Neurexins are cell-surface molecules that bind neuroligins to form a heterophilic, Ca 2؉ -dependent complex at central synapses. This transsynaptic complex is required for efficient neurotransmission and is involved in the formation of synaptic contacts. In addition, both molecules have been identified as candidate genes for autism. Here we performed mutagenesis experiments to probe for essential components of the neurexin/neuroligin binding interface at the single-amino acid level. We found that in neurexins the contact area is sharply delineated and consists of hydrophobic residues of the LNS domain that surround a Ca 2؉ binding pocket. Point mutations that changed electrostatic and shape properties leave Ca 2؉ coordination intact but completely inhibit neuroligin binding, whereas alternative splicing in ␣-and ␤-neurexins and in neuroligins has a weaker effect on complex formation. In neuroligins, the contact area appears less distinct because exchange of a more distant aspartate completely abolished binding to neurexin but many mutations of predicted interface residues had no strong effect on binding. Together with calculations of energy terms for presumed interface hot spots that complement and extend our mutagenesis and recent crystal structure data, this study presents a comprehensive structural basis for the complex formation of neurexins and neuroligins and their transsynaptic signaling between neurons.calcium ͉ cell adhesion ͉ LNS domain ͉ neurotransmission ͉ synaptogenesis T he heterophilic complex formed by cell adhesion molecules neurexin (Nrxn) and neuroligin (Nlgn) reflects the asymmetric nature of the synapse with presynaptic and postsynaptic specializations (1, 2). Nrxns and Nlgns are essential molecules because they perform important functions in synaptic transmission (3, 4) and differentiation of synaptic contacts (5, 6), and both molecules have been identified as candidate genes for autism (7,8).Nrxns form a family of transmembrane proteins with variable extracellular sequences. Nrxn genes (Nrxn1-3) give rise to ␣-neurexins and shorter ␤-neurexins that contain five (␣-Nrxn) or two (␤-Nrxn) splice sites (SS1-5) (9). Although they share most sequences, the essential role of ␣-Nrxn in neurotransmission cannot be replaced by ␤-Nrxn (10), and one ligand exists for ␣-Nrxn that does not bind to ␤-Nrxn (11). In contrast, Nlgn was discovered by its interaction with ␤-Nrxn (12). The cholinesterase-like adhesion molecule (CAM) domain of Nlgn interacts with the extracellular domain of ␤-Nrxn in a Ca 2ϩ -dependent manner, and binding is facilitated by the splice variation of ␤-Nrxn that lacks an insert in SS4 (13). Nlgn mRNA is also susceptible to splicing, at two positions referred to as A and B (12), including splice variants with no insert in B that bind to all ␤-Nrxns and presumably ␣-Nrxn (14). Therefore, the Nrxn/Nlgn complex involves a domain shared by ␣-and ␤-Nrxns, and any structural characterization needs to account for the Ca 2ϩ dependence and regulation by alternative splicing (15-17).Extracell...

Vertebrate life critically depends on renal filtration and excretion of low molecular weight waste products. This process is controlled by a specialized cell-cell contact between podocyte foot processes: the slit diaphragm (SD). Using a comprehensive set of targeted KO mice of key SD molecules, we provided genetic, functional, and high-resolution ultrastructural data highlighting a concept of a flexible, dynamic, and multilayered architecture of the SD. Our data indicate that the mammalian SD is composed of NEPHRIN and NEPH1 molecules, while NEPH2 and NEPH3 do not participate in podocyte intercellular junction formation. Unexpectedly, homo- and heteromeric NEPHRIN/NEPH1 complexes are rarely observed. Instead, single NEPH1 molecules appear to form the lower part of the junction close to the glomerular basement membrane with a width of 23 nm, while single NEPHRIN molecules form an adjacent junction more apically with a width of 45 nm. In both cases, the molecules are quasiperiodically spaced 7 nm apart. These structural findings, in combination with the flexibility inherent to the repetitive Ig folds of NEPHRIN and NEPH1, indicate that the SD likely represents a highly dynamic cell-cell contact that forms an adjustable, nonclogging barrier within the renal filtration apparatus.

Background: Extracellular matrix dystroglycan has essential functions at the neuromuscular junction and at inhibitory synapses in the brain.Results: Brain dystroglycan competes with neurexophilin-1 and neuroligins for binding to presynaptic α-neurexins.Conclusion: Competition between α-neurexin ligands in combination with alternative splicing determines formation of important trans-synaptic complexes.Significance: This is the first analysis of binding interference in α-neurexin multiplexes.

