Lipid Hop Diffusion on the Plasma Membrane - a STED-FCS Investigation

Abstract

Currently two of the most important - as well as controversial - proposed membrane-organizing principles are the “lipid rafts” hypothesis, proposed by K. Simons, and the “picket-fence” model, proposed by A. Kusumi. To date, both hypotheses encounter obstacles for full acceptance due to technical limitations on the available superresolution and single molecule techniques and limitations on the correspondent probes utilized by such techniques applied to living cells.Trying to elucidate the lipid hop diffusion dilemma, which would be the underlying principle for the “picket-fence” model, we planned a series of STED-FCS experiments to probe the diffusion of phospholipids on the membrane of different cell types. The superresolution of STED microscopy allows FCS experiments to probe areas comparable in size to the compartments created by the actin cytoskeleton, arguably one of the major structures responsible for lipid and protein segregation in the cell membrane.Using STED-FCS, we were able to detect phospholipid hop diffusion in two of the cell lines studied (NRK and IA32) and free diffusion was observed for the other cell lines under consideration (Ptk2, Vero, Hela). Different treatments to deplete the actin cytoskeleton on IA32 and NRK cells resulted in the extinction or hindrance of phospholipid hop diffusion. The same result was observed in a mutant cell line Arp 2/3 depleted originated from IA32, evidencing the major role of actin cytoskeleton on the compartmentalization of lipids. Simulations connecting membrane content and actin cytoskeleton density provided insight into the reason for apparent different diffusion modes in different cells.Whereas lipid hop diffusion could still not be confirmed by SPT experiments using fluorescent tags as markers due to technical limitations, it was now for the first time evidenced by STED-FCS, which is the time-domain counterpart of that technique.