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Mechanism of filopodia initiation by reorganization of a dendritic network.

Svitkina TM, Bulanova EA, Chaga OY, Vignjevic DM, Kojima S, Vasiliev JM, Borisy GG - J. Cell Biol. (2003)

Bottom Line: Subsets of independently nucleated lamellipodial filaments elongated and gradually associated with each other at their barbed ends, leading to formation of cone-shaped structures that we term Lambda-precursors.The GFP-VASP foci were associated with Lambda-precursors, whereas Arp2/3 was not.We propose a convergent elongation model of filopodia initiation, stipulating that filaments within the lamellipodial dendritic network acquire privileged status by binding a set of molecules (including VASP) to their barbed ends, which protect them from capping and mediate association of barbed ends with each other.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Molecular Biology, Northwestern University Medical School, Chicago, Illinois 60611, USA. t-svitkina@northwestern.edu

ABSTRACT
Afilopodium protrudes by elongation of bundled actin filaments in its core. However, the mechanism of filopodia initiation remains unknown. Using live-cell imaging with GFP-tagged proteins and correlative electron microscopy, we performed a kinetic-structural analysis of filopodial initiation in B16F1 melanoma cells. Filopodial bundles arose not by a specific nucleation event, but by reorganization of the lamellipodial dendritic network analogous to fusion of established filopodia but occurring at the level of individual filaments. Subsets of independently nucleated lamellipodial filaments elongated and gradually associated with each other at their barbed ends, leading to formation of cone-shaped structures that we term Lambda-precursors. An early marker of initiation was the gradual coalescence of GFP-vasodilator-stimulated phosphoprotein (GFP-VASP) fluorescence at the leading edge into discrete foci. The GFP-VASP foci were associated with Lambda-precursors, whereas Arp2/3 was not. Subsequent recruitment of fascin to the clustered barbed ends of Lambda-precursors initiated filament bundling and completed formation of the nascent filopodium. We propose a convergent elongation model of filopodia initiation, stipulating that filaments within the lamellipodial dendritic network acquire privileged status by binding a set of molecules (including VASP) to their barbed ends, which protect them from capping and mediate association of barbed ends with each other.

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Actin kinetics during filopodia initiation. (A–C) Time-lapse sequences of GFP-actin–expressing B16F1 cells. Time in seconds; individual features marked by arrows and arrowheads. Nascent filopodia are marked starting from the frame preceding the appearance of the recognizable precursor. (A) Three established filopodia (0 s, arrowheads) fuse with each other (0–60 s). Two Λ-precursors (arrows) appear (20 s), and fuse with each other (40 s), forming a nascent filopodium that subsequently (60 s) joins the fusing older filopodia. (B) Several nascent filopodia form from Λ-precursors that appear within lamellipodium. (C) Two Λ-precursors existing at 0 s (arrows) fuse with each other (12 s), producing a nascent filopodium with a Λ-shaped root. The fusion point treadmills backward while the filopodium protrudes forward. (D) Gallery of Λ-precursors. First four examples represent enlarged and enhanced Λ-precursors from A (20 s), B (8 s), B (24 s), and C (0 s), respectively. Remaining examples are from other sequences. Bars, 2 μm.
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fig2: Actin kinetics during filopodia initiation. (A–C) Time-lapse sequences of GFP-actin–expressing B16F1 cells. Time in seconds; individual features marked by arrows and arrowheads. Nascent filopodia are marked starting from the frame preceding the appearance of the recognizable precursor. (A) Three established filopodia (0 s, arrowheads) fuse with each other (0–60 s). Two Λ-precursors (arrows) appear (20 s), and fuse with each other (40 s), forming a nascent filopodium that subsequently (60 s) joins the fusing older filopodia. (B) Several nascent filopodia form from Λ-precursors that appear within lamellipodium. (C) Two Λ-precursors existing at 0 s (arrows) fuse with each other (12 s), producing a nascent filopodium with a Λ-shaped root. The fusion point treadmills backward while the filopodium protrudes forward. (D) Gallery of Λ-precursors. First four examples represent enlarged and enhanced Λ-precursors from A (20 s), B (8 s), B (24 s), and C (0 s), respectively. Remaining examples are from other sequences. Bars, 2 μm.

Mentions: Filopodia in GFP-actin–expressing cells displayed a broad range of lengths and fluorescence intensities. Histories of large filopodia revealed that they were formed by fusion of smaller filopodia, which in turn were the result of fusion at an even finer scale (Fig. 2 A). Events of true filopodia initiation were recognized as the appearance of thin, faint nascent filopodia contained within the lamellipodial network. In most cases (81%, n = 124), they arose from fishtail-shaped actin densities within the lamellipodium (Fig. 2, B and C). These densities, which we will call Λ-precursors because of their shape, were just slightly brighter than the surrounding lamellipodium at their vertices, but gradually diffused into a lamellipodial network at their bases (Fig. 2 D). Although hardly distinguishable from the rest of the lamellipodial network, Λ-precursors could be consistently recognized, after contrast enhancement, by tracing back in time the sequences of newly formed filopodia. In the remaining cases of filopodial initiation (19%), Λ-precursors were not visible, possibly because of insufficient temporal resolution or contrast. Nascent filopodia subsequently fused with each other (Fig. 2, B and C) or with other filopodia, and thus increased in size. Fusion produced Λ-configurations of filopodial bundles reminiscent of the shape of Λ-precursors, but with more distinct individual branches. Over time, these Λ-shaped bundles treadmilled backward at the root of the fused filopodium (Fig. 2 C) until they disappeared in the course of depolymerization. The observed actin kinetics appears more consistent with the idea of network reorganization as a mechanism of filopodia initiation.


Mechanism of filopodia initiation by reorganization of a dendritic network.

