Synchronizing Protein Traffic to the Primary Cilium

The primary cilium is able to maintain a specific protein composition, which is critical for its function as a signaling organelle. Here we introduce a system to synchronize biosynthetic trafficking of ciliary proteins that is based on conditional aggregation domains (CADs). This approach enables to create a wave of ciliary proteins that are transported together, which opens novel avenues for visualizing and studying ciliary import mechanisms. By using somatostatin receptor 3 (SSTR3) as model protein we studied intracellular transport and ciliary import with high temporal and spatial resolution in epithelial Madin-Darby canine kidney (MDCK) cells. This yielded the interesting discovery that SSTR3, besides being transported to the primary cilium, is also targeted to the basolateral plasma membrane. In addition, we found a similar behavior for another ciliary protein, nephrocystin-3 (NPHP3), thus suggesting a potential correlation between ciliary and basolateral trafficking. Furthermore, our CAD-based system allowed assembling a large dataset in which apical and basolateral surface SSTR3 signals could be compared to ciliary SSTR3 signals on a single cell level. This enabled to generate novel complementary evidence for the previously proposed lateral import mechanism of SSTR3 into the cilium along the plasma membrane

ROCK1/2 signaling contributes to corticosteroid-refractory acute graft-versus-host disease

Patients with corticosteroid-refractory acute graft-versus-host disease (aGVHD) have a low one-year survival rate. Identification and validation of novel targetable kinases in patients who experience corticosteroid-refractory-aGVHD may help improve outcomes. Kinase-specific proteomics of leukocytes from patients with corticosteroid-refractory-GVHD identified rho kinase type 1 (ROCK1) as the most significantly upregulated kinase. ROCK1/2 inhibition improved survival and histological GVHD severity in mice and was synergistic with JAK1/2 inhibition, without compromising graft-versus-leukemia-effects. ROCK1/2-inhibition in macrophages or dendritic cells prior to transfer reduced GVHD severity. Mechanistically, ROCK1/2 inhibition or ROCK1 knockdown interfered with CD80, CD86, MHC-II expression and IL-6, IL-1β, iNOS and TNF production in myeloid cells. This was accompanied by impaired T cell activation by dendritic cells and inhibition of cytoskeletal rearrangements, thereby reducing macrophage and DC migration. NF-κB signaling was reduced in myeloid cells following ROCK1/2 inhibition. In conclusion, ROCK1/2 inhibition interferes with immune activation at multiple levels and reduces acute GVHD while maintaining GVL-effects, including in corticosteroid-refractory settings

Diverse molecular causes of unsolved autosomal dominant tubulointerstitial kidney diseases

Autosomal Dominant Tubulointerstitial Kidney Disease (ADTKD) is caused by mutations in one of at least five genes and leads to kidney failure usually in mid adulthood. Throughout the literature, variable numbers of families have been reported, where no mutation can be found and therefore termed ADTKD-not otherwise specified. Here, we aim to clarify the genetic cause of their diseases in our ADTKD registry. Sequencing for all known ADTKD genes was performed, followed by SNaPshot minisequencing for the dupC (an additional cytosine within a stretch of seven cytosines) mutation of MUC1. A virtual panel containing 560 genes reported in the context of kidney disease (nephrome) and exome sequencing were then analyzed sequentially. Variants were validated and tested for segregation. In 29 of the 45 registry families, mutations in known ADTKD genes were found, mostly in MUC1. Sixteen families could then be termed ADTKD-not otherwise specified, of which nine showed diagnostic variants in the nephrome (four in COL4A5, two in INF2 and one each in COL4A4, PAX2, SALL1 and PKD2). In the other seven families, exome sequencing analysis yielded potential disease associated variants in novel candidate genes for ADTKD; evaluated by database analyses and genome-wide association studies. For the great majority of our ADTKD registry we were able to reach a molecular genetic diagnosis. However, a small number of families are indeed affected by diseases classically described as a glomerular entity. Thus, incomplete clinical phenotyping and atypical clinical presentation may have led to the classification of ADTKD. The identified novel candidate genes by exome sequencing will require further functional validation

