The Warburg Micro Syndrome-associated Rab3GAP-Rab18 module promotes autolysosome maturation through the Vps34 Complex I.

Warburg micro syndrome (WMS) is a hereditary autosomal neuromuscular disorder in humans caused by mutations in Rab18, Rab3GAP1, or Rab3GAP2 genes. Rab3GAP1/2 forms a heterodimeric complex, which acts as a guanosine nucleotide exchange factor and activates Rab18. Although the genetic causes of WMS are known, it is still unclear whether loss of the Rab3GAP-Rab18 module affects neuronal or muscle cell physiology or both, and how. In this work, we characterize a Rab3GAP2 mutant Drosophila line to establish a novel animal model for WMS. Similarly to symptoms of WMS, loss of Rab3GAP2 leads to highly decreased motility in Drosophila that becomes more serious with age. We demonstrate that these mutant flies are defective for autophagic degradation in multiple tissues including fat cells and muscles. Loss of Rab3GAP-Rab18 module members leads to perturbed autolysosome morphology due to destabilization of Rab7-positive autophagosomal and late endosomal compartments and perturbation of lysosomal biosynthetic transport. Importantly, overexpression of UVRAG or loss of Atg14, two alternative subunits of the Vps34/PI3K (vacuole protein sorting 34/phosphatidylinositol 3-kinase) complexes in fat cells, mimics the autophagic phenotype of Rab3GAP-Rab18 module loss. We find that GTP-bound Rab18 binds to Atg6/Beclin1, a permanent subunit of Vps34 complexes. Finally, we show that Rab3GAP2 and Rab18 are present on autophagosomal and autolysosomal membranes and colocalize with Vps34 Complex I subunits. Our data suggest that the Rab3GAP-Rab18 module regulates autolysosomal maturation through its interaction with the Vps34 Complex I, and perturbed autophagy due to loss of the Rab3GAP-Rab18 module may contribute to the development of WMS.

Sex-specific regulation of aging in Caenorhabditis elegans.

A fascinating aspect of sexual dimorphism in various animal species is that the two sexes differ substantially in lifespan. In humans, for example, women's life expectancy exceeds that of men by 3-7 years. Whether this trait can be attributed to dissimilar lifestyles or genetic (regulatory) factors remains to be elucidated. Herein, we demonstrate that in the nematode Caenorhabditis elegans, the significantly longer lifespan of hermaphrodites-which are essentially females capable of sperm production-over males is established by TRA-1, the terminal effector of the sex-determination pathway. This transcription factor directly controls the expression of daf-16/FOXO, which functions as a major target of insulin/IGF-1 signaling (IIS) and key modulator of aging across diverse animal phyla. TRA-1 extends hermaphrodite lifespan through promoting daf-16 activity. Furthermore, TRA-1 also influences reproductive growth in a DAF-16-dependent manner. Thus, the sex-determination machinery is an important regulator of IIS in this organism. These findings provide a mechanistic insight into how longevity and development are specified unequally in the two genders. As TRA-1 is orthologous to mammalian GLI (glioma-associated) proteins, a similar sex-specific mechanism may also operate in humans to determine lifespan.

Drosophila Atg16 promotes enteroendocrine cell differentiation via regulation of intestinal Slit/Robo signaling.

Genetic variations of Atg16l1, Slit2 and Rab19 predispose to the development of inflammatory bowel disease (IBD), but the relationship between these mutations is unclear. Here we show that in Drosophila guts lacking the WD40 domain of Atg16, pre-enteroendocrine (pre-EE) cells accumulate that fail to differentiate into properly functioning secretory EE cells. Mechanistically, loss of Atg16 or its binding partner Rab19 impairs Slit production, which normally inhibits EE cell generation by activating Robo signaling in stem cells. Importantly, loss of Atg16 or decreased Slit/Robo signaling triggers an intestinal inflammatory response. Surprisingly, analysis of Rab19 and domain-specific Atg16 mutants indicates that their stem cell niche regulatory function is independent of autophagy. Our study reveals how mutations in these different genes may contribute to IBD.

Autophagy-from molecular mechanisms to clinical relevance.

