Genetic Manipulation of Glycogen Allocation Affects Replicative Lifespan in E. coli

In bacteria, replicative aging manifests as a difference in growth or survival between the two cells emerging from division. One cell can be regarded as an aging mother with a decreased potential for future survival and division, the other as a rejuvenated daughter. Here, we aimed at investigating some of the processes involved in aging in the bacterium Escherichia coli, where the two types of cells can be distinguished by the age of their cell poles. We found that certain changes in the regulation of the carbohydrate metabolism can affect aging. A mutation in the carbon storage regulator gene, csrA, leads to a dramatically shorter replicative lifespan; csrA mutants stop dividing once their pole exceeds an age of about five divisions. These old-pole cells accumulate glycogen at their old cell poles; after their last division, they do not contain a chromosome, presumably because of spatial exclusion by the glycogen aggregates. The new-pole daughters produced by these aging mothers are born young; they only express the deleterious phenotype once their pole is old. These results demonstrate how manipulations of nutrient allocation can lead to the exclusion of the chromosome and limit replicative lifespan in E. coli, and illustrate how mutations can have phenotypic effects that are specific for cells with old poles. This raises the question how bacteria can avoid the accumulation of such mutations in their genomes over evolutionary times, and how they can achieve the long replicative lifespans that have recently been reported.     

The Multi-PDZ domain protein MUPP1 as a lipid raft-associated scaffolding protein controlling the acrosome reaction in mammalian spermatozoa

The success of acrosomal exocytosis, a complex process with a variety of interrelated steps, relies on the coordinated interaction of participating signaling molecules. Since scaffolding proteins are known to spatially organize sequential signaling pathways, we examined whether the Multi-PDZ domain protein MUPP1, recently identified in mammalian spermatozoa, is functionally active in controlling acrosomal secretion in mammalian sperm cells. To address this question, permeabilized mouse sperm were loaded with inhibitory antibodies against MUPP1 as well as with a photosensitive Ca(2+) chelator which allows a controlled release of acrosomal Ca(2+). The results revealed that MUPP1 controls initial tethering and docking of the acrosomal vesicle, whereas syntaxin 2, a t-SNARE protein also expressed in the acrosomal cap of mammalian spermatozoa, appears to take part in the final process of acrosomal fusion. Interestingly, using immunogold electron microscopy, it was found that MUPP1 is detectable in the region of the periacrosomal membrane. Furthermore, in isolated detergent-insoluble glycolipid-enriched membrane domains from epididymal spermatozoa, MUPP1 was found to show a striking association with the Triton X-100 insoluble membrane fraction, which did not change significantly upon sperm capacitation or partial chemical extraction of cholesterol. This evidence points to a role of MUPP1 as a membrane raft-associated molecular organizer, and suggests that mammalian spermatozoa may use a scaffolding protein and distinct membrane subdomains to spatially organize components involved in the process of acrosomal exocytosis.     

Create and preserve: Proteostasis in development and aging is governed by Cdc48/p97/VCP

The AAA-ATPase Cdc48 (also called p97 or VCP) acts as a key regulator in proteolytic pathways, coordinating recruitment and targeting of substrate proteins to the 26S proteasome or lysosomal degradation. However, in contrast to the well-known function in ubiquitin-dependent cellular processes, the physiological relevance of Cdc48 in organismic development and maintenance of protein homeostasis is less understood. Therefore, studies on multicellular model organisms help to decipher how Cdc48-dependent proteolysis is regulated in time and space to meet developmental requirements. Given the importance of developmental regulation and tissue maintenance, defects in Cdc48 activity have been linked to several human pathologies including protein aggregation diseases. Thus, addressing the underlying disease mechanisms not only contributes to our understanding on the organism-wide function of Cdc48 but also facilitates the design of specific medical therapies. In this review, we will portray the role of Cdc48 in the context of multicellular organisms, pointing out its importance for developmental processes, tissue surveillance, and disease prevention. This article is part of a Special Issue entitled: Ubiquitin–Proteasome System. Guest Editors: Thomas Sommer and Dieter H. Wolf.     

