Finally we also show, for the first time, strong evidence of transport from the central region of the cell to the extremity of critical adhesion components, via vesicular trafficking. Finally, mapping other phospholipids and membrane components we observed that the PIP2 domains formation is correlated with sphingosine and cholesterol rafts. Among all the different available techniques in molecular biology, fluorescence microscopy (FM) is by far one of the most used1. This technique, in Fondaparinux Sodium fact, has the great advantage to obtain functional and structural information in a single experiment. Particularly, live cells imaging is becoming fundamental in the understanding of the dynamics of many biological processes. A typical strategy for carrying out live cell imaging include the use of fluorescent proteins (FP)2. The development Fondaparinux Sodium of molecular genetics and engineering has allowed proteins manipulation and, consequently, the creation of fluorescent protein libraries. These proteins can be expressed within live cells in specific sub-cellular compartments. In addition to the intrinsic limitations associated with genetic manipulations, fluorescence proteins cannot be used for any cellular components that have a post-translational origin. This includes most glycols as well as phospholipids, sphingosines and sterols. The latter are essential molecules in the life of a cell, as they control compartmentalization, in cooperation with membrane proteins, leading to the formation of fluid membrane bounded structures3. It is now accepted that the plasma membrane has a specific regulatory role in several signaling pathways which is directly controlled by rearrangement into raft domains, that results from the fluctuations of local composition and membrane spontaneous curvature4,5,6,7,8. These micro- Rabbit Polyclonal to UNG and nano- domains, composed of specific phospholipids and proteins9, have a central role in the regulation of many cellular functions such as signalling pathways, membrane shaping, cell motility and polarization4,8,10. Among the different membrane components, one of the most studied phospholipids is Phosphatidylinositol 4,5-bisphosphate (PIP2)11,12,13,14,15,16. PIP2 works as an anchoring points for several proteins whose function is to control membrane deformation. These include several GTPases belonging to the Rho family (e.g., Rho, Rac and Cdc4215,17) as well as Fondaparinux Sodium several actin and cytoskeleton regulators (e.g., ERM-proteins, Talin, WAVE/WASP, Gelsolin capping, ADF/Cofilin, Profilin and Twinfilin14,16,18). Although progress has been made to elucidate the different pathways and proteins involved in membrane/cytoskeleton interaction19, there are still lot of queries on (i) how PIP2 is transported along the F-actin polymerization sites, (ii) how the PIP2 membrane organization is related to the local lipid composition and (iii) how its function is controlled by other membrane components. Herein we show that we can access these important membrane components using effective intracellular delivery of fluorescently labelled phospholipids and actin probes. We have recently developed a nanotechnological platform to introduce probes within the cells without affecting their metabolic activity hence allowing live cell imaging. This is based on the use of synthetic vesicles (called polymersomes) formed by pH sensitive copolymers20,21,22,23. Here we use these to deliver, separately and simultaneously six different probes, two common phospholipids namely: 2-Decanoyl-1-(O-(11-(4,4-Difluoro-5,7-Dimethyl-4-Bora-3a,4a-Diaza-s-Indacene-3-Propionyl)amino)Undecyl)-sn-Glycero-3-Phosphocholine (PC), and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (PE), a cholesterol analogous: 22-(N-(7-Nitrobenz-2-Oxa-1,3-Diazol-4-yl)Amino)-23,24-Bisnor-5-Cholen-3-Ol (CHOL), a sphingosine: N-((4-(4,4-difluoro-5-(2-thienyl)-4-bora-3a, 4a-diaza-s-indacene-3-yl)phenoxy)acetyl)sphingosine (S), as well as the inositol: TopFluor? phosphatidylinositol 4,5-bisphosphate (PIP2) and the peptide Phalloidin-ATTO647 (ACTIN) to stain F-actin. We first investigated the correlation between the cytoskeleton structures and the PIP2 membrane domains, to understand their role in cell adhesion and spreading processes. In particular, Fondaparinux Sodium we explored the specific interactions between PIP2 membrane domains and the actin cytoskeleton, by means of real time imaging of living cells, during important processes such as cell adhesion and spreading. We complemented these studied by delivering three more membrane probes, in addition to PIP2 and ACTIN, specifically PC, CHOL and S. This has allowed us Fondaparinux Sodium to study their specific distribution and reciprocal interconnections within the cell, and to explore their topological localization with the membrane, particularly at the cell edge where cell adhesion and spreading processes initiate. Results Validation of methodology pH sensitive diblock copolymers poly(2-(methacryloyloxy)ethyl phosphorylcholine)-poly(2-(diisopropylamino)ethyl methacrylate) (PMPC-PDPA) are used to form polymersomes. The PMPC and the PDPA block convey two important functions to.