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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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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.
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fig3: 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.

Mentions: Total internal reflection fluorescence (TIRF) microscopy showed that the filopodia folding back into the cell entered the region of the evanescent wave within 200 nm of the coverslip surface, which corresponds to the level of the ventral cytoskeleton (Fig. 3 and Video 4, available at http://www.jcb.org/cgi/content/full/jcb.200709134/DC1). In Fig. 3 (A and B), the superimposed and simultaneous wide field (red) and TIRF images (green) of a cell transfected with EGFP-fascin demonstrate the lifting of filopodia before their entry into the base of the lamellipodium. And in Fig. 3 C, dual wavelength TIRF of mCherry-actin (pseudocolor green) and EGFP-fascin (pseudocolor red) likewise shows the contribution of filopodia to the formation of a stress fiber in the ventral cytoskeleton of the lamella.


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)

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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2290848&req=5

fig3: 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.
Mentions: Total internal reflection fluorescence (TIRF) microscopy showed that the filopodia folding back into the cell entered the region of the evanescent wave within 200 nm of the coverslip surface, which corresponds to the level of the ventral cytoskeleton (Fig. 3 and Video 4, available at http://www.jcb.org/cgi/content/full/jcb.200709134/DC1). In Fig. 3 (A and B), the superimposed and simultaneous wide field (red) and TIRF images (green) of a cell transfected with EGFP-fascin demonstrate the lifting of filopodia before their entry into the base of the lamellipodium. And in Fig. 3 C, dual wavelength TIRF of mCherry-actin (pseudocolor green) and EGFP-fascin (pseudocolor red) likewise shows the contribution of filopodia to the formation of a stress fiber in the ventral cytoskeleton of the lamella.

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