The biogenesis of integrin-dependent cell adhesions starts with the interaction of the glycocalyx embedded extracellular domain of integrins with ECM ligands. The interaction causes assembly of integrin-mediated adhesions with distinctive morphology, molecular
26 composition and dynamics, even in the same cell. Initially, activated ligand-bound integrins cluster to an early form of dot-shape integrin-mediated adhesions known as nascent FAs (NFAs; <0.1-0.5 µm2), that mainly assemble at the leading edge of migrating cells (Geiger and Yamada, 2011). They maturate to FCs and FAs (~1-5 µm2) by further integrin clustering and recruitment of actin-linker multiprotein complexes to the cytoplasmic platform of NFAs (Fig. 17). FAs that contain α5β1 integrin and are under intracellular actomyosine tensions can further develope into elongated fibrillar adhesions (FBs; >5 µm long) which are located in the central areas of cells.
Figure 17. Different type of integrin-based adhesions. (A) Nascent Focal adhesions (NFA), focal adhesions (FA) and fibrillar adhesions (FB) are visualized by Paxillin (green) and pY-Paxillin (red) immunostaining. (B)
Podosomes are visualized by Paxillin (red) and actin (green) immunostaining in osteoclasts. From (Geiger and Yamada, 2011).
Podosomes are another type of integrin-based adhesion that is found in smooth muscle cells, endothelial cells and cell types of monocytic origin (osteoclasts, machrophages and dendritic cells). They are ring-shaped adhesions that assemble around an F-actin core and contain matrix degrading enzymes. Podosomes are important for cell adhesion and degradation of matrixes such as bone by osteoclasts (Linder and Kopp, 2005).
So far, only a few in vivo integrin-mediated adhesions have been characterized including FAs of endothelial cells at sites of fluid shear stress in blood vessels and smooth muscle
27 dense plaque (Geiger and Yamada, 2011). The morphology, composition and signalling properties of integrin-based adhesions are considered to be different in 2D cell cultures and
in vivo environments (Fig. 18). Fibroblastic cells in 3D environment of tissue show distinct
morphology and are more biologically efficient during migration and proliferation when compared with 2D-matrix cell cultures (Cukierman et al., 2001).
Figure 18. In vivo integrin-based cell adhesions. Immunostaining of Paxillin (red) and α5 integrin (green) showed distinctive localization in vivo and in vitro. FAs (filled arrowheads) and FBs (open arrowheads) of
(A-E) FN-seeded NIH-3T3 mouse fibroblast in cell culture compared with (F-J) 3D-matrix adhesion (arrows) of craniofacial mesenchymes from transverse cryosections of E13.5 mouse embryo. Scale bar 5µm. From (Cukierman et al., 2001).
Recently, the ultra-structure -architecture of FAs has been revealed by cyro-electron tomography (Fig 19A-C). Patla et al. (2010) showed that the interaction of the plasma membrane with cytoskeleton in FAs of fibroblasts is mediated through hundreds of donut- like particles with a diameter of 20-30 nm. F-actin connections to particles were in different orientations (about 25 different structural classes; Fig 19D) proposing diversity in the molecular composition within FAs (Patla et al., 2010). It has been shown that many of FA proteins recruited to FAs bind integrin tails directly (Zaidel-Bar and Geiger, 2010). As cytoplasmic tails of integrins are short, the simultaneous interaction of all these molecules with the same integrin heterodimer is not possible and integrin tail-bound multiprotein complexes peobaly contain different composition in the same adhesion site.
FA proteins can be divided into four classes based on their binding/scaffolding properties: 1) Integrin-associated proteins that directly bind to F-actin cytoskeleton, such as talin, filamin and α-actinin; 2) Integrin-associated proteins that indirectly bind to/regulate the F-
28 actin cytoskeleton, such as Paxillin, FAK, ILK and kindlins; 3) Actin-binding proteins that are not associated with integrins directly, such as vinculin and Parvins; and 4) scaffolding/signalling molecules that tune integrin signals but are not associated with integrin/actin (Legate and Fassler, 2009).
Figure 19. Ultra-structure of FAs. Correlated microscopy, combining fluorescence microscopy and cryo- electron tomography. (A) Fibroblasts expressing YFP-Paxillin to visualize FAs (yellow and red arrows). Scale bar is 20 µm. (B) A cryo-tomogram slice of the same FA in panel A (yellow arrow) and (C) its surface rendering view with F-actin (brown), plasma membrane (blue) and donut-like adhesion-related particles (green). (D) Higher magnification of adhesion-related particles (20-30 nm diameters; green) linked to differentially oriented F-actin (brown). Adopted from (Patla et al., 2010).
3.1. Integrin-linked kinase (ILK)
ILK was initially found in a yeast-two hybrid screen as a direct binding partner of β1 and
β3 integrin tails (Hannigan et al., 1996; Pasquet et al., 2002). ILK consists of five N- terminal Ankyrin homology domains (ANK), a Plekestrin homology (PH) domain and a C- terminal pseudokinase (PK) domain. ILK predominantly localizes to FAs, although there have been reports on ILK recruitment to nucleus, centrosomes and cell-cell adhesions (Widmaier et al, 2011). ILK forms an ILK-Pinch-Parvin (IPP) complex in the cytoplasm by interacting with Lin-11, Isl-1 and Mec-3 (LIM) domain of Pinch and the second Caplonin homology (CH) doman of Parvins (Widmaier et al, 2011). The formation of the IPP complex prevents the proteasomal degradation of its components. IPP serves as a signalling platform that recruits many other molecules. Post translational modification of IPP components and alternative assembly of Pinch and Parvin isoforms give rises to distinctive signalling outputs that are cell type-, tissue- and developmental stage-specific.
29 ILK was originally considered to be a Ser/Thr protein kinase; however, X-ray crystallography and genetic studies in D. melanogaster, C. elegans and mice demonstrated that the ILK kinase domain is catalytically inactive (Wickstrom et al., 2010). ILK controls cell polarity, proliferation, differentiation, stability of microtubule tips at the cell cortex and the organization of the F-actin cytoskeleton through signalling crosstalks with growth GFRs (Grashoff et al., 2003; Lange et al., 2009; Legate et al., 2006; Lorenz et al., 2007; Mackinnon et al., 2002; Wang et al., 2008; Wickstrom et al., 2011). However, it remains unclear how ILK co-operates with GFR to control F-actin remodeling and downstream signalling pathways. For more information the reader is referred to a review article presented in the appendix of this thesis (Widmaier et al, 2011).