Imaging plasma membrane domains in signal transduction pathways

Pezzarossa, A. (2012)

Doctoral Thesis

The cell membrane acts as a barrier that controls the passage of substances from the outside to the inside of a cell. It is composed of various lipids organized in a bilayer with proteins embedded. Experimental data suggested that lipids are organized in nanometer-sized structures called membrane domains. I study the existence and the role of domains in living cells through single-molecule fluorescence microscopy. This technique allows pinpointing the position of each molecule with high spatial accuracy. I apply it to study the distribution of a membrane-anchored protein, HRas, in the inner leaflet of the membrane. From the single-molecule positions a map of protein distribution is reconstructed. Statistical analysis revealed dynamic partitioning in membrane domains. A different approach relies on tracking single proteins diffusion in the membrane. With this method I studied the influence of domains in the assembly of a two-component receptor, type I interferon receptor. I observed confinements of the components in small domains, which makes assembly faster and more efficient. Further, I present an advanced technique, to track proteins at microsecond time scale. After validating the technique on DNA, I applied it to GPI-anchor protein diffusion. These data confirmed the existence of theoretically proposed, complex diffusive modes.

• Single-molecule fluorescence microscopy
• Diffusion
• Membrane structuring
• Microsecond tracking
• Ras protein
• Interferon receptor

• All authors: Pezzarossa, A.
• Supervisor: Schmidt, T.
• Committee: Piehler, J.; Peterman, E.J.G.; Eliel, E.R.; Orrit, M.; Noort, S.J.T. van
• Qualification: Doctor (dr.)
• Awarding Institution: Leiden Institute of Physics (LION), Faculty of Science, Leiden University
• Date: 2012-03-13
• ISBN (print): 9789085931171
• Court: LEI Universiteit Leiden
• Sponsorship: Stichting voor Fundamenteel Onderzoek der Materie (FOM), die financieel wordt gesteund door de Nederlandse Organisatie voor Wetenschappelijk Onderzoek (NWO)