Expression of a ULK1/2 binding-deficient ATG13 variant can partially restore autophagic activity in ATG13-deficient cells

Autophagy describes an intracellular process responsible for the lysosome-dependent degradation of cytosolic components. The ULK1/2 complex comprising the kinase ULK1/2 and the accessory proteins ATG13, RB1CC1, and ATG101 has been identified as a central player in the autophagy network, and it represents the main entry point for autophagy-regulating kinases such as MTOR and AMPK. It is generally accepted that the ULK1 complex is constitutively assembled independent of nutrient supply. Here we report the characterization of the ATG13 region required for the binding of ULK1/2. This binding site is established by an extremely short peptide motif at the C terminus of ATG13. This motif is mandatory for the recruitment of ULK1 into the autophagy-initiating high-molecular mass complex. Expression of a ULK1/2 binding-deficient ATG13 variant in ATG13-deficient cells resulted in diminished but not completely abolished autophagic activity. Collectively, we propose that autophagy can be executed by mechanisms that are dependent or independent of the ULK1/2-ATG13 interaction.     

Prey-race drives differentiation of biotypes in ant-eating spiders

1.?Disruptive natural selection resulting from specialization on different hosts is recognized as one of the most important driving forces in the diversification of herbivores and parasites. It has been proposed that a similar mechanism could apply to carnivorous predators too, although the evidence is still lacking. 2.?Here, we show that the differentiation of biotypes of specialized ant-eating spiders of the genus Zodarion has probably been induced by prey-shifting. We focused on two forms of one species Z.?styliferum from the Iberian Peninsula that presumably represent ecological races. We conducted geographic, ecological, venom-oriented, reproductive and genetic divergence analysis among multiple populations collected at a number of sites across Portugal and Madeira. 3.?Geographic analysis revealed that the two forms occur in mosaic sympatry. Each form was found to associate in nature with a different ant species in a different habitat. Specifically, the styliferum form hunted predominantly Messor ants, and the extraneum form hunted mainly Camponotus ants. Laboratory experiments revealed that the two forms exhibit a significant preference for attacking focal ants, demonstrating higher paralysis efficiency, and also show different venom composition. Cross-mating of the two forms was significantly less likely than between pairs of the same form, suggesting moderate assortative mating. Phylogenetic analyses indicate low genetic differentiation of the two forms and parallel-repeated evolution of biotypes. 4.?Adaptive prey-shifting correlated with habitat preference are at present the most valid explanations for biotype formation in Zodarion. The speciation of ant-eating Zodarion spiders thus appears to follow a scenario similar to that of host-shifting in parasites and herbivores.     

Artificial ubiquitylation is sufficient for sorting of a plasma membrane ATPase to the vacuolar lumen of Arabidopsis cells

Sorting of transmembrane proteins into the inner vesicles of multivesicular bodies for subsequent delivery to the vacuole/lysosome can be induced by attachment of a single ubiquitin or K63-linked ubiquitin chains to the cytosolic portion of the cargo in yeast and mammals. In plants, large efforts have been undertaken to elucidate the mechanisms of vacuolar trafficking of soluble proteins. Sorting of transmembrane proteins, by contrast, is still largely unexplored. As a proof of principle, that ubiquitin is involved in vacuolar sorting in plants we show that a translational fusion of a single ubiquitin to the Arabidopsis plasma membrane ATPase PMA-EGFP is sufficient to induce its endocytosis and sorting into the vacuolar lumen. Sorting of the artificial reporter is not dependent on ubiquitin chain formation, but involves ubiquitin's hydrophobic patch and can be inhibited by coexpression of a dominant-negative version of the ESCRT (endosomal sorting complex required for transport) related protein AtSKD1 (SUPPRESSOR OF K+ TRANSPORT GROWTH DEFECT1). Our results suggest that ubiquitin can in principle act as vacuolar sorting signal in plants.     

Margareta Ryberg (1946–2012): a personal tribute

We pay tribute to the life and work of Margareta Ryberg (1946-2012). She was an expert on the different forms of protochlorophyll(ide), their protein partners, and their transformations in angiosperms; on the structural aspects, and the nature of prolamellar bodies, as well as on the localization of light-dependent NADPH:protochlorophyllide oxido-reductase. She was a great teacher, who also loved gardening and handicraft. But above all, she was a beloved wife, mother, grandmother, and friend who will be deeply missed.     

