Ultrastructural reinvestigation of the trophosome in adults of Riftia pachyptila (Annelida, Siboglinidae)

Abstract. The trophosome of adults of Riftia pachyptila (Vestimentifera) was reinvestigated using 3-dimensional ultrastructural reconstruction and quantitative morphological analysis. The symbionts make up 24.1%, the symbiont-containing cells (bacteriocytes) are 70.5% of the trophosome's volume. The trophosome is composed of lobules that have a central axial blood vessel surrounded by a myoepithelium containing bacteriocytes, in turn surrounded by an apolar tissue of bacteriocytes. Part of the splanchnic peritoneum lining the trunk coelom encases the bacteriocytes and forms a ramifying network of peripheral blood vessels. Based on the morphology and ultrastructure of the adult, we hypothesize a mesodermal rather than endodermal origin of trophosome and its constitute bacteriocytes. Some of the central bacteriocytes are part of the myoepithelium surrounding the axial blood vessel and act as stem cells for a proliferating tissue produced in the center and ultimately degraded at the periphery of each lobule. Similarly, the rod-shaped symbionts in the center act as stem cells and exhibit a simple cell cycle. Differentiation into cocci takes place in the median and peripheral zone. Lysis of cocci occurs in the degenerative zone.     

Microarray phenotyping places cyclase associated protein CAP at the crossroad of signaling pathways reorganizing the actin cytoskeleton in Dictyostelium

Large-scale gene expression analysis has been applied recently to uncover groups of genes that are co-regulated in particular processes. Here we undertake such an analysis on CAP, a protein that participates in the regulation of the actin cytoskeleton and in cAMP signaling in Dictyostelium. microarray analysis revealed that loss of CAP altered the expression of many cytoskeletal components. One of these, the Rho GDP-dissociation inhibitor RhoGDI1, was analyzed further. RhoGDI1 null cells expressed lower amounts of CAP, which failed to accumulate predominantly at the cell cortex. To further position CAP in the corresponding signal transduction pathways we studied CAP localization and cellular functioning in mutants that have defects in several signaling components. CAP showed correct localization and dynamics in all analyzed strains except in mutants with deficient cAMP dependent protein kinase A activity, where CAP preferentially accumulated in crown shaped structures. Ectopic expression of CAP improved the efficiency of phagocytosis in Gβ-deficient cells and restored the pinocytosis, morphology and actin distribution defects in a PI3 kinase double mutant (pi3k1/2 null). Our results show that CAP acts at multiple crossroads and links signaling pathways to the actin cytoskeleton either by physical interaction with cytoskeletal components or through regulation of their gene expression.     

Reference map for liquid chromatography–mass spectrometry-based quantitative proteomics

The accurate mass and time (AMT) tag strategy has been recognized as a powerful tool for high-throughput analysis in liquid chromatography–mass spectrometry (LC–MS)-based proteomics. Due to the complexity of the human proteome, this strategy requires highly accurate mass measurements for confident identifications. We have developed a method of building a reference map that allows relaxed criteria for mass errors yet delivers high confidence for peptide identifications. The samples used for generating the peptide database were produced by collecting cysteine-containing peptides from T47D cells and then fractionating the peptides using strong cationic exchange chromatography (SCX). LC–tandem mass spectrometry (MS/MS) data from the SCX fractions were combined to create a comprehensive reference map. After the reference map was built, it was possible to skip the SCX step in further proteomic analyses. We found that the reference-driven identification increases the overall throughput and proteomic coverage by identifying peptides with low intensity or complex interference. The use of the reference map also facilitates the quantitation process by allowing extraction of peptide intensities of interest and incorporating models of theoretical isotope distribution.     

