Trans Interactions Research Articles

Correction: A refined model of claudin-15 tight junction paracellular architecture by molecular dynamics simulations.

[This corrects the article DOI: 10.1371/journal.pone.0184190.].

Direct Evidence of APLP1 Trans Interactions in Cell-Cell Adhesion Platforms Investigated via Fluorescence Fluctuation Spectroscopy

Direct Evidence of APLP1 Trans Interactions in Cell-Cell Adhesion Platforms Investigated via Fluorescence Fluctuation Spectroscopy

The dynamic dimer structure of the chaperone Trigger Factor

The chaperone Trigger Factor (TF) from Escherichia coli forms a dimer at cellular concentrations. While the monomer structure of TF is well known, the spatial arrangement of this dimeric chaperone storage form has remained unclear. Here, we determine its structure by a combination of high-resolution NMR spectroscopy and biophysical methods. TF forms a symmetric head-to-tail dimer, where the ribosome binding domain is in contact with the substrate binding domain, while the peptidyl-prolyl isomerase domain contributes only slightly to the dimer affinity. The dimer structure is highly dynamic, with the two ribosome binding domains populating a conformational ensemble in the center. These dynamics result from intermolecular in trans interactions of the TF client-binding site with the ribosome binding domain, which is conformationally frustrated in the absence of the ribosome. The avidity in the dimer structure explains how the dimeric state of TF can be monomerized also by weakly interacting clients.

Direct evidence of amyloid precursor–like protein 1 trans interactions in cell–cell adhesion platforms investigated via fluorescence fluctuation spectroscopy

The amyloid precursor-like protein 1 (APLP1) is a type I transmembrane protein that plays a role in synaptic adhesion and synaptogenesis. Past investigations indicated that APLP1 is involved in the formation of protein-protein complexes that bridge the junctions between neighboring cells. Nevertheless, APLP1-APLP1 trans interactions have never been directly observed in higher eukaryotic cells. Here, we investigated APLP1 interactions and dynamics directly in living human embryonic kidney cells using fluorescence fluctuation spectroscopy techniques, namely cross-correlation scanning fluorescence correlation spectroscopy and number and brightness analysis. Our results show that APLP1 forms homotypic trans complexes at cell-cell contacts. In the presence of zinc ions, the protein forms macroscopic clusters, exhibiting an even higher degree of trans binding and strongly reduced dynamics. Further evidence from giant plasma membrane vesicles suggests that the presence of an intact cortical cytoskeleton is required for zinc-induced cis multimerization. Subsequently, large adhesion platforms bridging interacting cells are formed through APLP1-APLP1 trans interactions. Taken together, our results provide direct evidence that APLP1 functions as a neuronal zinc-dependent adhesion protein and allow a more detailed understanding of the molecular mechanisms driving the formation of APLP1 adhesion platforms.

Structural inhibition of dynamin-mediated membrane fission by endophilin.

Dynamin, which mediates membrane fission during endocytosis, binds endophilin and other members of the Bin-Amphiphysin-Rvs (BAR) protein family. How endophilin influences endocytic membrane fission is still unclear. Here, we show that dynamin-mediated membrane fission is potently inhibited in vitro when an excess of endophilin co-assembles with dynamin around membrane tubules. We further show by electron microscopy that endophilin intercalates between turns of the dynamin helix and impairs fission by preventing trans interactions between dynamin rungs that are thought to play critical roles in membrane constriction. In living cells, overexpression of endophilin delayed both fission and transferrin uptake. Together, our observations suggest that while endophilin helps shape endocytic tubules and recruit dynamin to endocytic sites, it can also block membrane fission when present in excess by inhibiting inter-dynamin interactions. The sequence of recruitment and the relative stoichiometry of the two proteins may be critical to regulated endocytic fission.

A refined model of claudin-15 tight junction paracellular architecture by molecular dynamics simulations.

