The two Dictyostelium discoideum autophagy 8 proteins have distinct autophagic functions.

Autophagy is a highly conserved cellular degradation pathway which is crucial for various cellular processes. The autophagic process is subdivided in the initiation, autophagosome maturation and lysosomal degradation phases and involves more than forty core and accessory autophagy-related (ATG) proteins. Autophagy 8 (ATG8, in mammals LC3) is a well-established marker of autophagy and is linked to the autophagic membrane from initiation until fusion with the lysosome. We generated single and double knock-out mutants of the two Dictyostelium paralogues, ATG8a and ATG8b, as well as strains that expressed RFP-ATG8a and/or GFP-ATG8b, RFP-ATG8b, RFP-GFP-ATG8a or RFP-GFP-ATG8b in different knock-out mutants. The ATG8b¯ mutant displayed only subtle phenotypic changes in comparison to AX2 wild-type cells. In contrast, deletion of ATG8a resulted in a complex phenotype with delayed development, reduced growth, phagocytosis and cell viability, an increase in ubiquitinylated proteins and a concomitant decrease in proteasomal activity. The phenotype of the ATG8a¯/b¯ strain was, except for cell viability, in all aforementioned aspects more severe, showing that both proteins function in parallel during most analysed cellular processes. Immunofluorescence analysis of knock-out strains expressing either RFP-GFP-ATG8a or RFP-GFP-ATG8b suggests a crucial function for ATG8b in autophagosome-lysosome fusion. Quantitative analysis of strains expressing RFP-ATG8a, RFP-ATG8b, or RFP-ATG8a and GFP-ATG8b revealed that ATG8b generally localised to small and large vesicles, whereas ATG8a preferentially co-localised with ATG8b on large vesicles, indicating that ATG8b associated with nascent autophagosomes before ATG8a, which is supported by previous results (Matthias et al., 2016). Deconvoluted confocal fluorescence images showed that ATG8b localised around ATG8a and was presumably mainly present on the outer membrane of the autophagosome while ATG8a appears to be mainly associated with the inner membrane. In summary, our data show that ATG8a and ATG8b have distinct functions and are involved in canonical as well as non-canonical autophagy. The data further suggest that ATG8b predominantly acts as adapter for the autophagy machinery at the outer and ATG8a as cargo receptor at the inner membrane of the autophagosome.

The C-Terminal SynMuv/DdDUF926 Domain Regulates the Function of the N-Terminal Domain of DdNKAP.

NKAP (NF-κB activating protein) is a highly conserved SR (serine/arginine-rich) protein involved in transcriptional control and splicing in mammals. We identified DdNKAP, the Dictyostelium discoideum ortholog of mammalian NKAP, as interacting partner of the nuclear envelope protein SUN-1. DdNKAP harbors a number of basic RDR/RDRS repeats in its N-terminal domain and the SynMuv/DUF926 domain at its C-terminus. We describe a novel and direct interaction between DdNKAP and Prp19 (Pre mRNA processing factor 19) which might be relevant for the observed DdNKAP ubiquitination. Genome wide analysis using cross-linking immunoprecipitation-high-throughput sequencing (CLIP-seq) revealed DdNKAP association with intergenic regions, exons, introns and non-coding RNAs. Ectopic expression of DdNKAP and its domains affects several developmental aspects like stream formation, aggregation, and chemotaxis. We conclude that DdNKAP is a multifunctional protein, which might influence Dictyostelium development through its interaction with RNA and RNA binding proteins. Mutants overexpressing full length DdNKAP and the N-terminal domain alone (DdN-NKAP) showed opposite phenotypes in development and opposite expression profiles of several genes and rRNAs. The observed interaction between DdN-NKAP and the DdDUF926 domain indicates that the DdDUF926 domain acts as negative regulator of the N-terminus.

The two Dictyostelium autophagy eight proteins, ATG8a and ATG8b, associate with the autophagosome in succession.

Autophagy is an ancient cellular pathway that is conserved from yeast to man. It contributes to many physiological and pathological processes and plays a major role in the degradation of proteins and/or organelles in response to starvation and stress. In the autophagic process cytosolic material is captured into double membrane-bound vesicles, the autophagosomes. After fusion with lysosomes, the cargo is degraded in the generated autolysosomes and then recycled for further use. Autophagy 8 (ATG8, in mammals LC3), a well-established marker of autophagy, is covalently linked to phosphatidylethanolamine on the autophagic membrane during autophagosome formation. Bioinformatic analysis of the Dictyostelium genome revealed two atg8 genes which encode the ATG8a and ATG8b paralogs. They are with around 14kDa similar in size, 54 % identical to one another and more closely related to the corresponding proteins in fungi and plants than in animals. For ATG8a we found a strong up-regulation throughout the 24h developmental time course while ATG8b expression was highest in vegetative cells followed by a moderate reduction during early development. Confocal microscopy of fluorescently tagged ATG8a and ATG8b in vegetative AX2 wild-type and in ATG9(-) cells showed that both proteins mainly co-localized on vesicular structures with a diameter above 500nm while those smaller than 500nm were predominantly positive for ATG8b. In ATG9(-) cells we found a strong increase in the relative abundance of ATG8a-positive large vesicular structures and of total ATG8b-positive structures per cell indicating autophagic flux problems in this mutant. We also found that vesicular structures positive for ATG8a and/or ATG8b were also positive for ubiquitin. Live cell imaging of AX2 and ATG9(-) cells co-expressing combinations of red and green tagged ATG8a, ATG8b or ATG9 revealed transient co localizations of these proteins. Our results suggest that ATG8b associates with nascent autophagosomes before ATG8a. We further find that the process of autophagosome formation in Dictyostelium is highly dynamic. We infer from our data that Dictyostelium ATG8a and ATG8b have distinct functions in autophagosome formation and that ATG8b is the functional orthologue of the mammalian LC3 subfamily and ATG8a of the GABARAP subfamily.