Trypsin promotes efficient influenza vaccine production in MDCK cells by interfering with the antiviral host response

Trypsin is commonly used in Madin–Darby canine kidney (MDCK) cell culture-based influenza vaccine production to facilitate virus infection by proteolytic activation of viral haemagglutinin, which enables multi-cycle replication. In this study, we were able to demonstrate that trypsin also interferes with pathogen defence mechanisms of host cells. In particular, a trypsin concentration of 5 BAEE U/mL (4.5 μg/mL porcine trypsin) used in vaccine manufacturing strongly inhibited interferon (IFN) signalling by proteolytic degradation of secreted IFN. Consequently, absence of trypsin during infection resulted in a considerably stronger induction of IFN signalling and apoptosis, which significantly reduced virus yields. Under this condition, multi-cycle virus replication in MDCK cells was not prevented but clearly delayed. Therefore, incomplete infection can be ruled out as the reason for the lower virus titres. However, suppression of IFN signalling by overexpression of viral IFN antagonists (influenza virus PR8-NS1, rabies virus phosphoprotein) partially rescued virus titres in the absence of trypsin. In addition, virus yields could be almost restored by using the influenza strain A/WSN/33 in combination with fetal calf serum (FCS). For this strain, FCS enabled trypsin-independent fast propagation of virus infection, probably outrunning cellular defence mechanisms and apoptosis induction in the absence of trypsin. Overall, addition of trypsin provided optimal conditions for high yield vaccine production in MDCK cells by two means. On the one hand, proteolytic degradation of IFN keeps cellular defence at a low level. On the other hand, enhanced virus spreading enables viruses to replicate before the cellular response becomes fully activated.     

Compartmentalization and regulation of iron metabolism proteins protect male germ cells from iron overload

The universal importance of iron, its high toxicity, and complex chemistry present a challenge to biological systems in general and to protected compartments in particular. The high mitotic rate and avid mitochondriogenesis of developing male germ cells imply high iron requirements. Yet access to germ cells is tightly regulated by the blood-testis barrier that protects the meiotic and postmeiotic germ cells. To elucidate how iron is supplied to developing male germ cells, we analyzed iron deposition and iron transport proteins in testes of mice with iron overload and with genetic ablation of the iron regulators Hfe and iron regulatory protein 2. Iron accumulated mainly around seminiferous tubules, and only small amounts localized within the seminiferous tubules. The localization and regulation of proteins involved in iron import, storage, and export such as transferrin, transferrin receptor, the divalent metal transporter-1, cytosolic ferritin, and ferroportin strongly support a model of a largely autonomous iron cycle within seminiferous tubules. We show evidence that ferritin secretion from Sertoli cells may play an important role in iron acquisition of primary spermatocytes. During spermatogenic development iron is carried along from primary spermatocytes to spermatids, and from spermatids iron is recycled to the apical compartment of Sertoli cells, which traffic it back to a new generation of spermatocytes. Losses are replenished by the peripheral circulation. Such an internal iron cycle essentially detaches the iron homeostasis within the seminiferous tubule from the periphery and protects developing germ cells from iron fluctuations. This model explains how compartmentalization can optimize cellular and systemic nutrient homeostasis.     

Distinct priming kinases contribute to differential regulation of collapsin response mediator proteins by glycogen synthase kinase-3 in vivo

Collapsin response mediator proteins (CRMPs) are a family of neuron-enriched proteins that regulate neurite outgrowth and growth cone dynamics. Here, we show that Cdk5 phosphorylates CRMP1, CRMP2, and CRMP4, priming for subsequent phosphorylation by GSK3 in vitro. In contrast, DYRK2 phosphorylates and primes CRMP4 only. The Cdk5 and DYRK2 inhibitor purvalanol decreases the phosphorylation of CRMP proteins in neurons, whereas CRMP1 and CRMP2, but not CRMP4, phosphorylation is decreased in Cdk5(-/-) cortices. Stimulation of neuroblastoma cells with IGF1 or TPA decreases GSK3 activity concomitantly with CRMP2 and CRMP4 phosphorylation. Conversely, increased GSK3 activity is not sufficient to increase CRMP phosphorylation. However, the growth cone collapse-inducing protein Sema3A increases Cdk5 activity and promotes phosphorylation of CRMP2 (but not CRMP4). Therefore, inhibition of GSK3 alters phosphorylation of all CRMP isoforms; however, individual isoforms can be differentially regulated by their respective priming kinase. This is the first GSK3 substrate found to be regulated in this manner and may explain the hyperphosphorylation of CRMP2 observed in Alzheimer's disease.     

