Building the actin cytoskeleton: filopodia contribute to the construction of contractile bundles in the lamella - PubMed (original) (raw)

Building the actin cytoskeleton: filopodia contribute to the construction of contractile bundles in the lamella

Maria Nemethova et al. J Cell Biol. 2008.

Abstract

Filopodia are rodlike extensions generally attributed with a guidance role in cell migration. We now show in fish fibroblasts that filopodia play a major role in generating contractile bundles in the lamella region behind the migrating front. Filopodia that developed adhesion to the substrate via paxillin containing focal complexes contributed their proximal part to stress fiber assembly, and filopodia that folded laterally contributed to the construction of contractile bundles parallel to the cell edge. Correlated light and electron microscopy of cells labeled for actin and fascin confirmed integration of filopodia bundles into the lamella network. Inhibition of myosin II did not subdue the waving and folding motions of filopodia or their entry into the lamella, but filopodia were not then integrated into contractile arrays. Comparable results were obtained with B16 melanoma cells. These and other findings support the idea that filaments generated in filopodia and lamellipodia for protrusion are recycled for seeding actomyosin arrays for use in retraction.

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Figures

Figure 1.

Figure 1.

Examples of actin cytoskeleton phenotypes in fish fibroblasts showing differences in the lamella region (LM) behind the anterior zone containing lamellipodia (LP) and filopodia (FP). Cells were transfected with mCherry-actin. Images show single frames from video sequences. (A) Lamella with bundles parallel to the cell front, together with arc shaped segments (AC). (B) Lamella with stress fiber bundles (SF) mainly perpendicular to the cell front. See Videos 1 and 2 (available at

http://www.jcb.org/cgi/content/full/jcb.200709134/DC1

). Bar, 10 μm.

Figure 2.

Figure 2.

Variable fates of filopodia. (A and B) Development of actin bundles in the lamella from lateral folding of filopodia into the base of the lamellipodium (transitions marked with arrows). Cells were transfected with mCherry-actin (red) and EGFP-fascin (green; see Video 3, available at

http://www.jcb.org/cgi/content/full/jcb.200709134/DC1

). Times are given in minutes and seconds. (C) Folding into, kinking (top arrow), and withdrawal (bottom arrow) of filopodia into lamella. The same cell as in B is shown. See Video 3. (D) Transition of radially oriented filopodia into stress fiber bundles. The filopodia marked 1–4 at time 5:40 remain essentially stationary as the cell front advances and finally appear as bundles in the lamella (30:00). Arrowheads mark equivalent positions on the filopodia through the sequence. At the position marked, the filopodia become kinked and separate from the lamellipodium/filopodium boundary, except filopodium 2, which continues extending and contributes a further bundle to the lamella. Filopodium 5 (5:40) fuses with two other filopodia; the resulting filopodium subsequently bends in two places and flows into the lamella (26:20; see Video 1). Bars, 5 μm.

Figure 3.

Figure 3.

Entry of filopodia into ventral layer of the cytoskeleton. (A and B) Simultaneous wide-field (red) and TIRF (green) microscopy of a cell expressing EGFP-fascin. Filopodia fold down into the zone of the evanescent wave to within 200 nm from the substrate. (A) Filopodium marked with arrowhead folds upwards and then backward into the cell; filopodia marked with arrows fold laterally and down into the cell edge. (B) The numbered filopodia fold in opposite directions into the cell edge. See Video 4 (available at

http://www.jcb.org/cgi/content/full/jcb.200709134/DC1

). (C) Periphery of a cell transfected with EGFP-fascin (pseudocolor red) and mCherry-actin (pseudocolor green) imaged simultaneously by TIRF microscopy. Note the generation of a ventral stress fiber bundle (arrow) from filopodia that fold bilaterally into the cell edge. Times are given in minutes and seconds. Bars, 5 μm.

Figure 4.

Figure 4.

Correlative live cell imaging and electron microscopy of filopodia transitions. (A) Image of a cell transfected with EGFP-fascin (green) and mCherry-actin (red) after fixation at the end of the video sequence. (B) Electron micrograph of region shown in A after negative staining. Boxes and arrows in A and B indicate regions corresponding to the video sequences shown in C and D. Arrows in C and D indicate transition steps of filopodia into lamella, with the eventual depletion of fascin; final panels show actin label alone in the fixed cell. Times are given in minutes and seconds. See Video 5 (available at

http://www.jcb.org/cgi/content/full/jcb.200709134/DC1

).

