Live fish fibroblast that expresses cyan fluorescent protein (CFP) -fascin (blue), yellow fluorescent protein (YFP) -actin (green) and mCherry-paxillin (red). Courtesy of M. Nemethova and V. Small, Austrian Academy of Sciences (IMBA) Austria.
Are you not eager to see how your favourite molecule behaves in a cell? With the current microscopy technologies and the availability of fluorescent probes such as green fluorescent protein (GFP), the visualization of molecules in living cells has become routine. However, until the late 1970s, experimental designs relied mostly on static tools such as electron microscopy and immunofluorescence techniques, which were inappropriate to analyse dynamic processes.
Cell biology drastically changed with the development of 'molecular cytochemistry', whereby purified cellular components are covalently labelled with fluorescent probes and, after being tested for their function in vitro, are reintroduced into living cells. This experimental approach was first introduced by Taylor and Wang, who used a reactive fluorescent dye — 5-iodoacetamido-fluorescein (IAF) — to label purified actin, and then directly microinjected the actin derivative into cells. IAF-labelled actin was shown to behave similarly to unlabelled actin both in vitro and in vivo — it polymerized normally, activated myosin ATPase and was incorporated into contracted pellets in motile cell extracts to the same extent as endogenous actin. Furthermore, the ability of labelled actin to form filamentous bundles when microinjected into the slime mold Physarum polycephalum demonstrated that, besides retaining its biological activity, the modified actin could be incorporated into normal structures. Another important contribution of the work by Taylor and Wang consisted of defining the controls that are necessary for the use of fluorescent analogues; these include comparing the biochemical activity, subcellular localization and in vivo stability of the analogue with the properties of native molecule, all of which were considered when developing modern probes such as GFP.
These papers opened up the study of the dynamics of cytoskeletal proteins in living cells.
Gregg Gundersen
These experiments opened up the study of cytoskeleton dynamics, leading to exciting findings, such as actin treadmilling — the continuous removal of actin monomers from the pointed ends of filaments and their reincorporation at barbed ends — at the leading edge of cells and microtubules exhibiting dynamic instability in vivo. However, the use of different fluorescent probes combined with later advances in microscopy proved molecular cytochemistry to be a tool that is more generally applicable to the analysis of different structures and processes in living cells. Importantly, this also led to the development of additional techniques to measure protein dynamics.