Inhibition of Arp2/3 Complex after ADP-Ribosylation of Arp2 by Binary Clostridioides Toxins

Clostridioides bacteria are responsible for life threatening infections. Here, we show that in addition to actin, the binary toxins CDT, C2I, and Iota from Clostridioides difficile, botulinum, and perfrigens, respectively, ADP-ribosylate the actin-related protein Arp2 of Arp2/3 complex and its additional components ArpC1, ArpC2, and ArpC4/5. The Arp2/3 complex is composed of seven subunits and stimulates the formation of branched actin filament networks. This activity is inhibited after ADP-ribosylation of Arp2. Translocation of the ADP-ribosyltransferase component of CDT toxin into human colon carcinoma Caco2 cells led to ADP-ribosylation of cellular Arp2 and actin followed by a collapse of the lamellipodial extensions and F-actin network. Exposure of isolated mouse colon pieces to CDT toxin induced the dissolution of the enterocytes leading to luminal aggregation of cellular debris and the collapse of the mucosal organization. Thus, we identify the Arp2/3 complex as hitherto unknown target of clostridial ADP-ribosyltransferases

ACDS: Adapting Computational Data Streams for High Performance

Data-intensive, interactive applications are an important class of metacomputing (Grid) applications. They are characterized by large data flows between data providers and consumers, like scientific simulations and remote visualization clients of simulation output. Such data flows vary at runtime, due to changes in consumers' data needs, changes in the nature of the data being transmitted, or changes in the availability of computing resources used by flows. The topic of this paper is the runtime adaptation of data streams, in response to changes in resource availability and/or in end user requirements, with the goal of continually providing to consumers data at the levels of quality they require. Our approach is one that associates computational objects with data streams. These objects offer services like data filtering and transformation. Runtime adaptation is achieved by adjusting objects' actions on streams, by splitting and merging objects, and by migrating them (and the streams on which they operate) across machines and network links. The resulting adaptive computational data streams maintain high performance by responding to changes in the needs of data consumers, as exemplified by variations in the resolution or rate at which they desire to receive data. Adaptive streams also react to changes in resource availability detected by online monitoring. The experimental demonstrations presented in this paper utilize computational data streams emanating from a global atmospheric simulation model and/or from stored model outputs, consumed by visualization clients that display this data. Experiments are performed on heterogeneous cluster machines and visualization clients connected by LAN or WAN networks

Lu/BCAM Adhesion Glycoprotein Is a Receptor for Escherichia coli Cytotoxic Necrotizing Factor 1 (CNF1)

The Cytotoxic Necrotizing Factor 1 (CNF1) is a protein toxin which is a major virulence factor of pathogenic Escherichia coli strains. Here, we identified the Lutheran (Lu) adhesion glycoprotein/basal cell adhesion molecule (BCAM) as cellular receptor for CNF1 by co-precipitation of cell surface molecules with tagged toxin. The CNF1-Lu/BCAM interaction was verified by direct protein-protein interaction analysis and competition studies. These studies revealed amino acids 720 to 1014 of CNF1 as the binding site for Lu/BCAM. We suggest two cell interaction sites in CNF1: first the N-terminus, which binds to p37LRP as postulated before. Binding of CNF1 to p37LRP seems to be crucial for the toxin's action. However, it is not sufficient for the binding of CNF1 to the cell surface. A region directly adjacent to the catalytic domain is a high affinity interaction site for Lu/BCAM. We found Lu/BCAM to be essential for the binding of CNF1 to cells. Cells deficient in Lu/BCAM but expressing p37LRP could not bind labeled CNF1. Therefore, we conclude that LRP and Lu/BCAM are both required for toxin action but with different functions. Author Summary We study a crucial virulence factor produced by pathogenic Escherichia coli strains, the Cytotoxic Necrotizing Factor 1 (CNF1). More than 80% of urinary tract infections (UTIs), which are counted among the most common bacterial infections of humans, are caused by Uropathogenic Escherichia coli (UPEC) strains. We and others elucidated the molecular mechanism of the E. coli toxin CNF1. It constitutively activates Rho GTPases by a direct covalent modification. The toxin enters mammalian cells by receptor-mediated endocytosis. Here, we identified the protein receptor for CNF1 by co-precipitation of cell surface molecules with the tagged toxin and subsequent Maldi-TOF analysis. We identified the Lutheran (Lu) adhesion glycoprotein/basal cell adhesion molecule (BCAM) as receptor for CNF1 and located its interaction site to the C-terminal part of the toxin. We performed direct protein-protein interaction analysis and competition studies. Moreover, cells deficient in Lu/BCAM could not bind labeled CNF1. The identification of a toxin's cellular receptor and receptor binding region is an important task for understanding the pathogenic function of the toxin and, moreover, to make the toxin accessible for its use as a cellbiological and pharmacological tool, for example for the generation of immunotoxins

