, including the investigation of background suppression factors for the described setup. These studies have demonstrated the feasibility of determining the shift and width of the kaonic deuterium atom 1s state with the desired accuracy of 60 eV and 140 eV.Comment: 12 pages, 9 figure