7. Actin polymerization drives protrusion
Protrusion is based on the unidirectional growth (polymerisation) of actin filaments, whereas retraction of the cell rear is a contractile process based on the interaction of filaments of actin and myosin in bundles of actin filaments, or stress fibres (Fig. 3-1). We gain an impression of these two processes of protrusion and retraction from the video in Fig.7-1, which shows the actin cytoskeleton in the melanoma cell from Fig. 2-1.
Figure 7-1. The actin cytoskeleton in the melanoma cell from Fig. 2-1. The cell was transfeced with a cDNA construct of actin tagged with green fluorescent protein (actin-GFP) allowing visualisation of actin in the fluorescent channel.
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Note that the breadth of the lamellipodium network at the cell front remains constant as the cell moves forward. This feature is explained by a so-called “treadmilling” of actin filaments, whereby the addition of actin monomer at the front of the lamellipodium is balanced by the release of monomer at the rear (Fig. 7-2, Pantaloni et al., 2001; Carlier et al., 2003).
Figure 7-2. Schematic illustration of the treadmilling of actin monomers during polymerisation and depolymerisation at steady state in vitro. Addition of monomer occurs preferentially at the “plus end” and release of monomer at the “minus end” (from Pantaloni and Carlier, 2001).
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The treadmilling of actin in the lamellipodium can be demonstrated in different ways, two of which are shown in Figures 7-3,4 and 7-5.
Figure 7-3. Treadmilling of actin as illustrated by the recovery of actin fluorescence after photobleach (FRAP). The figure shows time-lapse images of the lamellipodium region of a B16 melanoma cell before (pre) and after photobleach. Number at top right indicates time in seconds. From Lai et al. 2008.
Figure 7-4. The video shows a B16 melanoma cell that was transfected with actin-GFP. In contrast to the regime in Fig. 7-3, a scanning laser beam was used to photobleach the fluorescence of actin in the region behind the lamellipodium marked by the square. This is immediately followed by incorporation of the bleached molecules at the lamellipodium tip. Note: the changes in fluorescence observed correspond to the fluorescence from polymerised filaments; monomeric actin diffuses very rapidly and adds only a weak homogeneous background to the image. From Lai et al., 2008.
In the method of speckle microscopy introduced by Clare Waterman-Storer (Fig.7-5) small amounts of fluorescently-labelled actin are injected into cells so that only a fraction of the actin filaments are labelled, giving a “speckled signal”(Compare with the more complete labelling of actin with GFP actin in Fig. 7-3,4). The “retrograde flow” of speckles is consistent with the continuous incorporation of new actin monomer at the front of the lamellipodium.
Figure 7-5. Treadmilling of actin in the lamellipodium of a migrating cell as shown by speckle microscopy. Video was produced by Clare Waterman-Storer.
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