Procedure for the identification of Escherichia coli mutants affected in components containing glycerol derived from phospholipid turnover: Isolation of mutants lacking glycerol in membrane-derived oligosaccharides (MDO)

Abstract A mutant screening procedure is described which allows the identification of mutants carrying lesions in lipoprotein, membrane-derived oligosac-charides (MDO), and other compounds of the E. coli cell envelope containing glycerol derived from phospholipid metabolism. Two mutants lacking glycerol in MDO and one mutant devoid of lipoprotein demonstrate the usefulness of the procedure.     

The seeds of Loasaceae subfam. Loasoideae (Cornales) II: Seed morphology of “South Andean Loasas” (Loasa, Caiophora, Scyphanthus and Blumenbachia)

South Andean Loasas (Blumenbachia, Caiophora, Loasa, Scyphanthus) are a monophyletic group of taxa within Loasaceae subfam. Loasoideae, comprising some 100 species, 49 of which are investigated here. They retain a many-layered testa in the mature seeds and usually have passive transfer testas with complex, spongiose wall outgrowths. Additional modifications concern the undulations of the testa epidermis, presence or absence of the outer periclinal wall, secondary sculpturing, the presence or absence of spines, warts and finally spongiose structures on the anticlinal walls of the testa epidermis and the inner periclinal wall. Seeds of the widespread “deeply pitted” type are plesiomorphic, while various subclades of South Andean Loasas have derivations underscoring their relationships and confirming the relationships found with molecular markers and other morphological characters. The genus Blumenbachia has either seeds with a many-layered testa forming longitudinal lamellae (sect. Angulatae), or balloon seeds with a loose outer testa layer and spongiose wall outgrowths on the inner periclinal walls (sect. Blumenbachia and sect. Gripidea) and is clearly monophyletic. Loasa s.str. (ser. Loasa, ser. Macrospermae, ser. Floribundae, ser. Deserticolae) is characterized by the presence of a subterminal hilum or hilar scar and one subgroup (ser. Loasa, ser. Macrospermae) by very large and heavy seeds with a collapsed testa. L. ser. Pinnatae, ser. Acaules, ser. Volubiles, Scyphanthus and Caiophora share more or less one seed types with minor modifications. Within Caiophora various derivations are observed, of which the gradual loss of the secondary sculpture of the inner periclinal wall is the most striking one. Anemochoria is the most widespread dispersal mechanism in South Andean Loasas and is achieved in at least five structurally different ways.     

A novel proteomic screen for peptide-protein interactions

Regulated interactions between short, unstructured amino acid sequences and modular protein domains are central to cell signaling. Here we use synthetic peptides in "active" (e.g. phosphorylated) and "control" (e.g. non-phosphorylated) forms as baits in affinity pull-down experiments to determine such interactions by quantitative proteomics. Stable isotope labeling by amino acids in cell culture distinguishes specific binders directly by the isotope ratios determined by mass spectrometry (Blagoev, B., Kratchmarova, I., Ong, S.-E., Nielsen, M., Foster, L. J., and Mann, M. (2003) Nat. Biotechnol. 21, 315-318). A tyrosine-phosphorylated peptide of the epidermal growth factor receptor specifically retrieved the Src homology domain (SH) 2- and SH3 domain-containing adapter protein Grb2. A proline-rich sequence of Son of Sevenless also specifically bound Grb2, demonstrating that the screen maintains specificity with low affinity interactions. The proline-rich Sos peptide retrieved only SH3 domain containing proteins as specific binding partners. Two of these, Pacsin 3 and Sorting Nexin 9, were confirmed by immunoprecipitation. Our data are consistent with a change in the role of Sos from Ras-dependent signaling to actin remodeling/endocytic signaling events by a proline-SH3 domain switch.     

A Novel Antibody against Human Properdin Inhibits the Alternative Complement System and Specifically Detects Properdin from Blood Samples

