31 research outputs found

    Force Dependent Changes in Non-Erythroid Spectrin and Ankyrins

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    The Cellular Content of Non-Erythroid Spectrins and Ankyrins is Modulated by External Forces

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    Electrical Response of Bilayers to the Bee Venom Toxin Melittin

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    THE NON-ERYTHROID MEMBRANE SKELETON AND ITS ROLE IN MECHANOTRANSDUCTION

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    The thesis is concerned with questions regarding cellular bio-mechanics and cell surface interactions. A particular focus is thereby on the role of the spectrin membrane skeleton in transducing forces to and from non-erythroid mammalian cells. This spectrin based membrane skeleton has been the focus of much study in the context of red blood cells, as it determines their mechanical properties due to the lack of an extended actin based cytoskeleton. In stark contrast, the corresponding structure in non-erythroids is much less studied and understood, although it seems to play important roles in organization of membrane associated proteins, cellular mechanics, adhesion, traction and possibly mechanotransduction. In this work, I was able to determine the amount -down to the average copy number per cell- of the main protein components of the non-erythroid membrane skeleton, in model cell lines commonly used in cell-mechanics studies. The results of the measurements provided by combining a variety of optical microscopic and biochemical techniques, demonstrate that proteins associated with the membrane skeleton constitute a large (s10%) fraction of cellular proteins. These results are then compared with the respective quantities after mechanical stimulation of the cells. It is found that external forces result in both an up to 60% changes of the overall amounts of proteins as well as the protein composition of the membrane skeleton itself. In addition, it was established that the fraction of polyubiquitnated spectrin has significantly increased due to stimulation. The work helps to establish the fact that the spectrin based membrane skeleton, while often overlooked in non-erythroids, is indeed a verily generic and important system in mammalian cells that is also quite sensitive to external forces. Thus, the skeleton should be taken into account when studying cellular mechanics, membrane structure or composition. Furthermore, I present my successful work on integrating several light based methods to simultaneously measure traction forces of adherent cells as well as internal strains in their membrane skeleton. For the proof of principle experiments and optimization procedures, I used NIH-3T3 fibroblast and H9c2 (2 -1) cardiomyocyte cell lines, both of which are known to be mechanically active. Using my method, I demonstrate that the internal strains in the membrane skeleton of fibroblasts are correlated with the polyacrylamide substrate stiffness. The later has also a measurable impact on the generated cellular traction forces. These findings open up a first glimpse on the questions that can now be addressed with this method and it promises to help to refine our still rudimentary understanding of the interrelated mechanisms of cellular mechanotransduction and force generation machinery. Cells are exposed not only to mechanical forces but also to electrical, chemical and magnetic forces found in their environment. In a separate series of experiments, I observed that cellular behavior of 3T3 fibroblasts on glass supported lipid bilayers depend on the detail of lipid charge mixtures in the bilayer as well as the head group compositions. Live cell-supported lipid membrane hybrids are frequently used in dissecting membrane based inter-cellular communication. This work indicates that in the interpretation of the observed cell behavior on this hybrid system, the lipid bilayer itself and not only the protein augmentation can play an important role

    Regulatory polymorphisms in the bovine Ankyrin 1 gene promoter are associated with tenderness and intramuscular fat content

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    peer-reviewedRecent QTL and gene expression studies have highlighted ankyrins as positional and functional candidate genes for meat quality. Our objective was to characterise the promoter region of the bovine ankyrin 1 gene and to test polymorphisms for association with sensory and technological meat quality measures. Results Seven novel promoter SNPs were identified in a 1.11 kb region of the ankyrin 1 promoter in Angus, Charolais and Limousin bulls (n = 15 per breed) as well as 141 crossbred beef animals for which meat quality data was available. Eighteen haplotypes were inferred with significant breed variation in haplotype frequencies. The five most frequent SNPs and the four most frequent haplotypes were subsequently tested for association with sensory and technological measures of meat quality in the crossbred population. SNP1, SNP3 and SNP4 (which were subsequently designated regulatory SNPs) and SNP5 were associated with traits that contribute to sensorial and technological measurements of tenderness and texture; Haplotype 1 and haplotype 4 were oppositely correlated with traits contributing to tenderness (P < 0.05). While no single SNP was associated with intramuscular fat (IMF), a clear association with increased IMF and juiciness was observed for haplotype 2. Conclusion The conclusion from this study is that alleles defining haplotypes 2 and 4 could usefully contribute to marker SNP panels used to select individuals with improved IMF/juiciness or tenderness in a genome-assisted selection framework.Department of Agriculture, Fisheries and Food, Ireland, through the Food Institutional Research Measur

