Cadherin-directed actin assembly: E-cadherin physically associates with the Arp2/3 complex to direct actin assembly in nascent adhesive contacts

Cadherin cell adhesion molecules are major determinants of tissue patterning which function in cooperation with the actin cytoskeleton. In the context of stable adhesion, cadherin/catenin complexes are often envisaged to passively scaffold onto cortical actin filaments. However, cadherins also form dynamic adhesive contacts during wound healing and morphogenesis. Here actin polymerization has been proposed to drive cell surfaces together, although F-actin reorganization also occurs as cell contacts mature. The interaction between cadherins and actin is therefore likely to depend on the functional state of adhesion. We sought to analyze the relationship between cadherin homophilic binding and cytoskeletal activity during early cadherin adhesive contacts. Dissecting the specific effect of cadherin ligation alone on actin regulation is difficult in native cell-cell contacts, due to the range of juxtacrine signals that can arise when two cell surfaces adhere. We therefore activated homophilic ligation using a specific functional recombinant protein. We report the first evidence that E-cadherin associates with the Arp2/3 complex actin nucleator and demonstrate that cadherin binding can exert an active, instructive influence on cells to mark sites for actin assembly at the cell surface.     

Cellular senescence mediated by p16INK4A-coupled miRNA pathways

p16 is a key regulator of cellular senescence, yet the drivers of this stable state of proliferative arrest are not well understood. Here, we identify 22 senescence-associated microRNAs (SA-miRNAs) in normal human mammary epithelial cells. We show that SA-miRNAs-26b, 181a, 210 and 424 function in concert to directly repress expression of Polycomb group (PcG) proteins CBX7, embryonic ectoderm development (EED), enhancer of zeste homologue 2 (EZH2) and suppressor of zeste 12 homologue (Suz12), thereby activating p16. We demonstrate the existence of a tight positive feedback loop in which SA-miRNAs activate and re-enforce the expression of other SA-miRNA members. In contrast, PcG members restrain senescence by epigenetically repressing the expression of these SA-miRNAs. Importantly, loss of p16 leads to repression of SA-miRNA expression, intimately coupling this effector of senescence to the SA-miRNA/PcG self-regulatory loop. Taken together, our findings illuminate an important regulatory axis that underpins the transition from proliferation to cellular senescence.     

The influence of spatial pulsed magnetic field application on neuropathic pain after tibial nerve transection in rat

The purpose of the study was to examine the influence of the spatial variable magnetic field (induction: 150–300?µT, 80–150?µT, 20–80?µT; frequency 40?Hz) on neuropathic pain after tibial nerve transection. The experiments were carried out on 64 male Wistar C rats. The exposure of animals to magnetic field was performed 1?d/20?min., 5?d/week, for 28?d. Behavioural tests assessing the intensity of allodynia and sensitivity to mechanical and thermal stimuli were conducted 1?d prior to surgery and 3, 7, 14, 21 and 28?d after the surgery. The extent of autotomy was examined. Histological and immunohistochemical analysis was performed. The use of extremely low-frequency magnetic fields of minimal induction values (20–80?µT/40?Hz) decreased pain in rats after nerve transection. The nociceptive sensitivity of healthy rats was not changed following the exposition to the spatial magnetic field of the low frequency. The results of histological and immunohistochemical investigations confirm those findings. Our results indicate that extremely low-frequency magnetic field may be useful in the neuropathic pain therapy.     

Variable spatial magnetic field influences peripheral nerves regeneration in rats

Generator of spatial magnetic field is one of most recent achievements among the magnetostimulators. This apparatus allows to obtain the rotating magnetic field. This new method may be more effective than other widely used techniques of magnetostimulation and magnetotherapy. We investigated the influence of alternating, spatial magnetic field on the regeneration of the crushed rat sciatic nerves. Functional and morphological evaluations were used. After crush injury of the right sciatic nerve, Wistar C rats (n?=?80) were randomly divided into four groups (control and three experimental). The experimental groups (A, B, C) were exposed (20?min/day, 5?d/week, 4 weeks) to alternating spatial magnetic field of three different intensities. Sciatic Functional Index (SFI) and tensometric assessments were performed every week after nerve crush. Forty-eight hours before the sacrificing of animals, DiI (1,1’-di-octadecyl-3,3,3’,3’-tetramethyloindocarbocyanine perchlorate) was applied 5?mm distally to the crush site. Collected nerves and dorsal root ganglia (DRG) were subjected to histological and immunohistochemical staining. The survival rate of DRG neurons was estimated. Regrowth and myelination of the nerves was examined. The results of SFI and tensometric assessment showed improvement in all experimental groups as compared to control, with best outcome observed in group C, exposed to the strongest magnetic field. In addition, DRG survival rate and nerve regeneration intensity were significantly higher in the C group. Above results indicate that strong spatial alternating magnetic field exerts positive effect on peripheral nerve regeneration and its application could be taken under consideration in the therapy of injured peripheral nerves.     

