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

Nemethova M, Auinger S, Small JV - J. Cell Biol. (2008)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Biotechnology, Austrian Academy of Sciences, Vienna 1030, Austria.

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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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.
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fig2: 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.

Mentions: We focused in the present analysis on cells in which the front moved significantly during the video sequence so as to deduce developments relevant to the migration process. In cells expressing mCherry-actin, several fates of filopodia could be recognized. First, concave bundles seen at the base of lamellipodia received the major part of their building material from laterally translating filopodia that folded into the cell edge (Video 1). This activity of filopodia was especially highlighted in cells cotransfected with mCherry-actin and EGFP-fascin (Fig. 2, A and B; and Video 3, available at http://www.jcb.org/cgi/content/full/jcb.200709134/DC1). Measurements indicated that fascin transfection had no significant influence on the number of filopodia expressed at the cell edge. The formation of concave bundles at the base of lamellipodia involved one or several filopodia translating in the same or opposite directions (see Videos 1 and 3). The same bundles either dispersed into the lamella, or survived there as bundles that extended in length and interconnected with adjacent bundles (Fig. 2, A and B; and Video 3). Second, radially oriented filopodia were observed to persist in the same position as the cell front advanced over them and to merge as bundles into the stress fiber network (Fig. 2 D and Video 2). A significant part of this activity was the separation of the top part of the filopodium that was still embedded in the lamellipodium at a point sometimes corresponding to a kink in the filopodium bundle (Fig. 2 D, arrowheads; and Video 2). In such cases, the position of the kink corresponded to the end of the stress fiber bundle that later developed; four examples of filopodia that underwent these transitions are shown in Fig. 2 D (filopodia 1–4 at time 5:40). We shall return to this activity of filopodia in the context of adhesion formation at the cell front. More dramatic kinking of filopodia sometimes occurred as a consequence of retrograde flow or retraction of the lamellipodium, and multiple kinks gave rise to filopodium fragments that dissolved in the lamella as the cell front advanced (Fig. 2 C, top arrow; and Video 3). Finally, filopodia were observed to fold upwards and back into the lamella (Video 3) or, in cells where lamellipodia were depleted, to fold back directly into stress fiber bundles that extended to the cell edge (unpublished data).


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

Nemethova M, Auinger S, Small JV - J. Cell Biol. (2008)

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.
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Related In: Results  -  Collection

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fig2: 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.
Mentions: We focused in the present analysis on cells in which the front moved significantly during the video sequence so as to deduce developments relevant to the migration process. In cells expressing mCherry-actin, several fates of filopodia could be recognized. First, concave bundles seen at the base of lamellipodia received the major part of their building material from laterally translating filopodia that folded into the cell edge (Video 1). This activity of filopodia was especially highlighted in cells cotransfected with mCherry-actin and EGFP-fascin (Fig. 2, A and B; and Video 3, available at http://www.jcb.org/cgi/content/full/jcb.200709134/DC1). Measurements indicated that fascin transfection had no significant influence on the number of filopodia expressed at the cell edge. The formation of concave bundles at the base of lamellipodia involved one or several filopodia translating in the same or opposite directions (see Videos 1 and 3). The same bundles either dispersed into the lamella, or survived there as bundles that extended in length and interconnected with adjacent bundles (Fig. 2, A and B; and Video 3). Second, radially oriented filopodia were observed to persist in the same position as the cell front advanced over them and to merge as bundles into the stress fiber network (Fig. 2 D and Video 2). A significant part of this activity was the separation of the top part of the filopodium that was still embedded in the lamellipodium at a point sometimes corresponding to a kink in the filopodium bundle (Fig. 2 D, arrowheads; and Video 2). In such cases, the position of the kink corresponded to the end of the stress fiber bundle that later developed; four examples of filopodia that underwent these transitions are shown in Fig. 2 D (filopodia 1–4 at time 5:40). We shall return to this activity of filopodia in the context of adhesion formation at the cell front. More dramatic kinking of filopodia sometimes occurred as a consequence of retrograde flow or retraction of the lamellipodium, and multiple kinks gave rise to filopodium fragments that dissolved in the lamella as the cell front advanced (Fig. 2 C, top arrow; and Video 3). Finally, filopodia were observed to fold upwards and back into the lamella (Video 3) or, in cells where lamellipodia were depleted, to fold back directly into stress fiber bundles that extended to the cell edge (unpublished data).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Biotechnology, Austrian Academy of Sciences, Vienna 1030, Austria.

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.

Show MeSH
Related in: MedlinePlus