    Unraveling the interactions of 14-3-3 with the neuronal proteins L1 and alpha II spectrin

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    ENAC REGULATION IN THE KIDNEY: THE ROLE OF ANKYRIN G

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    The epithelial sodium channel (ENaC) is the limiting entry point for Na+ reabsorption in the distal kidney nephron and is regulated by numerous hormones, including the mineralocorticoid hormone aldosterone. Previously we identified ankyrin G (AnkG), a cytoskeletal protein involved in vesicular transport, as a novel aldosterone-induced protein that can alter Na+ transport in mouse cortical collecting duct cells. AnkG is highly expressed in the kidney, particularly in the distal nephron. Increasing AnkG expression increases ENaC activity while depleting AnkG reduces ENaC-mediated Na+ transport. The underlying mechanism presiding over this effect; however, was unknown. Here we report that AnkG expression directly regulates Na+ transport by altering ENaC activity in the apical membrane. These changes are due to a change in ENaC directly rather than through alterations to the Na+ driving force created by Na+K+ATPase. Using a constitutively open mutant of ENaC and surface biotinylation, we demonstrate that the augmentation of Na+ transport is caused predominantly by increasing the number of ENaCs at the surface rather than alterations to open probability. To determine the mechanism of AnkG action on ENaC surface number, changes in rates of internalization, recycling, and membrane delivery were investigated. AnkG did not alter ENaC delivery to the membrane from biosynthetic pathways or removal by endocytosis; however, AnkG did alter ENaC insertion from constitutive recycling pathways. We also investigated the potential role of a putative AnkG binding domain in the C-terminus of ╬▓ENaC, and whether single-site mutations of a charged residue and two regulatory phosphorylation sites could disrupt AnkG augmentation of ENaC current. We did not find any significant evidence that this region is essential for AnkG-ENaC interaction. These findings provide a mechanism to account for the role of AnkG in the regulation of Na+ transport in the distal kidney nephron

    The Effect of Traumatic Brain Injury on Expression Levels of Ankyrin-G in the Corpus Callosum and Cerebral Cortex

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    The ankyrins comprise a family of proteins serving as components of the membrane cytoskeleton, and participate in a diverse set of associations with multiple binding partners including the cytoplasmic domains of transporters, ion channels, some classes of receptors, and cell adhesion proteins. Moreover, evidence is accumulating that ankyrin participates in defining functionally distinct subcellular regions. The complex functional and structural roles of ankyrins indicate they are likely to play essential roles in the pathology of traumatic axonal injury. The current study examined changes in ankyrin-G expression following a moderate central fluid percussion injury administered to adult rats. At 1d, 3d, and 7d postinjury (or following a sham control injury), protein levels of ankyrin-G in the corpus callosum and cerebral cortex were assessed using Western Blot analysis. Three immunopositive bands were identified in both brain regions as 220,212, and 75 kD forms of ankyrin-G. Time-dependent changes in ankyrin-G were observed in the corpus callosum. At 1d injury-induced elevations were observed in the callosal 220 kD (+147% relative to sham levels) and in the 212 kD (+73%) forms of ankyrin-G, but in both cases the expression decreased to control levels by 3d and 7d. In contrast, the 75 kD form showed moderate increases at 1d postinjury, but was significantly below control levels at 3d (-54%) and at 7d (-41%). Ankyrin-G expression in the cerebral cortex was only slightly affected by the injury, with a significant decrease in the `220 kD form occurring between 1d and 3d. These data suggest that the 220 and 212 kD changes probably represent postinjury proteolytic fragments derived from intact ankyrin-G isoforms of 480 andor 270 kD, while the 75 kD effects are likely breakdown products of intact 190 kD ankyrin-G. These results were discussed as they relate to prior findings of differential vulnerabilities of callosal myelinated and unmyelinated axons to injury. In this context, the 220,212 kD changes may reflect pathology within myelinated axons, and alterations to the 75 kD form may reflect more persistent pathology affecting unmyelinated callosal fibers

    Dysfunction in the ╬▓II Spectrin-Dependent Cytoskeleton Underlies Human Arrhythmia.

