Cells employ active measures to restrict infection by pathogens, even prior to responses from the innate and humoral immune defenses. In this context selective autophagy is activated upon pathogen induced membrane rupture to sequester and deliver membrane fragments and their pathogen contents for lysosomal degradation. Adenoviruses, which breach the endosome upon entry, escape this fate by penetrating into the cytosol prior to autophagosome sequestration of the ruptured endosome. We show that virus induced membrane damage is recognized through Galectin-8 and sequesters the autophagy receptors NDP52 and p62. We further show that a conserved PPxY motif in the viral membrane lytic protein VI is critical for efficient viral evasion of autophagic sequestration after endosomal lysis. Comparing the wildtype with a PPxY-mutant virus we show that depletion of Galectin-8 or suppression of autophagy in ATG5-/- MEFs rescues infectivity of the PPxY-mutant virus while depletion of the autophagy receptors NDP52, p62 has only minor effects. Furthermore we show that wildtype viruses exploit the autophagic machinery for efficient nuclear genome delivery and control autophagosome formation via the cellular ubiquitin ligase Nedd4.2 resulting in reduced antigenic presentation. Our data thus demonstrate that a short PPxY-peptide motif in the adenoviral capsid permits multi-layered viral control of autophagic processes during entry.

Nucleolin is an abundant protein of the nucleolus. Nucleolar proteins structurally related to nucleolin are found in organisms ranging from yeast to plants and mammals. The association of several structural domains in nucleolin allows the interaction of nucleolin with different proteins and RNA sequences. Nucleolin has been implicated in chromatin structure, rDNA transcription, rRNA maturation, ribosome assembly and nucleo-cytoplasmic transport. Studies of nucleolin over the last 25 years have revealed a fascinating role for nucleolin in ribosome biogenesis. The involvement of nucleolin at multiple steps of this biosynthetic pathway suggests that it could play a key role in this highly integrated process.

Aims/hypothesis Glucose and incretins regulate beta cell function, gene expression and insulin exocytosis via calcium and cAMP. Prolonged exposure to elevated glucose (also termed glucotoxicity) disturbs calcium homeostasis, but little is known about cAMP signalling. We therefore investigated long-term effects of glucose on this pathway with special regard to the incretin glucagon-like peptide 1 (GLP-1). Methods We exposed INS-1E cells and rat or human islets to different levels of glucose for 3 days and determined functional responses in terms of second messengers (cAMP, Ca 2+ ), transcription profiles, activation of cAMP-responsive element (CRE) and secretion by measuring membrane capacitance. Moreover, we modulated directly the abundance of a calcium-sensitive adenylyl cyclase (ADCY8) and GLP-1 receptor (GLP1R).Results GLP-1-or forskolin-mediated increases in cytosolic calcium, cAMP-levels or insulin secretion were largely reduced in INS-1E cells cultured at elevated glucose (>5.5 mmol/l). Statistical analysis of transcription profiles identified cAMP pathways as major targets regulated by glucose. Quantitative PCR confirmed these findings and unravelled marked downregulation of the calcium-sensitive adenylyl cyclase ADCY8 also in rat and in human islets. Re-expression of ADCY8, but not of the GLP1R, recovered GLP-1 signalling in glucotoxicity in INS-1E cells and in rat islets. Moreover, knockdown of this adenylyl cyclase showed that GLP-1-induced cAMP generation, calcium signalling, activation of the downstream target CRE and direct amplification of exocytosis by cAMP-raising agents (evaluated by capacitance measurement) proceeds via ADCY8. Conclusions/interpretation cAMP-mediated pathways are modelled by glucose, and downregulation of the calcium-B. Roger and J. Papin contributed equally to this study. Electronic supplementary material The online version of this article

These results provide the first demonstration that the MAP2c and tau MTBD domains exhibit distinct properties, diverging in actin binding and neurite initiation activities. These results implicate a novel actin function for MAP2c in neuronal morphogenesis and furthermore suggest that actin interactions could contribute to functional differences between MAP2 and tau in neurons.

