Dawe, H.; (2004) The role of ADF/cofilin in the acquisition and maintenance of cell polarity during fibroblast migration. Doctoral thesis , University of London.

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Abstract

To migrate normally, a cell must establish morphological polarity and continuously protrude a single cell margin, termed the lamellipodium, polarised in the direction of migration. Previous data from our laboratory showed that actin filament disassembly was necessary for protrusion of the lamellipodium during fibroblast migration but was not required for non-polarised lamellipodial protrusion in non-migrating cells. DNase I staining of actin monomer levels in the lamellipodium showed that this was because actin monomer was highly limiting in the lamellipodium of polarised migrating cells. As ADF/cofilin (AC) proteins are essential for the catalysis of filament disassembly in cells, their role in polarised cell migration was assessed. The spatial distribution of AC and inactive, phosphorylated AC (pAC) was compared in the lamellipodium of polarised migrating cells. AC, but not pAC, localised to the lamellipodium. Adenoviral-mediated gene transfer was used to manipulate AC activity levels in cells. Locally maintaining active AC at the leading edge was required for maintaining cell polarity during fibroblast migration. When pAC was forced into the lamellipodium by introduction of a constitutively active form of LIM kinase, cells lost both their morphological polarity and their ability to migrate. This polarity loss could be prevented by expression of a non-phosphorylatable form of AC. Furthermore; AC activity was necessary for the acquisition of morphological polarity. Fibroblasts polarised in a distinct series of sub-steps. The first step in polarity acquisition was organisation of actin from a circumferential organisation to an oriented array. This was required to specify position of the cell tail. Both jasplakinolide treatment and introduction of either constitutively active LIM kinase or dominant negative AC blocked formation of oriented actin bundles; actin remained circumferentially oriented and the cell failed to polarise. Blocking AC and actin filament disassembly did not affect later steps in acquisition of polarity. Stabilisation of the cell tail was dependent on myosin II. Blocking myosin using either methyl-blebbistatin or Y-27632 produced abnormally crescent-shaped cells as the tail encroached into the cell body. Microtubules were not required for polarity acquisition, however blocking microtubule dynamics led to de-stabilisation of the lamellipodium and a loss of migratory capability.

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