Journal of Cell Science Functional analysis of parvin and different modes of IPP-complex assembly at integrin sites during Drosophila development Katerina M. Vakaloglou*, Maria Chountala* and Christos G. Zervas ` Biomedical Research Foundation, Academy of Athens (BRFAA), Division of Genetics, Soranou Efessiou 4, 11527 Athens, Greece *These authors contributed equally to this work ` Author for correspondence (czervas@bioacademy.gr) Accepted 5 March 2012 Journal of Cell Science 125, 3221–3232 ß 2012. Published by The Company of Biologists Ltd doi: 10.1242/jcs.102384 Summary Integrin-linked kinase (ILK), PINCH and parvin constitute the tripartite IPP complex that maintains the integrin–actin link at embryonic muscle attachment sites (MASs) in Drosophila. Here we showed that parvin null mutants in Drosophila exhibit defects in muscle adhesion, similar to ILK and PINCH mutants. Furthermore, the identical muscle phenotype of the triple mutant, which for the first time in any organism removed the entire IPP-complex function, genetically demonstrated that parvin, ILK and PINCH function synergistically. This is consistent with the tight localization of the tripartite complex at sites of integrin adhesion, namely MASs in the developing embryo and focal-contact-like structures in the wing epithelium. Parvin contains tandem unconventional calponin-homology (CH) domains separated by a linker sequence, and a less-well conserved N-terminal region. In vivo structure–function analysis revealed that all the domains are essential for parvin function, whereas recruitment at integrin adhesion sites is mediated by two localization signals: one located within the CH2 domain as previously reported, and a second novel signal within the CH1 domain. Interestingly, this site is masked by the linker region between the two CH domains, suggesting a regulatory mechanism to control parvin localization. Finally, whereas in muscles only ILK controls the stability and localization of both PINCH and parvin, in the wing epithelium the three proteins mutually depend on each other. Thus molecular differences exist in the assembly properties of IPP complex in specific tissues during development, where differential modulation of the integrin connection to the cytoskeleton is required. Key words: ILK, PINCH, Muscle attachment sites, Adhesion, Actin Introduction During animal development cells assemble into tissues and establish specific adhesion sites with the surrounding extracellular matrix (ECM). ECM enables the stable attachment of cells and their separation in distinct layers permitting tissue morphogenesis. The integrin family of transmembrane proteins mediate physical contact of cells with the ECM. This connection requires direct binding of the extracellular domain of both the a- and the b-subunit of integrin to their ECM ligands, whereas their small cytoplasmic tails bind a network of proteins to form the integrin adhesome and mediate the link to the actin cytoskeleton (Geiger and Yamada, 2011). The molecular composition of the integrin adhesome network is diverse, highly dynamic and largely determined by the cell type and the physical strength required by the local developmental microenvironment (Wolfenson et al., 2009; Zaidel-Bar et al., 2007). The tripartite IPP complex containing integrin-linked kinase (ILK), PINCH and parvin is central to the integrin adhesome network (Wickstro ¨m et al., 2010; Wu and Dedhar, 2001). In invertebrates, the IPP-complex components are encoded by single genes, in contrast to the two PINCH genes and three parvin genes (a-, b- and c-parvin) in mammals (Legate et al., 2006). The early evolutionary appearance of ancestral IPP-complex components suggest that the IPPb complex may have been one of the first molecular machines used by integrins (Sebe ´-Pedro ´s et al., 2010). ILK plays a central role in the formation of the IPP complex. It contains five tandem ankyrin repeats (ANKRs) followed by a kinase-like domain, and interacts with the b1 and b3 integrin cytoplasmic tails (Hannigan et al., 1996). The ANKRs of ILK bind to PINCH, an adaptor protein containing five LIM domains, whereas the kinase-like domain of ILK binds to parvin, which in turn binds to actin (Legate et al., 2006). Thus, a simple model of IPP-complex function envisages either direct or indirect anchoring of ILK to integrins, with parvin mediating the link to actin. This model is supported by studies showing linear assembly of IPP complex in invertebrates. In Caenorhabditis elegans, ILK is required for both parvin and PINCH subcellular localization but not the reverse, and in Drosophila ILK is similarly required for PINCH stability and recruitment, although parvin has not been tested (Lin et al., 2003; Norman et al., 2007; Zervas et al., 2011). In mammalian cells, genetic elimination of one component of the IPP complex results in a significant decrease of the other two members, although some compensation has been observed for parvins and PINCH (Wickstro ¨m et al., 2011). Moreover, distinct IPP complexes are formed within cells containing different parvin and PINCH members, resulting in different functional properties (Wu, 2004). However, it is unknown whether removing all IPP components enhances the defects of single deletions, and thus whether all components function together or have other individual functions. Research Article 3221