��-Helical Domains Promote Translocation of Intrinsically Disordered Polypeptides into the Endoplasmic Reticulum

Co-translational import into the endoplasmic reticulum (ER) is primarily controlled by N-terminal signal sequences that mediate targeting of the ribosome-nascent chain complex to the Sec61/translocon and initiate the translocation process. Here we show that after targeting to the translocon the secondary structure of the nascent polypeptide chain can significantly modulate translocation efficiency. ER-targeted polypeptides dominated by unstructured domains failed to efficiently translocate into the ER lumen and were subjected to proteasomal degradation via a co-translocational/preemptive pathway. Productive ER import could be reinstated by increasing the amount of α-helical domains, whereas more effective ER signal sequences had only a minor effect on ER import efficiency of unstructured polypeptides. ER stress and overexpression of p58^IPK promoted the co-translocational degradation pathway. Moreover polypeptides with unstructured domains at their N terminus were specifically targeted to proteasomal degradation under these conditions. Our study indicates that extended unstructured domains are signals to dispose ER-targeted proteins via a co-translocational, preemptive quality control pathway.To ensure cellular homeostasis and to preclude toxic effects of aberrant protein conformers quality control mechanisms have evolved to recognize and degrade non-functional and misfolded proteins. In the cytosol the ubiquitin-proteasome system is the main pathway for regulated protein turnover (for reviews, see Refs. 1–3). Moreover the proteasome is part of a quality control system designated endoplasmic reticulum (ER)⁵-associated degradation (ERAD), which mediates post-translational degradation of non-native proteins generated in the ER. ERAD is the primary response to eliminate non-native ER proteins. It involves recognition of non-native polypeptides by ER-resident proteins and retrograde transport to the cytosol where proteasomal degradation occurs (for reviews, see Refs. 4–6). In case ERAD substrates accumulate in the ER lumen intracellular signaling pathways are induced that are collectively called the unfolded protein response (for reviews, see Refs. 7 and 8). Recently a preemptive, co-translocational quality control pathway was described that operates before translocation into the ER is completed (9, 10). Regulated translocation could act as an early quality control step to prevent an overload of the ER with non-native proteins. This regulation relies on an interplay between intrinsic features of the polypeptides and accessory factors able to modulate the translocation efficiency (for a review, see Ref. 11). Although numerous factors are known to be involved in ERAD less is known about mediators of the preemptive, co-translocational quality control pathway. In one study p58^IPK was identified as a key regulator (9), whereas in another it was shown that ER translocation during conditions of acute ER stress is controlled by the signal peptide (10). Indeed the signal peptide is an important intrinsic determinant. Although an exceptionally diverse set of sequences can target proteins for ER import it has been demonstrated that the translocation efficiency is modulated in a signal peptide sequence-specific manner (for reviews, see Refs. 12–14). Another attractive candidate for an intrinsic factor to regulate translocation is the folding state of the nascent polypeptide chain. Formation of secondary structure occurs already in the ribosome exit tunnel (15–18). Moreover it was shown that the polypeptide structure within the ribosomal exit tunnel can modulate translocation of distal parts of the nascent chain (19).The mammalian prion protein (PrP) is a suitable model protein to study whether formation of secondary structure could modulate translocation efficiency because it has an intriguing modular composition: the N-terminal domain spanning 120 amino acids is flexibly disordered followed by a highly structured C-terminal domain of ∼110 amino acids. This autonomously folding domain contains three α-helical regions and a short, two-stranded β-sheet (20–22). Notably folding of the C-terminal domain is one of the most rapid folding reactions measured in vitro (23). Interestingly previous studies indicated that the C-terminal folded domain of PrP is necessary and sufficient to promote ER import. In cultured cells and neurons of transgenic animals PrP-S230X (also known as PrPΔGPI or GPI⁻PrP), a mutant devoid of the C-terminal glycosylphosphatidylinositol (GPI) anchor signal peptide, is efficiently imported into the ER and secreted (24–27). However, by deleting parts of the α-helical domains located in the C-terminal domain a fraction is directed to proteasomal degradation in the cytosol (28).We have now analyzed the underlying mechanisms of this impaired ER import and show that ER-targeted proteins require a certain amount of structured domains to be imported into the ER. In addition, our study indicates that extended unstructured domains are signals for a preemptive/co-translocational degradation pathway.     