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    Background: The cardiac cytoskeleton plays key roles in maintaining myocyte structural integrity in health and disease. In fact, human mutations in cardiac cytoskeletal elements are tightly linked with cardiac pathologies including myopathies, aortopathies, and dystrophies. Conversely, the link between cytoskeletal protein dysfunction in cardiac electrical activity is not well understood, and often overlooked in the cardiac arrhythmia field. Methods and Results: Here, we uncover a new mechanism for the regulation of cardiac membrane excitability. We report that ╬▓II spectrin, an actin-associated molecule, is essential for the post-translational targeting and localization of critical membrane proteins in heart. ╬▓II spectrin recruits ankyrin-B to the cardiac dyad, and a novel human mutation in the ankyrin-B gene disrupts the ankyrin-B/╬▓II spectrin interaction leading to severe human arrhythmia phenotypes. Mice lacking cardiac ╬▓II spectrin display lethal arrhythmias, aberrant electrical and calcium handling phenotypes, and abnormal expression/localization of cardiac membrane proteins. Mechanistically, ╬▓II spectrin regulates the localization of cytoskeletal and plasma membrane/sarcoplasmic reticulum protein complexes that include the Na/Ca exchanger, RyR2, ankyrin-B, actin, and ╬▒II spectrin. Finally, we observe accelerated heart failure phenotypes in ╬▓II spectrin-deficient mice. Conclusions: Our findings identify ╬▓II spectrin as critical for normal myocyte electrical activity, link this molecule to human disease, and provide new insight into the mechanisms underlying cardiac myocyte biology

    Protease inhibitors-based therapy induces acquired spherocytic-like anemia and ineffective erythropoiesis in chronic HCV patients

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    Background & Aims: The addition of protease inhibitors, boceprevir (BOC)or telaprevir (TRV), to peg-interferon and ribavirin (PR) increases the incidenceof anaemia in patients with chronic hepatitis C virus (HCV) infection.Although genetic variants in inosine triphosphatase (ITPA) gene have beenlinked to the haemolytic anaemia induced by PR, the mechanism sustainingsevere anaemia during triple therapy is still unknown. This study aims to elucidatethe molecular mechanisms underlying anaemia in chronic HCVpatients with combined therapy. Methods: We studied 59 patients withchronic HCV genotype-1: 29 treated with TRV/PR and 30 with BOC/PR. Weevaluated biochemical and haematological parameters, red cell index at baseline,4, 12, 16 and 24 weeks of treatment; in a subgroup, we performed functionalstudies: osmotic fragility, red cell membrane protein separation, massspectrometry analysis, quantification of erythroid microparticles release.IL28B and ITPA polymorphisms were also evaluated. Results: We foundearly acute normochromic normocytic haemolytic anaemia (4\u20138 weeks) followedby a late macrocytic hypo-regenerative anaemia with inappropriatelow reticulocyte count (12\u201324 weeks). Studies on red cells revealed: (i) presenceof spherocytes; (ii) increased osmotic fragility; (iii) abnormalities in redcell membrane protein composition; (iv) reduced membrane-cytoskeletonstability; (v) increased release of erythroid microparticles. ITPA polymorphismsimpacted only the early phase of anaemia. Conclusions: The bimodalpattern of anaemia in chronic HCV patients on triple therapy might bebecause of acquired spherocytic-like anaemia in the early phase, followed byhyporegenerative anaemia, most likely related to the combined effects of PRand TRV or BOC on erythropoiesis
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