Tight-junctions between epithelial cells of biological barriers are specialized molecular structures that regulate the flux of solutes across the barrier, parallel to cell walls. The tight-junction backbone is made of strands of transmembrane proteins from the claudin family, but the molecular mechanism of its function is still not completely understood. Recently, the crystal structure of a mammalian claudin-15 was reported, displaying for the first time the detailed features of transmembrane and extracellular domains. Successively, a structural model of claudin-15-based paracellular channels has been proposed, suggesting a putative assembly that illustrates how claudins associate in the same cell (via cis interactions) and across adjacent cells (via trans interactions). Although very promising, the model offers only a static conformation, with residues missing in the most important extracellular regions and potential steric clashes. Here we present detailed atomic models of paracellular single and double pore architectures, obtained from the putative assembly and refined via structural modeling and all-atom molecular dynamics simulations in double membrane bilayer and water environment. Our results show an overall stable configuration of the complex with a fluctuating pore size. Extracellular residue loops in trans interaction are able to form stable contacts and regulate the size of the pore, which displays a stationary radius of 2.5–3.0 Å at the narrowest region. The side-by-side interactions of the cis configuration are preserved via stable hydrogen bonds, already predicted by cysteine crosslinking experiments. Overall, this work introduces an improved version of the claudin-15-based paracellular channel model that strengthens its validity and that can be used in further computational studies to understand the structural features of tight-junctions regulation.

Structural dynamics of RbmA governs plasticity of Vibrio cholerae biofilms.

Biofilm formation is critical for the infection cycle of Vibrio cholerae. Vibrio exopolysaccharides (VPS) and the matrix proteins RbmA, Bap1 and RbmC are required for the development of biofilm architecture. We demonstrate that RbmA binds VPS directly and uses a binary structural switch within its first fibronectin type III (FnIII-1) domain to control RbmA structural dynamics and the formation of VPS-dependent higher-order structures. The structural switch in FnIII-1 regulates interactions in trans with the FnIII-2 domain, leading to open (monomeric) or closed (dimeric) interfaces. The ability of RbmA to switch between open and closed states is important for V. cholerae biofilm formation, as RbmA variants with switches that are locked in either of the two states lead to biofilms with altered architecture and structural integrity.

Dendritic Eph organizes dendrodendritic segregation in discrete olfactory map formation in Drosophila.

Proper function of the neural network results from the precise connections between axons and dendrites of presynaptic and postsynaptic neurons, respectively. In the Drosophila olfactory system, the dendrites of projection neurons (PNs) stereotypically target one of ∼50 glomeruli in the antennal lobe (AL), the primary olfactory center in the brain, and form synapses with the axons of olfactory receptor neurons (ORNs). Here, we show that Eph and Ephrin, the well-known axon guidance molecules, instruct the dendrodendritic segregation during the discrete olfactory map formation. The Eph receptor tyrosine kinase is highly expressed and localized in the glomeruli related to reproductive behavior in the developing AL. In one of the pheromone-sensing glomeruli (DA1), the Eph cell-autonomously regulates its dendrites to reside in a single glomerulus by interacting with Ephrins expressed in adjacent PN dendrites. Our data demonstrate that the trans interaction between dendritic Eph and Ephrin is essential for the PN dendritic boundary formation in the DA1 olfactory circuit, potentially enabling strict segregation of odor detection between pheromones and the other odors.

Developmental Expression of Claudins in the Mammary Gland

Claudins are a large family of membrane proteins whose classic function is to regulate the permeability of tight junctions in epithelia. They are tetraspanins, with four alpha-helices crossing the membrane, two extracellular loops, a short cytoplasmic N-terminus and a longer and more variable C-terminus. The extracellular ends of the helices are known to undergo side-to-side (cis) interactions that allow the formation of claudin polymers in the plane of the membrane. The extracellular loops also engage in head-to-head (trans) interactions thought to mediate the formation of tight junctions. However, claudins are also present in intracellular structures, thought to be vesicles, with less well-characterized functions. Here, we briefly review our current understanding of claudin structure and function followed by an examination of changes in claudin mRNA and protein expression and localization through mammary gland development. Claudins-1, 3, 4, 7, and 8 are the five most prominent members of the claudin family in the mouse mammary gland, with varied abundance and intracellular localization during the different stages of post-pubertal development. Claudin-1 is clearly localized to tight junctions in mammary ducts in non-pregnant non-lactating animals. Cytoplasmic puncta that stain for claudin-7 are present throughout development. During pregnancy claudin-3 is localized both to the tight junction and basolaterally while claudin-4 is found only in sparse puncta. In the lactating mouse both claudin-3 and claudin-8 are localized at the tight junction where they may be important in forming the paracellular barrier. At involution and under challenge by lipopolysaccharide claudins −1, −3, and −4 are significantly upregulated. Claudin-3 is still colocalized with tight junction molecules but is also distributed through the cytoplasm as is claudin-4. These largely descriptive data provide the essential framework for future mechanistic studies of the function and regulation of mammary epithelial cell claudins.

