13. Coupling protrusion and retraction
We have seen that the active protrusion of lamellipodia and filopodia (as well as the microspike bundles that span the lamellipodium without protruding from the edge) results from the local induction of actin polymerisation at the cell membrane. Experiments in which fluorescent actin was microinjected into fibroblasts showed that actin is rapidly incorporated into these peripheral structures and only much later into the inner parts of the cytoskeleton. Consistent with this finding, studies on the movements of filopodia and microspikes indicate that these structures function not only in protrusion, but also in seeding the formation of contractile bundles in the main body of the cytoskeleton, behind the protruding zones. This can be appreciated in videos of fibroblasts and melanoma cells expressing fluorescent actin and also fluorescent fascin to label filopodia.
Figure 13-1. Video of a fish fibroblast expressing mCherry-actin and GFP-fascin (to label filopodia). Note that some filopodia fold bi-laterally into the cell edge, eventually giving rise to an actin bundle in the body of the cytoplasm. From Nemethova et al., (2008).
(Movie 6.56Mb) Click on picture to watch movie
After protrusion, filopodia can exhibit different fates resulting in the contribution of filaments to the cytoskeleton behind. They can fold laterally together with oppositely polarised filopodia into the cell edge, they can fold upwards and backwards into the cell body and can kink and fragment, leaving the fragments to flow into the cell body. All these activities result in the delivery to the cell body of filaments of mixed orientation ready to interact with myosin filaments to form contractile arrays (Movie 3-2, Actin-red, Myosin-green).
Figure 13-2. Schematic illustration of the different fates of filopodia. Integration of filopodia is coupled with the incorporation of myosin and actin cross-linkers into the bundle. Single filaments originating from the lamellipodium can also contribute to these bundles. For more details see Nemethova et al., 2008.
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As indicated in the section on adhesion, filopodia can also contribute to the formation of a contractile actomyosin bundle by initiating the formation of a focal adhesion. In this case the filopodium makes contact with the matrix at a point along its length and relinquishes the basal part to initiate stress fibre assembly.
Figure 13-3. Actin dynamics in a CAR fibroblast transfected with mCherry-actin. Two pairs of filopodia, the top one of each pair marked with an arrowhead, persist as the cell front advances and become integrated into the stress fibre network of the lamella. The sites of kinking of the filopodia mark the point of separation of the filopodia from the advancing front, except in one case for which a second filopodial extension adds onto the first, giving rise to a longer bundle in the lamella. Note also folding and bending of other filopodia into the lamella (Nemethova et al., 2008).
(Movie 9.85Mb) Click on picture to watch movie
The bilateral movements of microspikes, common in B16 melanoma cells, likewise gives rise to bipolar assemblies of actin that integrate with myosin at the base of the lamellipodium forming contractile arrays that flow rearwards contributing to an integrated contractile network.
Figure 13-4. Lateral flow of actin filament bundles (microspikes and filopodia) in migrating B16 melanoma cells. Actin was labeled with GFP in a-d and with mCherry in e-g. In f, the cell was also transfected with myosin-GFP: note the incorporation of myosin into the bundles that retreat behind the lamellipodium to produce contractile arrays. g, inhibition of myosin with blebbistatin does not inhibit lateral flow: this is driven by actin polymerization. For more details see Koestler et al., 2008. (Movie 6.47Mb) Click on picture to watch movie
Building retraction assemblies at the rear
How are contractile assemblies of actin and myosin developed at the cell rear? This is achieved by generating protrusions around the entire cell periphery at some point in the migration cycle. Cells do not move in straight lines, but in a zig-zag fashion, even when undergoing chemotaxis. They form protrusions around the whole cell perimeter at one time or another and it is the pole with persistant protrusion that determines the movement direction.
As we have seen above, the folding of filpodia and microspikes into the lamella behind the lamellipodium contribute to the formation of contractile arrays. Lamellipodia also fold upwards and backwards to form „ruffles“ (Abercrombie et al., Exp Cell Res 60; 437-44, 1970) that move rearwards and merge into the cell body. We suggest this is another way that the cell uses to deliver actin filaments into the cell body to seed the formation of contractile arrays (Figure 13-5).
Figure 13-5. A hypothetical migrating cell showing ruffles folding rearwards both at the front and transiently at the flanks and rear, contributing filaments for the assembly of contractile arrays. From Small, J.V. (2010). Encyclopedia of Biochemistry Vol.2, Elsevier.