Svitkina TM, Bulanova EA, Chaga OY, Vignjevic DM, Kojima S, Vasiliev JM, Borisy GG - J. Cell Biol. (2003)

Actin kinetics during filopodia initiation. (A–C) Time-lapse sequences of GFP-actin–expressing B16F1 cells. Time in seconds; individual features marked by arrows and arrowheads. Nascent filopodia are marked starting from the frame preceding the appearance of the recognizable precursor. (A) Three established filopodia (0 s, arrowheads) fuse with each other (0–60 s). Two Λ-precursors (arrows) appear (20 s), and fuse with each other (40 s), forming a nascent filopodium that subsequently (60 s) joins the fusing older filopodia. (B) Several nascent filopodia form from Λ-precursors that appear within lamellipodium. (C) Two Λ-precursors existing at 0 s (arrows) fuse with each other (12 s), producing a nascent filopodium with a Λ-shaped root. The fusion point treadmills backward while the filopodium protrudes forward. (D) Gallery of Λ-precursors. First four examples represent enlarged and enhanced Λ-precursors from A (20 s), B (8 s), B (24 s), and C (0 s), respectively. Remaining examples are from other sequences. Bars, 2 μm.
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Related In: Results  -  Collection

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fig2: Actin kinetics during filopodia initiation. (A–C) Time-lapse sequences of GFP-actin–expressing B16F1 cells. Time in seconds; individual features marked by arrows and arrowheads. Nascent filopodia are marked starting from the frame preceding the appearance of the recognizable precursor. (A) Three established filopodia (0 s, arrowheads) fuse with each other (0–60 s). Two Λ-precursors (arrows) appear (20 s), and fuse with each other (40 s), forming a nascent filopodium that subsequently (60 s) joins the fusing older filopodia. (B) Several nascent filopodia form from Λ-precursors that appear within lamellipodium. (C) Two Λ-precursors existing at 0 s (arrows) fuse with each other (12 s), producing a nascent filopodium with a Λ-shaped root. The fusion point treadmills backward while the filopodium protrudes forward. (D) Gallery of Λ-precursors. First four examples represent enlarged and enhanced Λ-precursors from A (20 s), B (8 s), B (24 s), and C (0 s), respectively. Remaining examples are from other sequences. Bars, 2 μm.
Mentions: Filopodia in GFP-actin–expressing cells displayed a broad range of lengths and fluorescence intensities. Histories of large filopodia revealed that they were formed by fusion of smaller filopodia, which in turn were the result of fusion at an even finer scale (Fig. 2 A). Events of true filopodia initiation were recognized as the appearance of thin, faint nascent filopodia contained within the lamellipodial network. In most cases (81%, n = 124), they arose from fishtail-shaped actin densities within the lamellipodium (Fig. 2, B and C). These densities, which we will call Λ-precursors because of their shape, were just slightly brighter than the surrounding lamellipodium at their vertices, but gradually diffused into a lamellipodial network at their bases (Fig. 2 D). Although hardly distinguishable from the rest of the lamellipodial network, Λ-precursors could be consistently recognized, after contrast enhancement, by tracing back in time the sequences of newly formed filopodia. In the remaining cases of filopodial initiation (19%), Λ-precursors were not visible, possibly because of insufficient temporal resolution or contrast. Nascent filopodia subsequently fused with each other (Fig. 2, B and C) or with other filopodia, and thus increased in size. Fusion produced Λ-configurations of filopodial bundles reminiscent of the shape of Λ-precursors, but with more distinct individual branches. Over time, these Λ-shaped bundles treadmilled backward at the root of the fused filopodium (Fig. 2 C) until they disappeared in the course of depolymerization. The observed actin kinetics appears more consistent with the idea of network reorganization as a mechanism of filopodia initiation.

Bottom Line: Subsets of independently nucleated lamellipodial filaments elongated and gradually associated with each other at their barbed ends, leading to formation of cone-shaped structures that we term Lambda-precursors.The GFP-VASP foci were associated with Lambda-precursors, whereas Arp2/3 was not.We propose a convergent elongation model of filopodia initiation, stipulating that filaments within the lamellipodial dendritic network acquire privileged status by binding a set of molecules (including VASP) to their barbed ends, which protect them from capping and mediate association of barbed ends with each other.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Molecular Biology, Northwestern University Medical School, Chicago, Illinois 60611, USA. t-svitkina@northwestern.edu

ABSTRACT
Afilopodium protrudes by elongation of bundled actin filaments in its core. However, the mechanism of filopodia initiation remains unknown. Using live-cell imaging with GFP-tagged proteins and correlative electron microscopy, we performed a kinetic-structural analysis of filopodial initiation in B16F1 melanoma cells. Filopodial bundles arose not by a specific nucleation event, but by reorganization of the lamellipodial dendritic network analogous to fusion of established filopodia but occurring at the level of individual filaments. Subsets of independently nucleated lamellipodial filaments elongated and gradually associated with each other at their barbed ends, leading to formation of cone-shaped structures that we term Lambda-precursors. An early marker of initiation was the gradual coalescence of GFP-vasodilator-stimulated phosphoprotein (GFP-VASP) fluorescence at the leading edge into discrete foci. The GFP-VASP foci were associated with Lambda-precursors, whereas Arp2/3 was not. Subsequent recruitment of fascin to the clustered barbed ends of Lambda-precursors initiated filament bundling and completed formation of the nascent filopodium. We propose a convergent elongation model of filopodia initiation, stipulating that filaments within the lamellipodial dendritic network acquire privileged status by binding a set of molecules (including VASP) to their barbed ends, which protect them from capping and mediate association of barbed ends with each other.

Show MeSH
Related in: MedlinePlus