PIPKIGKO · Targeted disruption of the type I phospatidylinositol phosphate kinase gamma gene and the effect on focal adhesion formation in a mouse genetic background
FP6 — Marie Curie Actions (Human Resources and Mobility)
- Duration
- 2004-10-01 → 2006-09-30
- EU contribution
- €158,198
- Participants
- 1
- Scheme
- IIF
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Results in brief
Final Activity Report Summary - PIPKIGKO (Targeted disruption of the type I phospatidylinositol phosphate kinase gamma gene and the effect on focal adhesion formation in a mouse genetic background)
Focal adhesions are sites of cell-extracellular matrix contact. The formation of such adhesions is thought to be mediated in part by the binding of specific focal adhesion proteins to an acidic phospholipid, phosphatidylinositol-4,5-bisphosphate (PIP2). Local production of PIP2 is likely accomplished by a lipid kinase, PIP kinase Type I gamma, which is found in focal adhesions. The prevailing hypothesis is that disruption of local PIP2 production will impair the formation, dynamics or disassembly of focal adhesions. To test this hypothesis, the focal adhesion targeting sequence of the PIP kinase Type I gamma gene was deleted in mice, and the consequences of this deletion were studied in both mice and cells. Surprisingly, mouse development was unaffected when the targeting of PIP kinase to focal adhesions was abrogated. Mice were viable, fertile and had a normal lifespan. Cells isolated from PIP kinase knockout mice showed no defect in adhesion to extracellular matrix, migration, proliferation or signalling pathways located downstream of focal adhesions, but early stages of spreading of PIP kinase -/- cells on fibronectin were delayed compared to genetically unaltered cells. Therefore we conclude that previous in vitro studies which suggest that local production of PIP2 at focal adhesions is important for focal adhesion integrity overemphasize the importance of PIP kinase Type I gamma, since the effect of its deletion are minor at best. However, a closer analysis of PIP kinase -/- mice has revealed a reduction in storage iron content in the spleen and liver, consistent with a defect in iron metabolism in these mice. This finding complements two recent studies which uncover a role for PIP kinase Type I gamma in clathrin-mediated endocytosis, the main route for iron uptake by cells. This finding is currently being examined in closer detail in cells as well as in mice by restricting their source of dietary iron. In summary, this project has revealed a greater in vivo role for PIP kinase Type I gamma in endocytosis in general, and iron homeostasis in particular, rather than in cell adhesion as previously thought.
Data: CORDIS, © European Union
Project objective
Phosphatidylinositol-4, 5-bisphosphate (PI-4,5-P2) is a lipid molecule that contributes to the formation and maintenance of focal adhesions, by promoting the formation of an indirect physical association between integrin molecules and the actin cytoskeleton.A splice variant of Type I phosphatidylinositol phosphate kinase gamma (PIPKIg), a key enzyme in the synthesis of PI-4.5-P2, has recently been demonstrated to localize to focal adhesions and may contribute to local synthesis of PI-4.5-P2 at the sites of focal adhesions.The goal of this proposal is to generate a targeted gene disruption of this splice variant of PIPKIg in a mouse genetic model, with the specific aim of studying the putative requirement for localized PI-4.5-P2 production in the formation and maintenance of focal adhesions.The phenotype of mice harbouring a disruption of the PIPKIg gene will be analysed using standard techniques, and their morphology and histology will be compared to mouse models containing disruptions of various focal adhes ion components.Most of the models that will be used for comparison have already been generated and characterized by the host laboratory. A cell biological approach will also be used, to study the formation and architecture of focal adhesions.Fibroblasts harbouring the targeted disruption of PIPKIg will be generated and the formation and composition of focal adhesions will be studied using immuno fluorescence, live cell imaging techniques and biochemical techniques.In this manner the specific PI-4,5-P2-depen dent steps leading to focal adhesion formation will be elucidated. An understanding of how focal adhesions are formed and maintained will advance our understanding of the processes leading to cancer formation and metastasis, immune cell malfunction and imp aired wound healing.
Original text from CORDIS.
Participants
- MAX-PLANCK-GESELLSCHAFT ZUR FOERDERUNG DER WISSENSCHAFTEN E.V. · MUENCHENCoordinatorGermany
Links
Data: CORDIS, © European Union