Prolonged exposure of beta-cells to high glucose (glucotoxicity) diminishes insulin secretion in response to glucose and has been linked to altered generation of metabolism-secretion coupling factors. We have investigated whether glucotoxicity may also alter calcium handling and late steps in secretion such as exocytosis. Clonal INS-1E beta-cells cultured at high glucose (20 or 30 mM vs. 5.5 mM) for 72 h exhibited elevated basal intracellular calcium ([Ca2+]i), which was KATP-channel dependent and due to long-term activation of protein kinase A. An increased amplitude and shortened duration of depolarization-evoked rises in [Ca2+]i were apparent. These changes were probably linked to the observed increased filling of intracellular stores and to short-term activation of protein kinase A. Insulin secretion was reduced not only by acute stimulation with either glucose or KCl but more importantly by direct calcium stimulation of permeabilized cells. These findings indicate a defect in the final steps of exocytosis. To confirm this, we measured expression levels of some 30 proteins implicated in trafficking/exocytosis of post-Golgi vesicles. Several proteins required for calcium-induced exocytosis of secretory granules were down-regulated, such as the soluble N-ethylmaleimide-sensitive factor-sensitive factor attachment receptor (SNARE) proteins VAMP-2 [vesicle (v)-SNARE, vesicle-associated membrane protein 2] and syntaxin 1 as well as complexin. VAMP-2 was also reduced in human islets. In contrast, cell immunostaining and expression levels of several fluorescent proteins suggested that other post-trans-Golgi trafficking steps and compartments are preserved and that cells were not degranulated. Thus, these studies indicate that, in addition to known metabolic changes, glucotoxicity impedes generation of signals for secretion and diminishes the efficiency of late steps in exocytosis.

The first processing event of the precursor ribosomal RNA (pre-rRNA) takes place within the 5 external transcribed spacer. This primary processing requires conserved cis-acting RNA sequence downstream from the cleavage site and several nucleic acids (small nucleolar RNAs) and proteins trans-acting factors including nucleolin, a major nucleolar protein. The specific interaction of nucleolin with the pre-rRNA is required for processing in vitro. Xenopus laevis and hamster nucleolin interact with the same pre-rRNA site and stimulate the processing activity of a mouse cell extract. A highly conserved 11-nucleotide sequence located 5-6 nucleotides after the processing site is required for the interaction of nucleolin and processing. In vitro selection experiments with nucleolin have identified an RNA sequence that contains the UCGA motif present in the 11-nucleotide conserved sequence. The interaction of nucleolin with pre-rRNA is required for the formation of an active processing complex. Our findings demonstrate that nucleolin is a key factor for the assembly and maturation of pre-ribosomal ribonucleoparticles.Ribosome biogenesis is a complex process that involves the transcription of a large pre-rRNA 1 precursor, its maturation, and assembly with ribosomal proteins (1, 2). Pre-rRNA undergoes multiple post-transcriptional nucleotide modification (3) and nucleolytic processing steps to yield the mature 18, 5.8, and 28 S rRNA species. The two first endonucleolytic cleavages occur in external transcribed spacers (ETS) and therefore do not lead to the formation of mature rRNA ends. The first cleavage also called early or primary processing occurs within the 5Ј-ETS (4 -6) is conserved from yeast (7) to human (8) and can be found at various positions within the 5Ј-ETS (5, 7-11).Despite its conservation, the role of this cleavage in ribosome biogenesis is still unknown, and only few factors involved in this process have been characterized. In yeast, the role of Rnt1p, an RNase III homologue remains unclear (12, 13). In higher eukaryotes, the major nucleolar protein nucleolin (15), an endonuclease (14) and several small nucleolar RNA are also involved (15, 16), but their exact function remains to be elucidated. In vitro UV cross-linking has identified a small number of proteins, including nucleolin, that interact with the RNA substrate (17, 18). The different factors assemble in a large 20 S complex (18) that could be visualized at the terminal ends of nascent pre-rRNA (terminal balls) observed on Miller's Christmas tree by electron microscopy (19,20). The formation of this complex seems necessary but not sufficient for processing (20,21).The sequence and RNA structural motif required for the processing have been extensively studied in vitro (21,22). In mouse pre-rRNA, an evolutionary conserved 11-nt motif (ϩ655 to ϩ666) located just 5-6 nt downstream from the cleavage site is absolutely required for the processing (21) and for formation of the complex observed at the 5Ј end of nascent pre-rRNA (20). A 27-nt RNA (ϩ645 to ϩ...

