Angiogenesis is the process when new blood vessels are formed from existing blood vessels. During angiogenesis, endothelial cells (ECs) generate a polarized leading edge through a process that involves the coordinated reorganization of the actin and microtubule cytoskeleton. Cytoskeletal reorganization and EC branching are necessary to generate physical contacts between the cell and the extracellular matrix (ECM). Together these processes help drive directional migration, a critical component that aids in the formation of new blood vessels. The phosphatidylinositol lipids have been identified as key mediators for directed motility. Upon stimulation, phosphatidylinositol 3,4,5-triphosphate (PI(3,4,5)P3) is synthesized from phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) by a family of phosphoinositide-3-kinases (PI3Ks) at the plasma membrane (PM). PM PI(3,4,5)P3 is important for actin polymerization at the leading edge of migrating cells through the activation of the small GTPase Rac1. PM PI(4,5)P2 is much more abundant when compared to PI(3,4,5)P3. PI(4,5)P2 is thought to be generated at the PM at sites of focal adhesions (FA) and has been implicated in promoting activation of various FA-associated proteins. While PM PI(4,5)P2 restricts cortical actin polymerization, it is unclear how PM PI(4,5)P2 is organized in the cell or how it regulates cell polarity and migration. We hypothesized that the regulation of PM PI(4,5)P2 levels functions to locally modulate cytoskeletal organization and EC-ECM adhesion. To test this hypothesis, we used an inositol polyphosphate 5-phosphatase fused rapamycin-triggered heterodimerization strategy (iRAP) to deplete PM PI(4,5)P2 levels in ECs. Cells co-expressing GFP-F-tractin/GFPPaxillin, and iRAP with PI(4,5)P2/ PI(3,4,5)P3 readouts were collected and analyzed by live cell confocal imaging. Experimental data reveal that PM PI(4,5)P2 depletion disrupted acto-myosin bundles, inhibited the assembly of nascent FA, increased the number of mature FAs, and 4 promoted oscillatory protrusions and retractions. Independent of PM PI(4,5)P2 localization, the PI(3,4,5)P3 biosensor localized to actin stress fibers associated with mature FA, and this localization was reduced by treatment with blebbistatin, supporting a role for myosin II contractility in the localization of PI(3,4,5)P3 near FAs. Thus, spatial and temporal dynamics of phosphoinositide levels modify EC morphology and polarity through actin reorganization and cell-ECM adhesion. Future investigations will focus on the role of both PI(4,5)P2 and PI(3,4,5)P3 in controlling EC polarization and FA assembly.
|School:||University of the Sciences in Philadelphia|
|School Location:||United States -- Pennsylvania|
|Source:||MAI 57/05M(E), Masters Abstracts International|
|Subjects:||Molecular biology, Cellular biology|
|Keywords:||Cell cytoskeleton, Cell migration, Focal adhesion dynamics, Phosphoinositides, Pip2 and pip3 signaling|
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