Characterization of β1 Integrin Cytoplasmic Domain Binding Proteins

The extracellular matrix (ECM) provides the structural frame for the development of tissues and organs. The ECM is bound by numerous membranous matrix-adhesion molecules and thereby triggers intracellular signals that control various cellular functions such as survival, polarity, proliferation and differentiation. Integrins represent an important family of ECM adhesion molecules which link the ECM with the intracellular actin-cytoskeleton. Integrin mediated adhesion structures also serve as important signaling platforms, although the integrin itself does not harbors any catalytic domains. Therefore integrin signaling depends on the recruitment of a number of cytoplasmic proteins that directly or indirectly bind to the short cytoplasmic integrin tails. During my PhD thesis I worked on three of these molecules, ILK, Kindlins and Palladin, and used the mouse as a model system to address their in vivo function. First, I investigated the role of integrin-linked kinase (ILK) in skeletal muscle. Loss of ILK expression in mice leads to peri-implantation lethality due to a cell polarization defect of the early embryo and abnormal actin accumulations. Studies in Caenorhabditis elegans and Drosophila melanogaster revealed an essential function for ILK in the attachment of actin filaments to the membrane of muscle cells and lack of ILK expression results in early lethality during embryogenesis. We generated mice with a skeletal muscle-restricted deletion of ILK that developed a mild, but progressive muscular dystrophy. This phenotype is predominantly restricted to myotendinous junctions (MTJs). Ultrastructural analyses showed muscle cell detachment from the basement membranes, and an accumulation of extracellular matrix. Endurance exercise training enhances the defect leading to disturbed subsarcolemmal myofiber architecture and an abrogation of the phosphorylation of Ser473 as well as Thr308 of protein kinase B (PKB)/Akt. The reduction in PKB/Akt activation is accompanied by an impaired insulin-like growth factor 1 receptor (IGF-1R) activation. Second, I studied the expression and in vivo function of a further integrin- and actin- associated protein, palladin. Palladin belongs to the palladin/myotilin/myopalladin protein family. Palladin represents a phosphoprotein which plays an important role in cell adhesion and motility. Initially, I characterized the gene structure and the expression pattern of palladin. The palladin gene spans about 400 kb, with 25 exons and 3 alternative promoters resulting in at least three different isoforms (200 kDa, 140 kDa and 90-92 kDa) in mice. Using RT-PCR and in situ hybridizations of embryonic and adult tissues, I could show that the 200kDa isoform is predominantly expressed in heart and skeletal muscle. In contrast, the 140kDa isoform is expressed in various tissues and represents the major palladin isoform of the brain. The 90-92 kDa isoform is almost ubiquitously expressed with highest levels in tissues rich in smooth muscle, like bladder, uterus, small intestine and colon. The expression of the 200kDa isoform was characterized in more detail with a polyclonal antibody showing that this isoform localizes to the Z-discs of heart and skeletal muscle cells. In vitro differentiation experiments with a mouse myoblast cell line revealed an induction of the 200kDa isoform during myoblast fusion and differentiation suggesting that the biggest palladin isoform may serve as a molecular scaffold during myogenesis. Third, I specifically inactivated the largest palladin isoform in mice. Lack of the 200 kDa palladin isoform has no impact on the development, viability and fertility of mice. However ultrastructural analyses by transmission electron microscopy (TEM) showed a mild cardiac myopathy due to disintegration of myofibrils. In collaboration with the group of Olli Carpén, we generated palladin 200 kDa isoform/ myotilin double knockout mice. Myotilin is also expressed in heart and skeletal muscle. Ablation of both myotilin and palladin 200 kDa isoform in mouse revealed in addition to the mild cardiac myopathy a structural and functional impairment of skeletal muscle. Finally, I was also involved in the characterization of the expression and subcellular localization of a novel family of integrin associated proteins: the Kindlins. The Kindlin family consists of three members, Kindlin-1, -2 and -3. Mutations in Kindlin-1 cause a human disease, called Kindler Syndrome, which represents a skin blistering disease affecting the actin cytoskeleton of basal keratinocytes. Kindlin gene expression was first analyzed at the mRNA level by RT-PCR and Northern Blot studies. In situ hybridizations showed that Kindlin-1 is preferentially expressed in epithelia. Kindlin-2 is expressed in all tissues with highest levels in striated and smooth muscle cells. While both localize to integrin-mediated adhesion sites in cultured keratinocytes Kindlin-2, but not Kindlin-1, colocalizes with E-cadherin to cell-cell contacts in differentiated keratinocytes. In contrast, Kindlin-3 expression is restricted to hematopoietic cells. Using a Kindlin-3-specific antiserum and an EGFP-tagged Kindlin-3 construct, we could show that Kindlin-3 is present in podosomes, which are specialized adhesion structures of hematopoietic cells

