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