Molecular imaging of the cytoskeleton using GFP-actin fluorescence microscopy

Fluorescence imaging of GFP-actin empowers the researcher with a unique tool to image changes in the organization and dynamics of the actin cytoskeleton in living cells over an extended period of time (>24 hours). While high levels of GFP-actin expression are deleterious, lower levels found in permanently transfected cell lines (5%) do not impair cell structure and function nor does this level alter dynamic properties of the actin cytoskeleton seen in motility and cytokinesis. The continuous expression and degradation of GFP-actin in cells allows the researcher to conduct fluorescence microscope based investigations of the cytoskeleton in single living cells for many days. GFP-actin is a faithful reporter of the actin cytoskeleton and it was shown to localize to stress fibers, focal adhesion contacts and in the cell cortex in a manner indistinguishable from endogenous actin in a variety of cell types. GFP-actin can be used in a quantitative manner to probe parameters underlying the regulation of actin filament turnover. In particular it is enriched in protroding lamellipodia, membrane ruffles, filopodia and in the comet tails that form behind motile Listeria bacteria. The decay of GFP-actin fluorescence signal in these dynamic regions decreases exponentially from the site of filament growth (Fig. 2A, 3A, [14, 17]. The continuous expression of the fusion protein in certain cells, together with the high photostability of the GFP-fluorophore, the lack of oxygen radical-mediated damage all contribute to the suitability of the probe to study long-term changes of the actin cytoskeleton during the EMT of NBT-II cells. Furthermore, the sensitivity of the fusion protein to the dynamics of actin filament assembly and disassembly makes it a suitable probe to image the regulation and role of actin polymerization/depolymerization in the protrusion of the leading edge and ultimately cell motility. © 2002 Elsevier B.V. All rights reserved.