Functional Characterization of an Extended Binding Component of the Actin-ADP-Ribosylating C2 Toxin Detected in Clostridium botulinum Strain (C) 2300 ▿

Clostridium botulinum C2 toxin consists of the binding component C2II and the enzyme component C2I, which ADP-ribosylates G-actin of eukaryotic cells. Trypsin-activated C2II (C2IIa) forms heptamers that mediate cell binding and translocation of C2I from acidic endosomes into the cytosol of target cells. By genome sequencing of C. botulinum strain (C) 2300, we found that C2II from this strain carries a C-terminal extension of 129 amino acids, unlike its homologous counterparts from strains (C) 203U28, (C) 468, and (D) 1873. This extension shows a high similarity to the C-terminal receptor-binding domain of C2II and is presumably the result of a duplication of this domain. The C2II extension facilitates the binding to cell surface receptors, which leads to an increased intoxication efficiency compared to that of C2II proteins from other C. botulinum strains

Real-Time Visualization in Distributed Computational Laboratories

Large data volumes cannot be transported, processed or displayed in real-time unless we apply to them general or application-specific compression and filtering techniques. In addition, when multiple end users inspect such data sets or when multiple programs access or consume them, data distribution and display should be performed differentially, in accordance with the queries generated by programs or end users. Finally, if dynamic access queries cannot be formulated precisely, then they must be refined as they progress in order to avoid unnecessary data retrievals, transfers, and information overload for programs or end users with uninteresting or unimportant data. The principal idea of our research is to create Active User Interfaces (AUIs) that continuously emit events describing their internal states and/or current information needs. Based on these events, we then develop methods for controlling the information streams directed at these interfaces, for single and for multiple, collaborating end users. The purposes of stream control are twofold. First, stream control is performed to deal with heterogeneous underlying hardware and software systems, where streams may originate at secondary storage media or may be generated dynamically, may have to be moved across the Internet or may utilize local area or high performance interconnects, and where collaborating user interfaces may range from low end PC-based displays to high end immersive visualization engines. Second, stream control aims to achieve scalability for user interfaces to large-scale, complex data streams directed at them, by offloading computations from visualizations to information generators or to information routing sites, to dynamically migrate such computations to appropriate locations, and to adapt these computations in order to effect tradeoffs in the amount of data moved across network links vs. the computations required when performing data rendering, compression, filtering, and routing actions

Computational Data Streams in Data-Intensive and Interactive Applications

Data-intensive, interactive applications are an important class of metacomputing or Grid applications. These applications are characterized by large data flows between data providers and active consumers; that is, consumers that actively request information. Event models such as the CORBA Event Service have been proposed as a communication middleware solution to such applications. Computational data streams are a means for embedding computational components into these large data flows. The service is provided as objects and libraries for (1) specifying computational component behavior as a set of event-action rules, (2) a run-time environment for a computational component, and (3) making active a consumer. Computational data streams are responsive to active interfaces, and adaptive in response to user information, resource information, or statistical data collected about the data stream. This paper demonstrates the performance benefits of computational data streams through experimental..