Nucleolin is one of the most abundant non-ribosomal proteins of the nucleolus. Several studies in vitro have shown that nucleolin is involved in several steps of ribosome biogenesis, including the regulation of rDNA transcription, rRNA processing, and ribosome assembly. However, the different steps of ribosome biogenesis are highly coordinated, and therefore it is not clear to what extent nucleolin is involved in each of these steps. It has been proposed that the interaction of nucleolin with the rDNA sequence and with nascent pre-rRNA leads to the blocking of RNA polymerase I (RNA pol I) transcription. To test this model and to get molecular insights into the role of nucleolin in RNA pol I transcription, we studied the function of nucleolin in Xenopus oocytes. We show that injection of a 2-4-fold excess of Xenopus or hamster nucleolin in stage VI Xenopus oocytes reduces the accumulation of 40 S pre-rRNA 3-fold, whereas transcription by RNA polymerase II and III is not affected. Direct analysis of rDNA transcription units by electron microscopy reveals that the number of polymerase complexes/ rDNA unit is drastically reduced in the presence of increased amounts of nucleolin and corresponds to the level of reduction of 40 S pre-rRNA. Transcription from DNA templates containing various combinations of RNA polymerase I or II promoters in fusion with rDNA or CAT sequences was analyzed in the presence of elevated amounts of nucleolin. It was shown that nucleolin leads to transcription repression from a minimal polymerase I promoter, independently of the nature of the RNA sequence that is transcribed. Therefore, we propose that nucleolin affects RNA pol I transcription by acting directly on the transcription machinery or on the rDNA promoter sequences and not, as previously thought, through interaction with the nascent pre-rRNA.The synthesis of functional ribosomes is a major task for the cell. Ribosomal gene transcription can account for as much as 40% of all cellular transcription and ribosomal RNA for about 80% of the RNA content of living cells (1). The different steps of ribosome biogenesis take place in a subcompartment of the nucleus called the nucleolus (2-4). The localization of the different steps of ribosome biogenesis in a single nuclear compartment probably allows an efficient coordination and regulation of ribosome assembly. The formation of mature ribosomes is one of the most complex assembly of ribonucleoparticles involving the interaction of four different RNAs and about 80 ribosomal proteins (5). In addition, several nucleolar non-ribosomal proteins are required for this process (6 -8). An ordered interaction of ribosomal and non-ribosomal proteins with pre-rRNA is probably required for the formation of functional ribosomes. The molecular details of this highly integrated process are still largely unknown.The non-ribosomal proteins fibrillarin and nucleolin as well as some ribosomal proteins have been detected on nascent prerRNA (9 -11) suggesting that they interact with the pre-rRNA during transcrip...

Despite the identification of numerous factors involved in ribosomal RNA synthesis and maturation, the molecular mechanisms of ribosome biogenesis, and in particular the relationship between the different steps, are still largely unknown. We have investigated the consequences of an increased amount of a major nucleolar non-ribosomal protein, nucleolin, in Xenopus laevisstage VI oocytes on the production of ribosomal subunits. We show that a threefold increase in nucleolin leads to the complete absence of pre-rRNA maturation in addition to significant repression of RNA polymerase I transcription. Observation of "Christmas trees" by electron microscopy and analysis of the sedimentation properties of 40S pre-ribosomal particles suggest that an increased amount of nucleolin leads to incorrect packaging of the 40S particle. Interestingly, nucleolin affects the maturation of the 40S particle only when it is present at the time of transcription. These results indicate that nucleolin participates in the co-transcriptional packaging of the pre-rRNA, and that the quality of this packaging will determine whether the 40S precursor undergoes maturation or is degraded. The interaction of nucleolin with nascent pre-rRNA could help the co-transcriptional assembly on pre-rRNA of factors necessary for the subsequent maturation of the pre-ribosomal particle containing the 40S pre-rRNA.