A network of cis and trans interactions is required for ParB spreading.

Most bacteria utilize the highly conserved parABS partitioning system in plasmid and chromosome segregation. This system depends on a DNA-binding protein ParB, which binds specifically to the centromere DNA sequence parS and to adjacent non-specific DNA over multiple kilobases in a phenomenon called spreading. Previous single-molecule experiments in combination with genetic, biochemical and computational studies have argued that ParB spreading requires cooperative interactions between ParB dimers including DNA bridging and possible nearest-neighbor interactions. A recent structure of a ParB homolog co-crystallized with parS revealed that ParB dimers tetramerize to form a higher order nucleoprotein complex. Using this structure as a guide, we systematically ablated a series of proposed intermolecular interactions in the Bacillus subtilis ParB (BsSpo0J) and characterized their effect on spreading using both in vivo and in vitro assays. In particular, we measured DNA compaction mediated by BsSpo0J using a recently developed single-molecule method to simultaneously visualize protein binding on single DNA molecules and changes in DNA conformation without protein labeling. Our results indicate that residues acting as hubs for multiple interactions frequently led to the most severe spreading defects when mutated, and that a network of both cis and trans interactions between ParB dimers is necessary for spreading.

NG2 Proteoglycan Enhances Brain Tumor Progression by Promoting Beta-1 Integrin Activation in both Cis and Trans Orientations.

By physically interacting with beta-1 integrins, the NG2 proteoglycan enhances activation of the integrin heterodimers. In glioma cells, co-localization of NG2 and α3β1 integrin in individual cells (cis interaction) can be demonstrated by immunolabeling, and the NG2-integrin interaction can be confirmed by co-immunoprecipitation. NG2-dependent integrin activation is detected via use of conformationally sensitive monoclonal antibodies that reveal the activated state of the beta-1 subunit in NG2-positive versus NG2-negative cells. NG2-dependent activation of beta-1 integrins triggers downstream activation of FAK and PI3K/Akt signaling, resulting in increased glioma cell proliferation, motility, and survival. Similar NG2-dependent cis activation of beta-1 integrins occurs in microvascular pericytes, leading to enhanced proliferation and motility of these vascular cells. Surprisingly, pericyte NG2 is also able to promote beta-1 integrin activation in closely apposed endothelial cells (trans interaction). Enhanced beta-1 signaling in endothelial cells promotes endothelial maturation by inducing the formation of endothelial junctions, resulting in increased barrier function of the endothelium and increased basal lamina assembly. NG2-dependent beta-1 integrin signaling is therefore important for tumor progression by virtue of its affects not only on the tumor cells themselves, but also on the maturation and function of tumor blood vessels.

Heterologous protein-DNA interactions lead to biased allelic expression of circadian clock genes in interspecific hybrids

Genomic interactions in allopolyploids create expression variation of homoeologous alleles through protein-protein and protein-DNA interactions. However, the molecular basis for this is largely unknown. Here we investigated the protein-protein and protein-DNA interactions among homoeologous transcription factors in the circadian-clock feedback loop, consisting of CCA1 HIKING EXPEDITION (CHE), CIRCADIAN CLOCK ASSOCIATED1 (CCA1), and TIMING OF CAB EXPRESSION1 (TOC1), plus the interaction with a chromatin factor, HISTONE DEACETYLASE1 (HD1). In the allotetraploids formed between A. thaliana (At) and Arabidopsis arenosa (Aa), AtCCA1 is expressed at lower levels than AaCCA1, which could alter clock output traits. The reduced AtCCA1 expressions in the allotetraploids are consistent with the biochemical data that AaCHE showed preferential binding to the AtCCA1 promoter, in which AaCHE interacts with a higher affinity to AtHD1 than AtCHE. AaCHE also showed a higher affinity to TOC1 than AtCHE, consistent with the effect of TOC1 on repressing CCA1. Thus, stronger AaCHE-TOC1 and AaCHE-AtHD1 interactions reduce AtCC1 allelic expression. Our current data suggest a biochemical basis for protein interactions in trans with a preference to the cis-acting elements in heterologous combinations to reduce AtCCA1 expression, while altered CCA1 expression has been shown to affect metabolic and biomass heterosis in interspecific hybrids or allotetraploids.

Combining genetic and biophysical approaches to probe the structure and function relationships of the notch receptor.