Figure 5.

Figure 5.

Integration of filopodia into lamella cytoskeleton. (A) Electron micrograph of region corresponding to the terminal frame in Fig. 4 C. (B) Enlargement of A (small box) showing central region of filopodium bundle in the lamella. (C) End region of the filopodia bundle (large box in A) showing splaying of filaments into adjacent bundles of the lamella (indicated by white arrows; also depicted in the inset). See Fig. S2 (available at

http://www.jcb.org/cgi/content/full/jcb.200709134/DC1

).

Figure 6.

Figure 6.

Integration of filopodia into lamella cytoskeleton. (A) Electron micrograph of the two bundles (1 and 2) marked by arrows in the terminal frame of Fig. 4 D. (B) Enlargement of the region boxed in A showing interconnection of the filopodium bundle with adjacent bundles of the lamella cytoskeleton. See also Fig. S3 (available at

http://www.jcb.org/cgi/content/full/jcb.200709134/DC1

).

Figure 7.

Figure 7.

Coupling with myosin in the lamella. (A) Withdrawal and integration of a filopodium into actomyosin network of lamella. Cell was transfected with mCherry-actin and EGFP-myosin regulatory light chain. Arrows indicate a filopodium that first became kinked and was then drawn into the lamella with the progressive accumulation of myosin and the eventual transition into a stress fiber bundle. Times are given in minutes and seconds. See Video 6 (available at

http://www.jcb.org/cgi/content/full/jcb.200709134/DC1

). (B) Inhibition of myosin contractility with 50 μM blebbistatin. CAR fibroblast transfected with mCherry-actin. Filopodia formation was not inhibited and filopodia translocated into the lamella (arrowheads) but did not form contractile bundles. See Video 7.

Figure 8.

Figure 8.

A subpopulation of filopodia are involved in the initiation of substrate adhesion. (A) Two video frames of a CAR fibroblast expressing mCherry-actin and EGFP-paxillin separated by 21 min and showing (marked on the right with arrowheads) those adhesion sites whose origin could be traced to a position along a filopodium. (B and C) Video frames of selected regions taken from the cell in A showing the appearance of focal complexes along filopodia (arrowheads; see Video 9). Small arrows in C indicate development of filopodia into bundles in the lamella, with focal adhesions at their ends. (D) Video frames of a cell transfected as in A and showing the bending of filopodia (marked 1–3) around adhesion foci (arrowheads). Times are given in minutes and seconds. Bars: (A) 10 μm; (B–D) 5 μm.

Figure 9.

Figure 9.

Contribution of microspikes in B16 melanoma cells to construction of actin bundles in the lamella network. (A–C) Video sequences of cells expressing GFP-fascin and mCherry-actin showing transition of fascin-positive microspikes (arrows) into radial (A and B) and transverse bundles (C, arcs) in the lamella. (D) Inhibition of myosin contractility by 30 μM blebbistatin in a B16 melanoma cell expressing GFP-fascin and mCherry-actin (see Video 10). The fascin images were obtained only at the given time points shown in the video sequence to avoid inactivation of blebbistastin. The arc-shaped bundles arising from the lateral translation of microspikes (arrows at time 0:00) dispersed in blebbistatin, but microspike segments continued to enter the lamella (times 15:00 and 40:00). Times are given in minutes and seconds.

Figure 10.

Figure 10.

Schematic illustration of the different fates of filopodia. Large arrow indicates direction of movement. (1 and 1′) Filopodia fold laterally in opposite directions into the cell edge, forming a bundle with antiparallel filaments that integrates into the lamella as the cell edge advances. Integration 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. (2) A few filopodia kink as a result of lamellipodial retrograde flow and can release fragments into the lamella. (3 and 3′). An early adhesion (focal complex) forms underneath a filopodium. The part of the filopodium distal to the adhesion retracts or folds away, finally separating from the point of adhesion. The proximal part of the filopodium links with oppositely polarized filaments in the lamella via interaction with myosin and actin cross-linkers, forming a contractile stress fiber and resulting in the maturation of the focal complex into a focal adhesion. Again, filaments originating from the lamellipodium can potentially be recruited into the bundle. (4 and 4′). A filopodium folds up and back into the lamella.

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