Investigation of the actin-membrane interaction at the leading edge and its influence on membrane diffusion.
PhD thesis, University of Glasgow.
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Actin polymerisation is a highly dynamic process which drives many cellular events, including endocytosis and cell motility. It is known that actin monomers are added to the filaments at the leading edge of a migrating cell and that this polymerisation is the driving force of protrusion. Much is known about the activation and regulation of this dynamic actin remodelling, but many questions about the exact nature of the interaction between the actin filaments and the plasma membrane remain. Weisswange et al (Weisswange et al., 2005) found that the leading edge of protruding fish keratocytes functions as a diffusion^M
barrier for lipid dyes.^M
The aim of the here presented thesis was to continue this project and to study the actin-membrane interaction at the leading edge, using the diffusion barrier^M
as an initial read-out method. First it was investigated whether this diffusion barrier is present in other cell types and could therefore be seen as a general^M
feature of protrusion. Using Fluorescence Recovery After Photo-bleaching (FRAP)in B16 F1 cells, it could be shown that the diffusion of membrane anchored GFP (GFP-F) is significantly inhibited at the leading edge compared to lamellar regions. No reduction in diffusion could be observed after destruction of the actin meshwork or at non-protruding sites of the lamellipodial periphery,^M
showing that the diffusion barrier depends on active protrusion. The diffusion of cytoplasmic GFP was not altered near the leading edge compared to in the^M
lamellipodium, indicating that only membrane bound proteins are affected. After showing that the diffusion barrier is a general feature of protrusion, the exact nature of the actin-membrane interaction causing this phenomenon was^M
investigated. No direct interaction between actin and the membrane could be observed using FLIM-FRET, but FRAP experiments on fixed cells and a correlation^M
between the strength of the diffusion barrier with the speed of protrusion indicate that the reason for the reduction in diffusion around the leading edge is^M
the force created by the actin filaments pushing against the membrane. Further FRAP experiments indicate that actin regulating proteins such as IRSp53 are influenced by the restricted diffusion zone at the leading edge. We propose that the lipid diffusion barrier traps regulatory proteins at the leading edge and can therefore be seen as a positive feedback mechanism for actin polymerisation.
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