Notch is a conserved cell signalling receptor regulating many aspects of development and tissue homeostasis. Notch is activated by ligand-induced proteolytic cleavages that release the Notch intracellular domain, which relocates to the nucleus to regulate gene transcription. Proteolytic activation first requires mechanical force to be applied to the Notch extracellular domain through an endocytic pulling mechanism transmitted through the ligand/receptor interface. This exposes the proteolytic cleavage site allowing the signal to be initiated following removal of the Notch extracellular domain. Ligands can also act, when expressed in the same cell, through non-productive cis-interactions to inhibit Notch activity. Furthermore, ligand selectivity and Notch activation are regulated by numerous post-translational modifications of the extracellular domain. Additional non-canonical trans and cis interactions with other regulatory proteins may modulate alternative mechanisms of Notch activation that depend on endocytic trafficking of the full-length receptor and proteolytic release of the intracellular domain from endo-lysosomal surface. Mutations of Notch, located in different regions of the protein, are associated with a spectrum of different loss and gain of function phenotypes and offer the possibility to dissect distinct regulatory interactions and mechanisms, particularly when combined with detailed structural analysis of Notch in complex with various regulatory partners.

On the tautomerisation of porphycene on copper (111): Finding the subtle balance between van der Waals interactions and hybridisation.

We use density-functional theory (DFT) to analyse the interaction of trans- and cis-porphycene with Cu(111) and their interconversion by intramolecular H-transfer. This tautomerisation reaction is characterised by small values for the reaction energy and barrier, on the order of ∼0.1 eV, where the trans configuration is thermodynamically more stable upon adsorption according to the experiments [J. N. Ladenthin et al., ACS Nano 9, 7287-7295 (2015)]. To gain even a qualitatively correct description of this reaction at the DFT level, an accurate treatment of dispersion interactions and a careful choice of the exchange contribution are required in order to predict the subtle energetics. Analysis of the electronic structure shows that adsorption is contributed by a van der Waals (vdW) interaction, mainly responsible for stabilising the polyaromatic fragments, and by a significant charge redistribution localised between Cu and the unsaturated N atoms of the molecule central cavity. We find that different vdW functionals can produce qualitatively different electronic structures, while yielding small trans vs. cis energy differences. Unlike other functionals surveyed here, vdW-DF with PBE exchange satisfactorily reproduces not only the experimental energetics but also the scanning tunneling microscopy images. This gives us confidence that this functional achieves a reliable balance between the two mechanisms contributing to the adsorption of porphycene.

Electrostatic Control of Polymer Translocation Speed through α‑Hemolysin Protein Pore.

The electrostatic origins behind the speed of translocation of a uniformly charged flexible macromolecule through α-hemolysin (αHL) protein pores under a voltage are investigated using variations in pH and electrolyte concentration. We have measured durations of successful threading of poly(styrenesulfonate) through αHL at two different pH conditions, pH 4.5 and pH 7.5, under various salt concentration conditions. Salt concentrations in the donor (cis) and the recipient (trans) compartments influence the polymer translocation dynamics differently, depending on pH. At both pH 4.5 and pH 7.5, decreasing the cis salt concentration, cs,cis , results in faster polymer translocations. On the other hand, a decrease in trans salt concentration, cs,trans , retards the polymer transport process at pH 4.5, while at pH 7.5 the translocation time is observed to be independent of cs,trans . We present a theoretical model to calculate the translocation times from the free energy of the polymer along the translocation process to describe our experimental results. We show that the charge density of the polymer inside the nanopore is significantly affected by cs,cis , explaining the cis salt effect on the speed of polymer translocation. The trans salt effects are attributed to the electrostatic interaction between the polymer and the exit portion of the αHL pore, which is determined by the pH of the trans compartment. At low pH where the net charge of the end of the αHL is positive, the attractive electrostatic interaction in trans becomes stronger, as cs,trans decreases, resulting in delays in translocation process.

Metabolic sialic acid blockade lowers the activation threshold of moDCs for TLR stimulation.

