Members of the autophagy class III phosphatidylinositol 3-kinase complex I interact with GABARAP and GABARAPL1 via LIR motifs

Autophagosome formation depends on a carefully orchestrated interplay between membraneassociated protein complexes. Initiation of macroautophagy/autophagy is mediated by the ULK1 (unc51 like autophagy activating kinase 1) protein kinase complex and the autophagy-specific class III phosphatidylinositol 3-kinase complex I (PtdIns3K-C1). The latter contains PIK3C3/VPS34, PIK3R4/ VPS15, BECN1/Beclin 1 and ATG14 and phosphorylates phosphatidylinositol to generate phosphatidylinositol 3-phosphate (PtdIns3P). Here, we show that PIK3C3, BECN1 and ATG14 contain functional LIR motifs and interact with the Atg8-family proteins with a preference for GABARAP and GABARAPL1. High resolution crystal structures of the functional LIR motifs of these core components of PtdIns3K-C1were obtained. Variation in hydrophobic pocket 2 (HP2) may explain the specificity for the GABARAP family. Mutation of the LIR motif in ATG14 did not prevent formation of the PtdIns3K-C1 complex, but blocked colocalization with MAP1LC3B/LC3B and impaired mitophagy. The ULK-mediated phosphorylation of S29 in ATG14 was strongly dependent on a functional LIR motif in ATG14. GABARAP-preferring LIR motifs in PIK3C3, BECN1 and ATG14 may, via coincidence detection, contribute to scaffolding of PtdIns3K-C1 on membranes for efficient autophagosome formation

Combination of in silico and in situ hybridisation approaches to identify potential Dll1 associated miRNAs during mouse embryogenesis.

MicroRNAs (miRNAs) have regulatory functions during vertebrate embryogenesis. They are short approximately 21bp long endogenously expressed single-stranded RNAs, which preferentially bind to complementary sequences in the 3' untranslated regions (UTR) of mRNAs and typically down-regulate the respective target mRNAs by translational repression or enhanced mRNA degradation. The Notch ligand Delta-like 1 (Dll1) is expressed in a highly dynamic pattern and has pleiotropic functions during embryogenesis and in adult tissues. Here, we report an interspecies in silico analysis to identify 16 miRNAs, which potentially bind to the mouse, human and chicken Dll1 3'UTRs. To analyze whether these miRNAs could regulate Dll1 gene expression during somitogenesis and neurogenesis, we performed a systematic whole mount in situ hybridisation screen, followed by radioactive in situ hybridisation on sections, using LNA modified DNA probes in mouse embryos. We find that 7 miRNAs (miR-34a, miR-103, miR-107, miR-130a, miR-130b, miR-449a and miR-449c) are expressed in developing somites, limbs, restricted regions of the brain and neural tube between 9.5 dpc and 12.5 dpc. This suggests that these miRNAs could possibly target the Dll1 3'UTR in these regions. The other miRNAs are not expressed or below the detection limit and thus are unlikely to regulate Dll1 at the analyzed embryonic stages

Use of Peptide Arrays for Identification and Characterization of LIR Motifs

The mammalian ATG8 proteins (LC3A-C/GABARAP, GABARAPL1, and GABARAPL2) are small ubiquitin-like proteins critically involved in macroautophagy. Their processed C-termini are posttranslationally conjugated to a phosphatidylethanolamine moiety, enabling their insertion into the lipid bilayers of both the inner and outer membranes of the forming autophagosomes. The ATG8s bind a diverse selection of proteins including cargo receptors for selective autophagy, members of the core autophagy machinery, and other proteins involved in formation, transport, and maturation (fusion to lysosomes) of autophagosomes. Protein binding to the ATG8s is in most cases mediated by short, conserved sequence motifs known as LC3-interacting regions (LIRs). Here, we present a protocol for identifying putative LIR motifs in a whole protein sequence using peptide arrays generated by SPOT synthesis on nitrocellulose membranes. The use of two-dimensional peptide arrays allows for further identification of specific residues critical for LIR binding

Mitochondria at the neuronal presynapse in health and disease

Synapses enable neurons to communicate with each other and are therefore a prerequisite for normal brain function. Presynaptically, this communication requires energy and generates large fluctuations in calcium concentrations. Mitochondria are optimized for supplying energy and buffering calcium, and they are actively recruited to presynapses. However, not all presynapses contain mitochondria; thus, how might synapses with and without mitochondria differ? Mitochondria are also increasingly recognized to serve additional functions at the presynapse. Here, we discuss the importance of presynaptic mitochondria in maintaining neuronal homeostasis and how dysfunctional presynaptic mitochondria might contribute to the development of disease