BRAF mutations are associated with aggressive, less-differentiated and therapy-resistant colorectal carcinoma. However, the underlying mechanisms for these correlations remain unknown. To understand how oncogenic B-Raf contributes to carcinogenesis, in particular to aspects other than cellular proliferation and survival, we generated three isogenic human colorectal carcinoma cell line models in which we can dynamically modulate the expression of the B-Raf V600E oncoprotein. Doxycyclin-inducible knockdown of endogenous B-Raf V600E decreases cellular motility and invasion in conventional and three-dimensional (3D) culture, whereas it promotes cell-cell contacts and induces various hallmarks of differentiated epithelia. Importantly, all these effects are recapitulated by B-Raf (PLX4720, vemurafenib, and dabrafenib) or MEK inhibitors (trametinib). Surprisingly, loss of B-Raf V600E in HT29 xenografts does not only stall tumor growth, but also induces glandular structures with marked expression of CDX2, a tumor-suppressor and master transcription factor of intestinal differentiation. By performing the first transcriptome profiles of PLX4720-treated 3D cultures of HT29 and Colo-205 cells, we identify several upregulated genes linked to epithelial differentiation and effector functions, such as claudin-1, a Cdx-2 target gene encoding a critical tight junction component. Thereby, we provide a mechanism for the clinically observed correlation between mutant BRAF and the loss of Cdx-2 and claudin-1. PLX4720 also suppressed several metastasis-associated transcripts that have not been implicated as targets, effectors or potential biomarkers of oncogenic B-Raf signaling so far. Together, we identify a novel facet of clinically applied B-Raf or MEK inhibitors by showing that they promote cellular adhesion and differentiation of colorectal carcinoma cells.

Mice with targeted genetic alterations are the most effective tools for deciphering organismal gene function. We generated an ENU-based parallel C3HeB/FeJ sperm and DNA archive characterized by a high probability to identify allelic variants of target genes as well as high efficiencies in allele retrieval and model revitalization. Our archive size of over 17,000 samples contains approximately 340,000 independent alleles (20 functional mutations per individual sample). Based on an estimated number of approximately 30,000 mouse genes, the parallel sperm/DNA archive should permit the identification and recovery of ten or more alleles per average target gene which translates to a calculated 99% success rate in the discovery of five allelic variants for any given average gene. The low rate of unrelated ENU-induced passenger mutations has no practical impact on the analysis of the allele-specific phenotype at the G3 generation because of dilution and free segregation of such unrelated passenger mutations. To date 39 mouse models representing 33 different genes have been recovered from our archive using in vitro fertilization techniques. The generation time for a murine model heterozygous for a mutation in a gene of interest is less than 2 months, i.e., three to four times faster compared with current embryonic stem-cell-based technologies. We conclude that ENU-based targeted mutagenesis is a powerful tool for the fast and high-throughput production of murine gene-specific models for biomedical research.

The nerve growth factor NGF has been shown to cause cell fate decisions toward either differentiation or proliferation depending on the relative activity of downstream pERK, pAKT, or pJNK signaling. However, how these protein signals are translated into and fed back from transcriptional activity to complete cellular differentiation over a time span of hours to days is still an open question. Comparing the time-resolved transcriptome response of NGF- or EGF-stimulated PC12 cells over 24 h in combination with protein and phenotype data we inferred a dynamic Boolean model capturing the temporal sequence of protein signaling, transcriptional response and subsequent autocrine feedback. Network topology was optimized by fitting the model to time-resolved transcriptome data under MEK, PI3K, or JNK inhibition. The integrated model confirmed the parallel use of MAPK/ERK, PI3K/AKT, and JNK/JUN for PC12 cell differentiation. Redundancy of cell signaling is demonstrated from the inhibition of the different MAPK pathways. As suggested in silico and confirmed in vitro, differentiation was substantially suppressed under JNK inhibition, yet delayed only under MEK/ERK inhibition. Most importantly, we found that positive transcriptional feedback induces bistability in the cell fate switch. De novo gene expression was necessary to activate autocrine feedback that caused Urokinase-Type Plasminogen Activator (uPA) Receptor signaling to perpetuate the MAPK activity, finally resulting in the expression of late, differentiation related genes. Thus, the cellular decision toward differentiation depends on the establishment of a transcriptome-induced positive feedback between protein signaling and gene expression thereby constituting a robust control between proliferation and differentiation.

The mechanistic target of rapamycin (mTOR) is a central regulatory pathway that integrates a variety of environmental cues to control cellular growth and homeostasis by intricate molecular feedbacks. In spite of extensive knowledge about its components, the molecular understanding of how these function together in space and time remains poor and there is a need for Systems Biology approaches to perform systematic analyses. In this work, we review the recent progress how the combined efforts of mathematical models and quantitative experiments shed new light on our understanding of the mTOR signaling pathway. In particular, we discuss the modeling concepts applied in mTOR signaling, the role of multiple feedbacks and the crosstalk mechanisms of mTOR with other signaling pathways. We also discuss the contribution of principles from information and network theory that have been successfully applied in dissecting design principles of the mTOR signaling network. We finally propose to classify the mTOR models in terms of the time scale and network complexity, and outline the importance of the classification toward the development of highly comprehensive and predictive models. WIREs Syst Biol Med 2017, 9:e1379. doi: 10.1002/wsbm.1379For further resources related to this article, please visit the WIREs website.

A straightforward, in situ calibration procedure for a vacuum ultraviolet (VUV) double-reflection polarization analyser is introduced which does not require knowledge of the optical properties of the reflecting materials or previously measured benchmark polarizations. The linear and circular polarization sensitivities of the analyser are determined from VUV light intensity measurements obtained for various mutual orientations of the two reflecting surfaces. As an example of the reliability of the calibration procedure, measurements of integrated Stokes parameters are presented and compared with the results of measurements carried out previously by other groups using different polarization analysers.