Sialic acid sugars cover the surface of dendritic cells (DCs) and have been suggested to impact several aspects of DC biology. Research into the role of sialic acids in DCs, however, is complicated by the limited number of tools available to modulate sialic acid expression. Here we report on a synthetic, fluorinated sialic acid mimetic, Ac53FaxNeu5Ac, which potently blocks sialic acid expression in human monocyte-derived DCs (moDCs). Sialic acid blockade enhanced the responsiveness of moDCs to Toll-like receptor (TLR) stimulation as measured by increased maturation marker expression and cytokine production. Consequently, the T-cell activation capacity of Ac53FaxNeu5Ac-treated moDCs was strongly increased. In addition to sialic acids, moDCs also expressed the sialic acid-binding immunoglobulin-like lectins (Siglecs) -3, -5, -7, -9 and -10, immune inhibitory receptors recognizing these sialic acids. Treatment with Ac53FaxNeu5Ac abrogated putative cis and trans interactions between sialic acids and Siglec-7/-9. Together, these data indicate that sialic acids limit the activation of moDCs via the TLR pathway, potentially by interacting with Siglec-7 or Siglec-9. Metabolic sialic acid blockade with Ac53FaxNeu5Ac could therefore potentially be used to generate more potent DC-based vaccines for induction of robust anti-viral or anti-tumor immune responses.

Molecular basis of sidekick-mediated cell-cell adhesion and specificity.

Sidekick (Sdk) 1 and 2 are related immunoglobulin superfamily cell adhesion proteins required for appropriate synaptic connections between specific subtypes of retinal neurons. Sdks mediate cell-cell adhesion with homophilic specificity that underlies their neuronal targeting function. Here we report crystal structures of Sdk1 and Sdk2 ectodomain regions, revealing similar homodimers mediated by the four N-terminal immunoglobulin domains (Ig1-4), arranged in a horseshoe conformation. These Ig1-4 horseshoes interact in a novel back-to-back orientation in both homodimers through Ig1:Ig2, Ig1:Ig1 and Ig3:Ig4 interactions. Structure-guided mutagenesis results show that this canonical dimer is required for both Sdk-mediated cell aggregation (via trans interactions) and Sdk clustering in isolated cells (via cis interactions). Sdk1/Sdk2 recognition specificity is encoded across Ig1-4, with Ig1-2 conferring the majority of binding affinity and differential specificity. We suggest that competition between cis and trans interactions provides a novel mechanism to sharpen the specificity of cell-cell interactions.

Force Bistability in Adhesion Switch

The lateral junctions of epithelial cells must display mechanical stability to form barriers, but also significant plasticity to allow for reorganization during development and repair. In this issue, Biswas et al. (1) extend the use of fluid supported-lipid bilayers (SLBs) to reconstitute function of the key adhesion molecule that initiates lateral junctions of epithelial cells, E-cadherin, and to further study the requirements for mechanical forces in activating the cytoplasmic complex that links E-cadherin to the cytoskeleton (1).

P8001 3D nuclear positioning of IGF2 alleles and trans interactions with imprinted genes

P8001 3D nuclear positioning of IGF2 alleles and trans interactions with imprinted genes

Adiposity-Dependent Regulatory Effects on Multi-tissue Transcriptomes

Obesity is a global epidemic that is causally associated with a range of diseases, including type 2 diabetes and cardiovascular disease, at the population-level. However, there is marked heterogeneity in obesity-related outcomes among individuals. This might reflect genotype-dependent responses to adiposity. Given that adiposity, measured by BMI, is associated with widespread changes in gene expression and regulatory variants mediate the majority of known complex trait loci, we sought to identify gene-by-BMI (G × BMI) interactions on the regulation of gene expression in a multi-tissue RNA-sequencing (RNA-seq) dataset from the TwinsUK cohort (n = 856). At a false discovery rate of 5%, we identified 16 cis G × BMI interactions (top cis interaction: CHURC1, rs7143432, p = 2.0 × 10−12) and one variant regulating 53 genes in trans (top trans interaction: ZNF423, rs3851570, p = 8.2 × 10−13), all in adipose tissue. The interactions were adipose-specific and enriched for variants overlapping adipocyte enhancers, and regulated genes were enriched for metabolic and inflammatory processes. We replicated a subset of the interactions in an independent adipose RNA-seq dataset (deCODE genetics, n = 754). We also confirmed the interactions with an alternate measure of obesity, dual-energy X-ray absorptiometry (DXA)-derived visceral-fat-volume measurements, in a subset of TwinsUK individuals (n = 682). The identified G × BMI regulatory effects demonstrate the dynamic nature of gene regulation and reveal a functional mechanism underlying the heterogeneous response to obesity. Additionally, we have provided a web browser allowing interactive exploration of the dataset, including of association between expression, BMI, and G × BMI regulatory effects in four tissues.