PhD Defense by Christoffer D. Florentsen

Title: Annexins in Plasma Membrane Repair

Abstract: Plasma membrane repair is a vital part of cell survival. Migrating tumors have a special need for an efficient plasma membrane repair (PMR) mechanism because invading cancer cells are exposed to mechanical stress as they manoeuvre through the extracellular matrix. How the PMR machinery is activated and how it functions is still not completely understood, and several mechanisms for efficient PMR have been proposed. The importance of a complete understanding of the function of the PMR machinery is highlighted by the fact that migrating cancer cells, known as metastatic cancer, are responsible for the majority of cancer deaths. Hence, as inhibition of metastases is a crucial part of combating cancer, knowledge of the PMR machinery is essential.

In this thesis, the role of annexins in PMR has been investigated. Annexins are a family of 12 different proteins, which are known for their Ca2+ dependent involvement in PMR. We have investigated the accumulation of several different annexins at damaged membrane sites. We have developed a technique for damaging the membrane using plamonic nanoparticles. This enables us to obtain microscopic lesions in the membrane and monitor the associated recruitment of proteins to the site of injury. Furthermore, to understand the recruitment mechanism in cells we have studied the biophysical properties of these same proteins in minimal model membrane systems with respect to mobility and membrane curvature affinity. This was investigated by pulling tethers, exhibiting high membrane curvature, from Giant Unilamellar lipid Vesicles (GUVs) and from plasma membranes isolated from living cells.

We show that annexins are recruited instantly following membrane damage, ex- hibiting a ring-formation around the wound. The binding of annexins seem stable, as the ring is visible for long time periods and three-dimensional z-stack analysis reveal that the hole radius is unchanged through the cell. Furthermore, the ringsize differs significantly between cells, which is attributed to the temperature of the nanoparticles as well as a difference in PMR efficiency between different cells.

Our investigations of the sorting of annexins indicate the existence of a heterogeneous binding efficiency within the annexin protein family. Several annexin family members are recruited to membrane areas with high curvature. We find that the sorting ability of these annexins is connected to their ability to oligomerize and show that annexins unable to oligomerize show less preference for curved membranes.

By Zoom: https://ucph-ku.zoom.us/j/631952145?pwd=U0FJNVdjd0RpdlV4WFdIbXBiOEFkdz09