Nectar chemistry is tailored for both attraction of mutualists and protection from exploiters

Plants produce nectar to attract pollinators in the case of floral nectar (FN) and defenders in the case of extrafloral nectar (EFN). Whereas nectars must function in the context of plant-animal mutualisms, their chemical composition makes them also attractive for non-mutualistic, exploiting organisms: nectar robbers and nectar-infesting microorganisms. We reviewed the chemical composition of both FNs and EFNs and found that nectar composition appears tailored to fulfil these ambivalent roles. Carbohydrates and amino acids usually function in the attraction of mutualists and appear adapted to the physiological needs of the respective mutualists. Volatiles are a further group of compounds that serves in the attractive function of nectars. By contrast, secondary compounds such as alkaloids and phenols serve the protection from nectar robbers, and most nectar proteins that have been characterised to date protect FN and EFN from microbial infestation. Nectar components serve both in attraction and the protection of nectar.Key words: extrafloral nectar, floral nectar, indirect defence, mutualism, pollination     

Is the combination of topsoil replacement and inoculation with plant material an effective tool for the restoration of threatened sandy grassland?

Question: Is it possible to restore dry calcareous inland sand ecosystems with their characteristic plant community structure within a 4‐yr period by means of combined abiotic–biotic techniques (topsoil replacement, inoculation with raked/mown plant material from target areas)? Location: Upper Rhine valley, Germany. Methods: Two 4‐year experiments were carried out on former arable land, each in the proximity of a reference area bearing a similar complex of threatened sandy grasslands (experiment 1: fine‐scale; experiment 2: landscape scale). In both experiments we used nutrient‐poor deep‐sand substrate (abiotic approach), raked/mown inoculation material from target areas and grazing as management tool (biotic and management approach). The vegetation of the restoration and donor areas was sampled once a year and analysed by non‐metric multidimensional scaling (NMDS) ordination and target‐species ratios. Mixed linear models were calculated to determine effects of grazing (experiment 1) and year (both experiments). Results: NMDS revealed a continuous development of the restored sites towards the corresponding donor sites. Similarly, target‐species ratios of the restored sites tended towards the ratios of the donor sites. To date, grazing effects have mainly been structural: reduction of a carpet‐forming pleurocarpous moss species and of litter. In addition, cover of target species in relation to total plant cover was significantly enhanced by grazing in the last two study years. Conclusions: The combination of nutrient‐poor substrate, inoculation with raked/mown plant material and grazing proved to be a very effective restoration method for dry base‐rich sand ecosystems. After 4 yr the restored plant communities serve as well‐developed parts of a habitat network.     

Loss of Dictyostelium ATG9 results in a pleiotropic phenotype affecting growth,development, phagocytosis and clearance and replication of Legionella pneumophila

Infection of Dictyostelium discoideum with Legionella pneumophila resulted in a large number of differentially regulated genes among them three core autophagy genes, ATG8, ATG9 and ATG16. Macroautophagy contributes to many physiological and pathological processes and might also constitute an important mechanism in cell‐autonomous immunity. For further studies we selected the highly conserved ATG9. In colocalization studies with GFP‐tagged ATG9 and different organelle marker proteins we neither observed colocalization with mitochondria, the ER nor lysosomes. However, there was partial colocalization with the Golgi apparatus and many ATG9‐GFP‐containing vesicles localized along microtubules and accumulated around the microtubule organizing centre. ATG9‐deficient cells had pleiotropic defects. In addition to growth defects they displayed severe developmental defects, consistent with the known role of autophagy in Dictyostelium development. Unexpectedly, the ATG9 mutant also had a strong phagocytosis defect that was particularly apparent when infecting the cells with L. pneumophila. However, those Legionellae that entered the host could multiply better in mutant than in wild‐type cells, because of a less efficient clearance in the early and a more efficient replication in the late phase of infection. We conclude that ATG9 and hence macroautophagy has a protective role during pathogen infection.