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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 filament organization in Λ-precursors. Correlative fluorescence and EM of the same cell. First row; fluorescence microscopy of the leading edge showing Texas Red-phalloidin labeled actin (left), GFP-fascin (middle), and merged image (right). White dots in the merged image mark putative Λ-precursors, and the arrowhead indicates a filopodium, which have been analyzed by EM. Second row; EM of the same region (right) overlaid with fluorescence images of actin in red (left) and fascin in green (middle). Boxed regions (a–c) are enlarged in the bottom panels, labeled respectively. Red outlines in enlarged panels denote areas of increased actin density in the fluorescence image that represent putative Λ-precursors or filopodia. Λ-precursors contain relatively long filaments, some of which are highlighted in cyan in A and C, along with short branching filaments highlighted in magenta; adjacent lamellipodium (C) contains mostly short branching filaments highlighted in magenta. Yellow outline denotes region enriched in fascin in the established filopodium. This region contains tightly bundled filaments. Bar, 0.2 μm.
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fig6: Actin filament organization in Λ-precursors. Correlative fluorescence and EM of the same cell. First row; fluorescence microscopy of the leading edge showing Texas Red-phalloidin labeled actin (left), GFP-fascin (middle), and merged image (right). White dots in the merged image mark putative Λ-precursors, and the arrowhead indicates a filopodium, which have been analyzed by EM. Second row; EM of the same region (right) overlaid with fluorescence images of actin in red (left) and fascin in green (middle). Boxed regions (a–c) are enlarged in the bottom panels, labeled respectively. Red outlines in enlarged panels denote areas of increased actin density in the fluorescence image that represent putative Λ-precursors or filopodia. Λ-precursors contain relatively long filaments, some of which are highlighted in cyan in A and C, along with short branching filaments highlighted in magenta; adjacent lamellipodium (C) contains mostly short branching filaments highlighted in magenta. Yellow outline denotes region enriched in fascin in the established filopodium. This region contains tightly bundled filaments. Bar, 0.2 μm.

Mentions: Fascin and VASP were used as filopodial markers. In GFP-fascin–expressing cells, putative Λ-precursors either did not contain significant amount of fascin (Fig. 4 A, arrows; also see Fig. 6), or they had fascin enriched only at the tip (Fig. 4 A, arrowheads), suggesting that fascin was recruited to the tips of the preformed Λ-precursors. In GFP-VASP–expressing cells (Fig. 4 B), VASP fluorescence at the leading edge became less uniform after extraction, perhaps revealing more strongly associated protein. The brightest VASP dots corresponded to established filopodia. The majority of Λ-precursors contained a distinct VASP dot at their vertex (Fig. 4 B, arrowheads). Weak VASP dots were also evident without recognizable Λ-precursors (Fig. 4 B, arrows). These may represent fluctuations in density of actin filament barbed ends within the lamellipodium or Λ-precursors not detected in the actin channel.


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 filament organization in Λ-precursors. Correlative fluorescence and EM of the same cell. First row; fluorescence microscopy of the leading edge showing Texas Red-phalloidin labeled actin (left), GFP-fascin (middle), and merged image (right). White dots in the merged image mark putative Λ-precursors, and the arrowhead indicates a filopodium, which have been analyzed by EM. Second row; EM of the same region (right) overlaid with fluorescence images of actin in red (left) and fascin in green (middle). Boxed regions (a–c) are enlarged in the bottom panels, labeled respectively. Red outlines in enlarged panels denote areas of increased actin density in the fluorescence image that represent putative Λ-precursors or filopodia. Λ-precursors contain relatively long filaments, some of which are highlighted in cyan in A and C, along with short branching filaments highlighted in magenta; adjacent lamellipodium (C) contains mostly short branching filaments highlighted in magenta. Yellow outline denotes region enriched in fascin in the established filopodium. This region contains tightly bundled filaments. Bar, 0.2 μm.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172658&req=5

fig6: Actin filament organization in Λ-precursors. Correlative fluorescence and EM of the same cell. First row; fluorescence microscopy of the leading edge showing Texas Red-phalloidin labeled actin (left), GFP-fascin (middle), and merged image (right). White dots in the merged image mark putative Λ-precursors, and the arrowhead indicates a filopodium, which have been analyzed by EM. Second row; EM of the same region (right) overlaid with fluorescence images of actin in red (left) and fascin in green (middle). Boxed regions (a–c) are enlarged in the bottom panels, labeled respectively. Red outlines in enlarged panels denote areas of increased actin density in the fluorescence image that represent putative Λ-precursors or filopodia. Λ-precursors contain relatively long filaments, some of which are highlighted in cyan in A and C, along with short branching filaments highlighted in magenta; adjacent lamellipodium (C) contains mostly short branching filaments highlighted in magenta. Yellow outline denotes region enriched in fascin in the established filopodium. This region contains tightly bundled filaments. Bar, 0.2 μm.
Mentions: Fascin and VASP were used as filopodial markers. In GFP-fascin–expressing cells, putative Λ-precursors either did not contain significant amount of fascin (Fig. 4 A, arrows; also see Fig. 6), or they had fascin enriched only at the tip (Fig. 4 A, arrowheads), suggesting that fascin was recruited to the tips of the preformed Λ-precursors. In GFP-VASP–expressing cells (Fig. 4 B), VASP fluorescence at the leading edge became less uniform after extraction, perhaps revealing more strongly associated protein. The brightest VASP dots corresponded to established filopodia. The majority of Λ-precursors contained a distinct VASP dot at their vertex (Fig. 4 B, arrowheads). Weak VASP dots were also evident without recognizable Λ-precursors (Fig. 4 B, arrows). These may represent fluctuations in density of actin filament barbed ends within the lamellipodium or Λ-precursors not detected in the actin channel.

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