Consistency of quasi-maximum likelihood for processes with asymMetric laplacian innovation

Strong consistency of the quasi-maximum likelihood estimator is given for a general class of multidimensional causal processes based on asyMmetric laplacian innovation

Clec16a is critical for autolysosome function and Purkinje cell survival

CLEC16A is in a locus genetically linked to autoimmune diseases including multiple sclerosis, but the function of this gene in the nervous system is unknown. Here we show that two mouse strains carrying independent Clec16a mutations developed neurodegenerative disease characterized by motor impairments and loss of Purkinje cells. Neurons from Clec16a-mutant mice exhibited increased expression of the autophagy substrate p62, accumulation of abnormal intra-axonal membranous structures bearing the autophagy protein LC3, and abnormal Golgi morphology. Multiple aspects of endocytosis, lysosome and Golgi function were normal in Clec16a-deficient murine embryonic fibroblasts and HeLa cells. However, these cells displayed abnormal bulk autophagy despite unimpaired autophagosome formation. Cultured Clec16a-deficient cells exhibited a striking accumulation of LC3 and LAMP-1 positive autolysosomes containing undigested cytoplasmic contents. Therefore Clec16a, an autophagy protein that is critical for autolysosome function and clearance, is required for Purkinje cell survival

Early endosomes and endosomal coatomer are required for autophagy

Autophagy, an intracellular degradative pathway, maintains cell homeostasis under normal and stress conditions. Nascent double-membrane autophagosomes sequester and enclose cytosolic components and organelles, and subsequently fuse with the endosomal pathway allowing content degradation. Autophagy requires fusion of autophagosomes with late endosomes, but it is not known if fusion with early endosomes is essential. We show that fusion of AVs with functional early endosomes is required for autophagy. Inhibition of early endosome function by loss of COPI subunits (β′, β, or α) results in accumulation of autophagosomes, but not an increased autophagic flux. COPI is required for ER-Golgi transport and early endosome maturation. Although loss of COPI results in the fragmentation of the Golgi, this does not induce the formation of autophagosomes. Loss of COPI causes defects in early endosome function, as both transferrin recycling and EGF internalization and degradation are impaired, and this loss of function causes an inhibition of autophagy, an accumulation of p62/SQSTM-1, and ubiquitinated proteins in autophagosomes

Contact mediated signalling during cell movement

EThOS - Electronic Theses Online ServiceGBUnited Kingdo

EM analysis of ULK1 knocked-down cells reveals larger melanosomes.

<p>Cells were treated with (A and C) control or (B) ULK1 siRNA and prepared for EM analysis. For quantification (D) of melanosome number and area, cells were blindly chosen. A series of images were taken within each cell to cover a similar area between all cells at a 49,000X magnification. Over 80 images from total of 15 cells per condition were collected. The number of melanosomes at different stages (shown in control cells C) was manually scored within each cell. (D) Average melanosome size of the melanosomes in each stage in control and ULK1 siRNA treated cells.</p

ULK1 knockdown increases melanin in MNT-1 cells.

<p>(A) MNT-1 cells were treated with a control siRNA, two ULK1-specific siRNAs and a TYR-specific siRNA pool as a positive control for changes in melanin levels. Three days after the second knockdown cells were harvested and their melanin content was analysed by measuring melanin/protein values for each sample. Significant changes relative to control were determined by a one-way ANOVA test based on three individual experiments performed in duplicates. * p<0.05; ** p<0.01; *** p<0.001; data are presented as the mean of values normalized to control within each experiment. (B) Western blot analysis of the siRNA-treated MNT-1 cells verifying the knockdowns. Samples are loaded in duplicates. Tubulin is used as a loading control.</p

The effect of ULK1 depletion on melanin levels is independent of mTORC1 or the ULK1-ATG13-FIP200 complex.

<p>(A) Cells were treated with ULK1, FIP200, ATG13, TYR or control siRNA. Note: the data from one duplex is shown for FIP200 and Atg13, and a second siRNA duplex for each had identical effects. siRNA transfections were performed on day 1 and 3 after plating, and melanin was quantified on day 6. Control and ULK1 knockdown cells were incubated with 100nM Rapamycin (Rapa, +) or DMSO (-) starting from day 1. The data shown represents at least three individual experiments performed in duplicates, and values are normalized to control samples within each experiment. p values for the differences between the treatments and control were determined by a paired student’s t-test. (B) Cells treated as described in (A) were subjected to western blot analysis. mTORC1 activity was monitored by S6K phosphorylation, showing no significant effect after ULK1 depletion. (C) siRNA depletion of cells treated as in (A) was verified by western blot analysis. (D) Changes in basal autophagy were monitored by LC3-I and LC3-II levels, indicating an inhibitory effect for ULK1, ATG13 and FIP200 depletion in these cells. LC3-I levels were quantified by ImageJ and normalised to actin. Data shown is the mean of values normalized to actin from 4 independent experiments. Errors are SEM.</p

Phosphorylation of the LIR Domain of SCOC Modulates ATG8 Binding Affinity and Specificity

Autophagy is a highly conserved degradative pathway, essential for cellular homeostasis and implicated in diseases including cancer and neurodegeneration. Autophagy-related 8 (ATG8) proteins play a central role in autophagosome formation and selective delivery of cytoplasmic cargo to lysosomes by recruiting autophagy adaptors and receptors. The LC3-interacting region (LIR) docking site (LDS) of ATG8 proteins binds to LIR motifs present in autophagy adaptors and receptors. LIR-ATG8 interactions can be highly selective for specific mammalian ATG8 family members (LC3A-C, GABARAP, and GABARAPL1-2) and how this specificity is generated and regulated is incompletely understood. We have identified a LIR motif in the Golgi protein SCOC (short coiled-coil protein) exhibiting strong binding to GABARAP, GABARAPL1, LC3A and LC3C. The residues within and surrounding the core LIR motif of the SCOC LIR domain were phosphorylated by autophagy-related kinases (ULK1-3, TBK1) increasing specifically LC3 family binding. More distant flanking residues also contributed to ATG8 binding. Loss of these residues was compensated by phosphorylation of serine residues immediately adjacent to the core LIR motif, indicating that the interactions of the flanking LIR regions with the LDS are important and highly dynamic. Our comprehensive structural, biophysical and biochemical analyses support and provide novel mechanistic insights into how phosphorylation of LIR domain residues regulates the affinity and binding specificity of ATG8 proteins towards autophagy adaptors and receptors