Development of Central Nervous System Autoimmunity Is Impaired in the Absence of Wiskott-Aldrich Syndrome Protein

Wiskott-Aldrich Syndrome protein (WASP) is a key regulator of the actin cytoskeleton in hematopoietic cells. Defective expression of WASP leads to multiple abnormalities in different hematopoietic cells. Despite severe impairment of T cell function, WAS patients exhibit a high prevalence of autoimmune disorders. We attempted to induce EAE, an animal model of organ-specific autoimmunity affecting the CNS that mimics human MS, in Was^−/− mice. We describe here that Was^−/− mice are markedly resistant against EAE, showing lower incidence and milder score, reduced CNS inflammation and demyelination as compared to WT mice. Microglia was only poorly activated in Was^−/− mice. Antigen-induced T-cell proliferation, Th-1 and -17 cytokine production and integrin-dependent adhesion were increased in Was^−/− mice. However, adoptive transfer of MOG-activated T cells from Was^−/− mice in WT mice failed to induce EAE. Was^−/− mice were resistant against EAE also when induced by adoptive transfer of MOG-activated T cells from WT mice. Was^+/− heterozygous mice developed an intermediate clinical phenotype between WT and Was^−/− mice, and they displayed a mixed population of WASP-positive and -negative T cells in the periphery but not in their CNS parenchyma, where the large majority of inflammatory cells expressed WASP. In conclusion, in absence of WASP, T-cell responses against a CNS autoantigen are increased, but the ability of autoreactive T cells to induce CNS autoimmunity is impaired, most probably because of an inefficient T-cell transmigration into the CNS and defective CNS resident microglial function.     

Interaction with Both Domain I and III of Albumin Is Required for Optimal pH-dependent Binding to the Neonatal Fc Receptor (FcRn)

Albumin is an abundant blood protein that acts as a transporter of a plethora of small molecules like fatty acids, hormones, toxins, and drugs. In addition, it has an unusual long serum half-life in humans of nearly 3 weeks, which is attributed to its interaction with the neonatal Fc receptor (FcRn). FcRn protects albumin from intracellular degradation via a pH-dependent cellular recycling mechanism. To understand how FcRn impacts the role of albumin as a distributor, it is of importance to unravel the structural mechanism that determines pH-dependent binding. Here, we show that although the C-terminal domain III (DIII) of human serum albumin (HSA) contains the principal binding site, the N-terminal domain I (DI) is important for optimal FcRn binding. Specifically, structural inspection of human FcRn (hFcRn) in complex with HSA revealed that two exposed loops of DI were in proximity with the receptor. To investigate to what extent these contacts affected hFcRn binding, we targeted selected amino acid residues of the loops by mutagenesis. Screening by in vitro interaction assays revealed that several of the engineered HSA variants showed decreased binding to hFcRn, which was also the case for two missense variants with mutations within these loops. In addition, four of the variants showed improved binding. Our findings demonstrate that both DI and DIII are required for optimal binding to FcRn, which has implications for our understanding of the FcRn-albumin relationship and how albumin acts as a distributor. Such knowledge may inspire development of novel HSA-based diagnostics and therapeutics.     

Refinement of the MHC Risk Map in a Scandinavian Primary Sclerosing Cholangitis Population

Background

Genetic variants within the major histocompatibility complex (MHC) represent the strongest genetic susceptibility factors for primary sclerosing cholangitis (PSC). Identifying the causal variants within this genetic complex represents a major challenge due to strong linkage disequilibrium and an overall high physical density of candidate variants. We aimed to refine the MHC association in a geographically restricted PSC patient panel.

Methodology/Principal Findings

A total of 365 PSC cases and 368 healthy controls of Scandinavian ancestry were included in the study. We incorporated data from HLA typing (HLA-A, -B, -C, -DRB3, -DRB1, -DQB1) and single nucleotide polymorphisms across the MHC (n = 18,644; genotyped and imputed) alongside previously suggested PSC risk determinants in the MHC, i.e. amino acid variation of DRβ, a MICA microsatellite polymorphism and HLA-C and HLA-B according to their ligand properties for killer immunoglobulin-like receptors. Breakdowns of the association signal by unconditional and conditional logistic regression analyses demarcated multiple PSC associated MHC haplotypes, and for eight of these classical HLA class I and II alleles represented the strongest association. A novel independent risk locus was detected near NOTCH4 in the HLA class III region, tagged by rs116212904 (odds ratio [95% confidence interval] = 2.32 [1.80, 3.00], P = 1.35×10⁻¹¹).

Conclusions/Significance

Our study shows that classical HLA class I and II alleles, predominantly at HLA-B and HLA-DRB1, are the main risk factors for PSC in the MHC. In addition, the present assessments demonstrated for the first time an association near NOTCH4 in the HLA class III region.