Histone hyperacetylation is associated with amelioration of experimental colitis in mice

Inhibitors of histone deacetylases (HDAC) are being studied for their antiproliferative effects in preclinical cancer trials. Recent studies suggest an anti-inflammatory role for this class of compounds. Because inflammatory bowel disease is associated with an increased risk of malignancies, agents with antiproliferative and anti-inflammatory properties would be of therapeutic interest. HDAC inhibitors from various classes were selected and evaluated for their in vitro capacity to suppress cytokine production and to induce apoptosis and histone acetylation. Valproic acid (VPA) and suberyolanilide hydroxamic acid (SAHA) were chosen for further studies in dextran sulfate sodium- and trinitrobenzene sulfonic acid-induced colitis in mice. In vitro, inhibition of HDAC resulted in a dose-dependent suppression of cytokine synthesis and apoptosis induction requiring higher concentrations of HDAC inhibitors for apoptosis induction compared with cytokine inhibition. Oral administration of either VPA or SAHA reduced disease severity in dextran sulfate sodium-induced colitis. The macroscopic and histologic reduction of disease severity was associated with a marked suppression of colonic proinflammatory cytokines. In parallel to the beneficial effect observed, a dose-dependent increase in histone 3 acetylation at the site of inflammation was shown under VPA treatment. Furthermore, SAHA as well as VPA treatment resulted in amelioration of trinitrobenzene sulfonic acid-induced colitis, which was associated with an increase of apoptosis of lamina propria lymphocytes. Inhibitors of HDAC reveal strong protective effects in different models of experimental colitis by inducing apoptosis and suppressing proinflammatory cytokines, thereby representing a promising class of compounds for clinical studies in human inflammatory bowel disease.     

Immunoproteasomes are essential for clearance of Listeria monocytogenes in nonlymphoid tissues but not for induction of bacteria-specific CD8+ T cells

Microbial infections induce the replacement of constitutive proteasomes by immunoproteasomes (I-proteasomes). I-proteasomes support efficient generation of MHC class I epitopes and influence immunodominance hierarchies of CD8(+) T cells. Recently, the function of I-proteasomes in antimicrobial responses was challenged by showing that the lack of I-proteasomes has no effect on induction and function of lymphocytic choriomeningitis virus-specific CD8(+) T cells. Here, we show that infection with Listeria monocytogenes rapidly induces I-proteasomes in nonlymphoid tissues, which leads to enhanced generation of protection relevant CD8(+) T cell epitopes. I-proteasome-deficient mice (beta5i(-/-) mice) exhibited normal frequencies of L. monocytogenes-specific CD8(+) T cells. However, clearance of L. monocytogenes in liver but not spleen was significantly impaired in I-proteasome-deficient mice. In summary, our studies demonstrate that induction of I-proteasomes is required for CD8(+) T cell-mediated elimination of L. monocytogenes from nonlymphoid but not lymphoid tissues.     

Ammonium Toxicity in Bacteria

Although an excellent nitrogen source for most bacteria, ammonium was—in analogy to plant and animal systems—assumed be detrimental to bacteria when present in high concentrations. In this study, we examined the effect of molar ammonium concentrations on different model bacteria, namely, Corynebacterium glutamicum, Escherichia coli, and Bacillus subtilis. The studied bacteria are highly resistant to ammonium. When growth was impaired upon addition of molar (NH₄)₂SO₄ concentrations, this was not caused by an ammonium-specific effect but was due to an enhanced osmolarity or increased ionic strength of the medium. Therefore, it was concluded that ammonium is not detrimental to C. glutamicum and other bacteria even when present in molar concentrations.