|M.Sc Student||Nir-Shapira Maayan|
|Subject||Cryo-EM Characterization of Mixed Lipid Bilayers|
|Department||Department of Nanoscience and Nanotechnology||Supervisor||PROFESSOR EMERITUS Yeshayahu Talmon|
|Full Thesis text|
Lipid segregation into membrane domains involves in a large variety of cellular functions and biological events, e.g., in various signal transduction pathways, in intracellular trafficking processes, and in viral entry and exit from cells. Until now these domains have been studied by direct imaging only on the micrometric, not on the nanosomic, scale.
Liposomes are spheroidal vesicles, composed of one or more lipid bilayers. They allow the easy and fairly realistic mimicking of bio-membranes; they can be built-up by one or just a few well-defined components, and hence allow better characterization of the physical principles underlying the self-organization processes observed in membranes.
Immunochemistry is a powerful tool, based on the concept that specific antigens can be detected by complementary antibodies. In the last few decades the use of colloidal gold in transmission electron microscopy (TEM) has grown at a fast rate, and has become virtually the only method worth considering for ultrastructural studies of cellular antigens. Immunogold labeling takes advantage of the high electron density of gold conjugated to antibodies, which, in turn, adsorb to a specific antigen. Although the probing molecules are often antibodies, other proteins of a specific affinity can be tagged.
Phosphatidylserine (PS) is a negatively charged phospholipid, found mostly in the inner leaflet of the cell membrane, facing the cytoplasm. Certain cell processes, such as apoptosis and microparticle (MP) shedding, involve PS migration to the outer leaflet. This phenomenon can be studied using the cellular protein, annexin V, which has high affinity to PS. Annexin V binding is Ca2 dependent, which should be taken into account during the procedure.
Thus far, most immunogold labeling of lipids has been performed in ways that eventually lead to room-temperature imaging by TEM or scanning electron microscopy (SEM). Our work presents immunogold labeling in cryogenic transmission electron microscopy (cryo-TEM). Cryo-TEM preserves the liposomes as close as possible to their native state, thus providing a more reliable view of the nanostructure and its morphology. We have attempted to label PS liposomes prepared by sonication and extrusion. Labeling was performed in solution, using biotinilated annexin V and gold-conjugated streptavidin, in a two-step process. We optimized the working conditions leading to extensive labeling of PS. The gold nanoparticles were not randomly spread in the solution, suggesting that the immunogold labeling in cryo-TEM was indeed successful. Apart from the excellent labeling we observed aggregation, which we attributed to the increased concentration of Ca2 ions in the vicinity of the highly charged liposomes. The successful labeling of liposomes allows the application of the methodology to synthetic mixed-lipid and natural systems, such as extracellular vesicles.