Autophagy is a lysosomal degradation pathway of eukaryotic cells that is highly conserved from yeast to mammals. During this process, cooperating protein complexes are recruited in a hierarchic order to the phagophore assembly site (PAS) to mediate the elongation and closure of double-membrane vesicles called autophagosomes, which sequester cytosolic components and deliver their content to the endolysosomal system for degradation. As a major cytoprotective mechanism, autophagy plays a key role in the stress response against nutrient starvation, hypoxia, and infections. Although numerous studies reported that impaired function of core autophagy proteins also contributes to the development and progression of various human diseases such as neurodegenerative disorders, cardiovascular and muscle diseases, infections, and different types of cancer, the function of this process in human diseases remains unclear. Evidence often suggests a controversial role for autophagy in the pathomechanisms of these severe disorders. Here, we provide an overview of the molecular mechanisms of autophagy and summarize the recent advances on its function in human health and disease.

MiniCORVET is a Vps8-containing early endosomal tether in Drosophila.

Yeast studies identified two heterohexameric tethering complexes, which consist of 4 shared (Vps11, Vps16, Vps18 and Vps33) and 2 specific subunits: Vps3 and Vps8 (CORVET) versus Vps39 and Vps41 (HOPS). CORVET is an early and HOPS is a late endosomal tether. The function of HOPS is well known in animal cells, while CORVET is poorly characterized. Here we show that Drosophila Vps8 is highly expressed in hemocytes and nephrocytes, and localizes to early endosomes despite the lack of a clear Vps3 homolog. We find that Vps8 forms a complex and acts together with Vps16A, Dor/Vps18 and Car/Vps33A, and loss of any of these proteins leads to fragmentation of endosomes. Surprisingly, Vps11 deletion causes enlargement of endosomes, similar to loss of the HOPS-specific subunits Vps39 and Lt/Vps41. We thus identify a 4 subunit-containing miniCORVET complex as an unconventional early endosomal tether in Drosophila.

[Role of the stroma in the initiation and progression of tumors]. / A stroma szerepe a tumorok kialakulásában és progressziójában.

In the last decade, growing attention was paid to the observation that tumors did not only consist of cancer cells, they are rather a complex tissue-like mixture of tumor and stromal cells, which are playing an important role in the course of the malignant disease. Their contribution is so essential that without them, tumors are not even able to grow. This short review summarizes how stromal cells can promote the cancerous transformation and early development of tumors, how chronic inflammation contributes to the progression of cancer and how the stroma takes part in the induction of angiogenesis. The main mechanisms by which tumors can escape the immune surveillance will be demonstrated as well as the complex contributions of stroma to the invasion, intravasation and metastasis of cancer cells. Finally, possible and promising therapies will be presented that aim at the stroma and its main effects on the progression of tumors.

Specialized Cortex Glial Cells Accumulate Lipid Droplets in Drosophila melanogaster.

Lipid droplets (LDs) are common organelles of the majority of eukaryotic cell types. Their biological significance has been extensively studied in mammalian liver cells and white adipose tissue. Although the central nervous system contains the highest relative amount and the largest number of different lipid species, neither the spatial nor the temporal distribution of LDs has been described. In this study, we used the brain of the fruitfly, Drosophila melanogaster, to investigate the neuroanatomy of LDs. We demonstrated that LDs are exclusively localised in glial cells but not in neurons in the larval nervous system. We showed that the brain's LD pool, rather than being constant, changes dynamically during development and reaches its highest value at the beginning of metamorphosis. LDs are particularly enriched in cortex glial cells located close to the brain surface. These specialized superficial cortex glial cells contain the highest amount of LDs among glial cell types and encapsulate neuroblasts and their daughter cells. Superficial cortex glial cells, combined with subperineurial glial cells, express the Drosophila fatty acid binding protein (Dfabp), as we have demonstrated through light- and electron microscopic immunocytochemistry. To the best of our best knowledge this is the first study that describes LD neuroanatomy in the Drosophila larval brain.

The role of the selective adaptor p62 and ubiquitin-like proteins in autophagy.

The ubiquitin-proteasome system and autophagy were long viewed as independent, parallel degradation systems with no point of intersection. By now we know that these degradation pathways share certain substrates and regulatory molecules and show coordinated and compensatory function. Two ubiquitin-like protein conjugation pathways were discovered that are required for autophagosome biogenesis: the Atg12-Atg5-Atg16 and Atg8 systems. Autophagy has been considered to be essentially a nonselective process, but it turned out to be at least partially selective. Selective substrates of autophagy include damaged mitochondria, intracellular pathogens, and even a subset of cytosolic proteins with the help of ubiquitin-binding autophagic adaptors, such as p62/SQSTM1, NBR1, NDP52, and Optineurin. These proteins selectively recognize autophagic cargo and mediate its engulfment into autophagosomes by binding to the small ubiquitin-like modifiers that belong to the Atg8/LC3 family.