Modulation of Mitochondria During Viral Infections

Mitochondria are organelles critical for cell survival because they produce ATP and modulate programmed cell death (PCD) pathways. PCD pathways are important in many clinical disorders, such as ischemia/reperfusion injuries, trauma, and toxic/metabolic syndromes, as well as in chronic neurodegenerative conditions, such as amyotrophic lateral sclerosis, Alzheimer’s disease, Parkinson’s disease, and Huntington’s disease. Moreover, many viruses and other pathogens target the mitochondria. Viruses induce the production of various proteins in their hosts that have proapoptotic and anti-apoptotic activities, depending on the cellular environment. More specifically, many viruses that target mitochondria regulate the balance between the anti- and proapoptotic Bcl-2 family proteins and thereby increase their own survival within the host cell. Recent studies indicated that mitochondria centralize several critical innate immune responses based on the presence of several important signaling proteins within the mitochondria: mitochondrial antiviral signaling (MAVS), stimulation of interferon genes (STING), and NLR family member X1. Therefore, mitochondria are not only vital because they regulate cell survival and death but also they have broad roles in the control of cell functions following pathogen invasion. This chapter highlights the tight interplay between viral infection and mitochondria

Nonequilibrium Pathways during Electrochemical Phase Transformations in Single Crystals Revealed by Dynamic Chemical Imaging at Nanoscale Resolution

© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. The energy density of current batteries is limited by the practical capacity of the positive electrode, which is determined by the properties of the active material and its concentration in the composite electrode architecture. The observation in dynamic conditions of electrochemical transformations creates the opportunity of identifying design rules toward reaching the theoretical limits of battery electrodes. But these observations must occur during operation and at multiple scales. They are particularly critical at the single-particle level, where incomplete reactions and failure are prone to occur. Here, operando full-field transmission X-ray microscopy is coupled with X-ray spectroscopy to follow the chemical and microstructural evolution at the nanoscale of single crystals of Li1+xMn2-xO4, a technologically relevant Li-ion battery electrode material. The onset and crystallographic directionality of a series of complex phase transitions are followed and correlated with particle fracture. The dynamic character of this study reveals the existence of nonequilibrium pathways where phases at substantially different potentials can coexist at short length scales. The results can be used to inform the engineering of particle morphologies and electrode architectures that bypass the issues observed here and lead to optimized battery electrode properties

More sustainable electricity generation in hot and dry fuel cells with a novel hybrid membrane of Nafion/nano-silica/hydroxyl ionic liquid

A new hybrid proton exchange membrane (PEM) has been prepared from hydroxyl functionalized imidazolium ionic liquid (IL-OH), Nafion and nano-SiO2. The IL-OH, with a hydroxyl group that acts as both a proton acceptor and donor, forms strong hydrogen bonds with both Nafion and nano-SiO2, resulting in an effective hydrogen bond network in the ternary membrane. Such an anhydrous hydrogen-bond network, which is unknown previously, endows the PEMs with higher proton conductivity, greater thermal stability and surprisingly a more robust mechanical performance than PEMs consisting of conventional ionic liquids. The resulting PEMs have a tensile strength that is more than twice as strong as recast Nafion and an anhydrous ionic conductivity of ∼55 mS cm−1 at temperatures above 160 °C, with a proton transfer number of ∼0.9. A laboratory assembled H2–O2 fuel cell employing this new PEM delivered a power density of 340 and 420 mW cm−2 at 160 and 180 °C, respectively

Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data