Changes in Lipid Composition During Manganese-Induced Apoptosis in PC12 Cells.

Lipid composition of membranes is fundamental to modulate signaling pathways relying on lipid metabolites and/or membrane proteins, thus resulting in the regulation of important cell processes such as apoptosis. In this case, membrane remodeling is an early event important for the activation of signaling leading to cell death and removal of apoptotic cells. In the present study, we analyzed phospholipid, cholesterol and fatty acid content during apoptosis induced by manganese in PC12 cells. Lipid analysis of whole cells and detergent-resistant membranes was carried out by HPLC/GC. Results showed that apoptosis is associated with changes in lipid composition detectable in whole cell extracts, namely cholesterol, phosphatidylserine and phosphatidylethanolamine decreases. Noteworthy, phosphatidylserine level reduction was detectable before to the detection of apoptosis, in correlation with our previous study carried out by radioactive labelling. By contrast, phosphatidylserine and phosphatidylethanolamine changes were not detected in detergent resistant membranes, which instead showed an altered composition in phosphatidylinositol, phosphatidylcholine and sphingomyelin in apoptotic cells.

Atomic force microscopy imaging of lipid rafts of human breast cancer cells.

Several studies suggest that the plasma membrane is composed of micro-domains of saturated lipids that segregate together to form lipid rafts. Lipid rafts have been operationally defined as cholesterol- and sphingolipid-enriched membrane micro-domains resistant to solubilization by non-ionic detergents at low temperatures. Here we report a biophysical approach aimed at investigating lipid rafts of MDA-MB-231 human breast cancer cells by coupling an atomic force microscopy (AFM) study to biochemical assays namely Western blotting and high performance thin layer chromatography. Lipid rafts were purified by ultracentrifugation on discontinuous sucrose gradient using extraction with Triton X-100. Biochemical analyses proved that the fractions isolated at the 5% and 30% sucrose interface (fractions 5 and 6) have a higher content of cholesterol, sphingomyelin and flotillin-1 with respect to the other purified fractions. Tapping mode AFM imaging of fraction 5 showed membrane patches whose height corresponds to the one awaited for a single lipid bilayer as well as the presence of micro-domains with lateral dimensions in the order of a few hundreds of nanometers. In addition, an AFM study using specific antibodies suggests the presence, in these micro-domains, of a characteristic marker of lipid rafts, the protein flotillin-1.

Heterogeneous and self-organizing mineralization of bone matrix promoted by hydroxyapatite nanoparticles.

The mineralization process is crucial to the load-bearing characteristics of the bone extracellular matrix. In this work, we have studied the spatiotemporal dynamics of mineral deposition by human bone marrow mesenchymal stem cells differentiating toward osteoblasts promoted by the presence of exogenous hydroxyapatite nanoparticles. At the molecular level, the added nanoparticles positively modulated the expression of bone-specific markers and enhanced calcified matrix deposition during osteogenic differentiation. The nucleation, growth and spatial arrangement of newly deposited hydroxyapatite nanocrystals have been evaluated using scanning micro X-ray diffraction and scanning micro X-ray fluorescence. As leading results, we have found the emergence of a complex scenario where the spatial organization and temporal evolution of the process exhibit heterogeneous and self-organizing dynamics. At the same time the possibility of controlling the differentiation kinetics, through the addition of synthetic nanoparticles, paves the way to empower the generation of more structured bone scaffolds in tissue engineering and to design new drugs in regenerative medicine.