Rab11 facilitates cross-talk between autophagy and endosomal pathway through regulation of Hook localization.

During autophagy, double-membrane autophagosomes deliver sequestered cytoplasmic content to late endosomes and lysosomes for degradation. The molecular mechanism of autophagosome maturation is still poorly characterized. The small GTPase Rab11 regulates endosomal traffic and is thought to function at the level of recycling endosomes. We show that loss of Rab11 leads to accumulation of autophagosomes and late endosomes in Drosophila melanogaster. Rab11 translocates from recycling endosomes to autophagosomes in response to autophagy induction and physically interacts with Hook, a negative regulator of endosome maturation. Hook anchors endosomes to microtubules, and we show that Rab11 facilitates the fusion of endosomes and autophagosomes by removing Hook from mature late endosomes and inhibiting its homodimerization. Thus induction of autophagy appears to promote autophagic flux by increased convergence with the endosomal pathway.

A novel role for the Drosophila epsin (lqf): involvement in autophagy.

Screening P-element-induced mutant collections, 52 lines were selected as potentially defected ones in endocytosis or autophagy. After excluding those which were rescued by 20-hydroxyecdysone treatment, the exact position of the inserted P-element was determined in the remaining lines. In the case of l(3)S011027 stock, the liquid facets (lqf) gene was affected which codes an epsin-homolog protein in Drosophila. We reveal that Lqf is essential to the receptor-mediated endocytosis of larval serum proteins (LSPs) in the larval fat body cells of Drosophila. In l(3)S011027 line, lack of Lqf fails the formation of autophagosomes thus leading to the arrest of destroying of trophocytes. Transgenic larvae carrying Lqf-RNAi construct were unable to generate endocytic and autophagic vacuoles and led to a prolonged larval stage. On the other hand, Lqf protein showed an exclusive colocalization with the LysoTracker Red- or GFP-Atg8a labeled autophagosomes. By using the antiserum generated against the fifth exon of lqf, we demonstrated that prior to the onset of developmental autophagy the Lqf protein was present in the nucleus of fat body cell, but thereafter the protein was localized in the territory of endocytic and autophagic vacuoles. The fact that the inhibition of the target of rapamycin (TOR) did not restore the autophagic process and the normal development in the case of lqf mutant larvae points to that the Lqf is downstream to the TOR, the central kinase of the autophagy pathway.

SNF4Agamma, the Drosophila AMPK gamma subunit is required for regulation of developmental and stress-induced autophagy.

In holometabolous insects including Drosophila melanogaster a wave of autophagy triggered by 20-hydroxyecdysone is observed in the larval tissues during the third larval stage of metamorphosis. We used this model system to study the genetic regulation of autophagy. We performed a genetic screen to select P-element insertions that affect autophagy in the larval fat body. Light and electron microscopy of one of the isolated mutants (l(3)S005042) revealed the absence of autophagic vesicles in their fat body cells during the third larval stage. We show that formation of autophagic vesicles cannot be induced by 20-hydroxyecdysone in the tissues of mutant flies and represent evidence demonstrating that the failure to form autophagic vesicles is due to the insertion of a P-element into the gene coding SNF4Agamma, the Drosophila homologue of the AMPK (AMP-activated protein kinase) gamma subunit. The ability to form autophagic vesicles (wild-type phenotype) can be restored by remobilization of the P-element in the mutant. Silencing of SNF4Agamma by RNAi suppresses autophagic vesicle formation in wild-type flies. We raised an antibody against SNF4Agamma and showed that this gene product is constitutively present in the wild-type larval tissues during postembryonal development. SNF4Agamma is nearly absent from the cells of homozygous mutants. SNF4Agamma translocates into the nuclei of fat body cells at the onset of the wandering stage concurrently with the beginning of the autophagic process. Our results demonstrate that SNF4Agamma has an essential role in the regulation of autophagy in Drosophila larval fat body cells.