Inter-kingdom Signaling by the Legionella Quorum Sensing Molecule LAI-1 Modulates Cell Migration through an IQGAP1-Cdc42-ARHGEF9-Dependent Pathway.

Small molecule signaling promotes the communication between bacteria as well as between bacteria and eukaryotes. The opportunistic pathogenic bacterium Legionella pneumophila employs LAI-1 (3-hydroxypentadecane-4-one) for bacterial cell-cell communication. LAI-1 is produced and detected by the Lqs (Legionella quorum sensing) system, which regulates a variety of processes including natural competence for DNA uptake and pathogen-host cell interactions. In this study, we analyze the role of LAI-1 in inter-kingdom signaling. L. pneumophila lacking the autoinducer synthase LqsA no longer impeded the migration of infected cells, and the defect was complemented by plasmid-borne lqsA. Synthetic LAI-1 dose-dependently inhibited cell migration, without affecting bacterial uptake or cytotoxicity. The forward migration index but not the velocity of LAI-1-treated cells was reduced, and the cell cytoskeleton appeared destabilized. LAI-1-dependent inhibition of cell migration involved the scaffold protein IQGAP1, the small GTPase Cdc42 as well as the Cdc42-specific guanine nucleotide exchange factor ARHGEF9, but not other modulators of Cdc42, or RhoA, Rac1 or Ran GTPase. Upon treatment with LAI-1, Cdc42 was inactivated and IQGAP1 redistributed to the cell cortex regardless of whether Cdc42 was present or not. Furthermore, LAI-1 reversed the inhibition of cell migration by L. pneumophila, suggesting that the compound and the bacteria antagonistically target host signaling pathway(s). Collectively, the results indicate that the L. pneumophila quorum sensing compound LAI-1 modulates migration of eukaryotic cells through a signaling pathway involving IQGAP1, Cdc42 and ARHGEF9.

VCP and PSMF1: Antagonistic regulators of proteasome activity.

Protein turnover and quality control by the proteasome is of paramount importance for cell homeostasis. Dysfunction of the proteasome is associated with aging processes and human diseases such as neurodegeneration, cardiomyopathy, and cancer. The regulation, i.e. activation and inhibition of this fundamentally important protein degradation system, is still widely unexplored. We demonstrate here that the evolutionarily highly conserved type II triple-A ATPase VCP and the proteasome inhibitor PSMF1/PI31 interact directly, and antagonistically regulate proteasomal activity. Our data provide novel insights into the regulation of proteasomal activity.

The phenotypes of ATG9, ATG16 and ATG9/16 knock-out mutants imply autophagy-dependent and -independent functions.

Macroautophagy is a highly conserved intracellular bulk degradation system of all eukaryotic cells. It is governed by a large number of autophagy proteins (ATGs) and is crucial for many cellular processes. Here, we describe the phenotypes of Dictyostelium discoideum ATG16(-) and ATG9(-)/16(-) cells and compare them to the previously reported ATG9(-) mutant. ATG16 deficiency caused an increase in the expression of several core autophagy genes, among them atg9 and the two atg8 paralogues. The single and double ATG9 and ATG16 knock-out mutants had complex phenotypes and displayed severe and comparable defects in pinocytosis and phagocytosis. Uptake of Legionella pneumophila was reduced. In addition, ATG9(-) and ATG16(-) cells had dramatic defects in autophagy, development and proteasomal activity which were much more severe in the ATG9(-)/16(-) double mutant. Mutant cells showed an increase in poly-ubiquitinated proteins and contained large ubiquitin-positive protein aggregates which partially co-localized with ATG16-GFP in ATG9(-)/16(-) cells. The more severe autophagic, developmental and proteasomal phenotypes of ATG9(-)/16(-) cells imply that ATG9 and ATG16 probably function in parallel in autophagy and have in addition autophagy-independent functions in further cellular processes.

Salmonella typhimurium is pathogenic for Dictyostelium cells and subverts the starvation response.

In unicellular amoebae, such as Dictyostelium discoideum, bacterial phagocytosis is a food hunting device, while in higher organisms it is the first defence barrier against microbial infection. In both cases, pathogenic bacteria exploit phagocytosis to enter the cell and multiply intracellularly. Salmonella typhimurium, the agent of food-borne gastroenteritis, is phagocytosed by both macrophages and Dictyostelium cells. By using cell biological assays and global transcriptional analysis with DNA microarrays covering the Dictyostelium genome, we show here that S. typhimurium is pathogenic for Dictyostelium cells. Depending on the degree of virulence, which in turn depended on bacterial growth conditions, Salmonella could kill Dictyostelium cells or inhibit their growth and development. In the early phase of infection in non-nutrient buffer, the ingested bacteria escaped degradation, induced a starvation-like transcriptional response but inhibited selectively genes required for chemotaxis and aggregation. This way differentiation of the host cells into spore and stalk cells was blocked or delayed, which in turn is likely to be favourable for the establishment of a replicative niche for Salmonella. Inhibition of the aggregation competence and chemotactic streaming of aggregation-competent cells in the presence of Salmonella suggests interference with cAMP signalling.