The complement system is an essential part of the innate immune system by acting as a first line of defense which is stabilized by properdin, the sole known positive regulator of the alternative complement pathway. Dysregulation of complement can promote a diversity of human inflammatory diseases which are treated by complement inhibitors. Here, we generated a novel blocking monoclonal antibody (mAb) against properdin and devised a new diagnostic assay for this important complement regulator. Mouse mAb 1340 specifically detected native properdin from human samples with high avidity. MAb 1340 inhibited specifically the alternative complement mediated cell lysis within a concentration range of 1–10 µg/mL. Thus, in vitro anti-properdin mAb 1340 was up to fifteen times more efficient in blocking the complement system as compared to anti-C5 or anti-Ba antibodies. Computer-assisted modelling suggested a three-dimensional binding epitope in a properdin-C3(H₂O)-clusterin complex to be responsible for the inhibition. Recovery of properdin in a newly established sandwich ELISA using mAb 1340 was determined at 80–125% for blood sample dilutions above 1∶50. Reproducibility assays showed a variation below 25% at dilutions less than 1∶1,000. Systemic properdin concentrations of healthy controls and patients with age-related macular degeneration or rheumatic diseases were all in the range of 13–30 µg/mL and did not reveal significant differences. These initial results encourage further investigation into the functional role of properdin in the development, progression and treatment of diseases related to the alternative complement pathway. Thus, mAb 1340 represents a potent properdin inhibitor suitable for further research to understand the exact mechanisms how properdin activates the complement C3-convertase and to determine quantitative levels of properdin in biological samples.     

Introducing “best single template” models as reference baseline for the Continuous Automated Model Evaluation (CAMEO)

Critical blind assessment of structure prediction techniques is crucial for the scientific community to establish the state of the art, identify bottlenecks, and guide future developments. In Critical Assessment of Techniques in Structure Prediction (CASP), human experts assess the performance of participating methods in relation to the difficulty of the prediction task in a biennial experiment on approximately 100 targets. Yet, the development of automated computational modeling methods requires more frequent evaluation cycles and larger sets of data. The “Continuous Automated Model EvaluatiOn (CAMEO)” platform complements CASP by conducting fully automated blind prediction evaluations based on the weekly pre-release of sequences of those structures, which are going to be published in the next release of the Protein Data Bank (PDB). Each week, CAMEO publishes benchmarking results for predictions corresponding to a set of about 20 targets collected during a 4-day prediction window. CAMEO benchmarking data are generated consistently for all methods at the same point in time, enabling developers to cross-validate their method's performance, and referring to their results in publications. Many successful participants of CASP have used CAMEO—either by directly benchmarking their methods within the system or by comparing their own performance to CAMEO reference data. CAMEO offers a variety of scores reflecting different aspects of structure modeling, for example, binding site accuracy, homo-oligomer interface quality, or accuracy of local model confidence estimates. By introducing the "bestSingleTemplate" method based on structure superpositions as a reference for the accuracy of 3D modeling predictions, CAMEO facilitates objective comparison of techniques and fosters the development of advanced methods.     

The rate of environmental fluctuations shapes ecological dynamics in a two‐species microbial system

Species interactions change when the external conditions change. How these changes affect microbial community properties is an open question. We address this question using a two‐species consortium in which species interactions change from exploitation to competition depending on the carbon source provided. We built a mathematical model and calibrated it using single‐species growth measurements. This model predicted that low frequencies of change between carbon sources lead to species loss, while intermediate and high frequencies of change maintained both species. We experimentally confirmed these predictions by growing co‐cultures in fluctuating environments. These findings complement more established concepts of a diversity peak at intermediate disturbance frequencies. They also provide a mechanistic understanding for how the dynamics at the community level emerges from single‐species behaviours and interspecific interactions. Our findings suggest that changes in species interactions can profoundly impact the ecological dynamics and properties of microbial systems.     

Restoration of normal stamen development and pollen formation by fusion of different cytoplasmic male-sterile cultivars of Nicotiana tabacum

Summary Fusion of two cytoplasmic male-sterile cultivars of Nicotiana tabacum, one with N. bigelovii cytoplasm and one with N. undulata cytoplasm, resulted in the restoration of male fertility in cybrid plants. All male-fertile cybrids exhibited fused corollas, which is characteristic for the cultivar with N. undulata cytoplasm, while their stamen structures varied from cybrid to cybrid, some producing stamens with anthers fused to petal-like appendages and one producing stamens of a normal appearance for N. tabacum. Restriction enzyme digestion and agarose gel electrophoresis of mitochondrial DNA showed that mitochondrial DNA of the fertile cybrids was more similar to the male-sterile cultivar with the cytoplasm of N. undulata than to the cultivar with N. bigelovii cytoplasm. Some restriction fragments were unique to the male-fertile cybrids. Comparisons between stamen structure and mitochondrial DNA for eight fertile progeny from one cybrid plant led to the identification of several restriction fragments that appeared at enhanced levels in connection with normal stamen development.