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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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Localization of filopodial and lamellipodial markers in Λ-precursors. Left column; actin revealed by Texas Red-phalloidin (A, B, and F) or by GFP-actin expression (C–E). Central column; actin-binding proteins (as indicated) revealed by expression of GFP-fusion proteins (A, B, and F) or by immunostaining (C–E). Right column; merged images. Λ-precursors are indicated by arrowheads. (A) Fascin is strongly enriched in established filopodia and localizes to the distal section of some Λ-precursors (wide arrowheads), but not others (narrow arrowheads). (B) VASP forms bright dots at the tips of filopodia and Λ-precursors. Additional dots can be seen along the leading edge (arrows), which apparently do not correspond to Λ-precursors. (C–E) Lamellipodial markers, Arp2/3 complex (C), cortactin (D), and capping protein (E), are excluded from filopodia and are partially depleted from Λ-precursors. (F) α-Actinin localizes to proximal parts of lamellipodia and filopodia. Bars, 2 μm.
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fig4: Localization of filopodial and lamellipodial markers in Λ-precursors. Left column; actin revealed by Texas Red-phalloidin (A, B, and F) or by GFP-actin expression (C–E). Central column; actin-binding proteins (as indicated) revealed by expression of GFP-fusion proteins (A, B, and F) or by immunostaining (C–E). Right column; merged images. Λ-precursors are indicated by arrowheads. (A) Fascin is strongly enriched in established filopodia and localizes to the distal section of some Λ-precursors (wide arrowheads), but not others (narrow arrowheads). (B) VASP forms bright dots at the tips of filopodia and Λ-precursors. Additional dots can be seen along the leading edge (arrows), which apparently do not correspond to Λ-precursors. (C–E) Lamellipodial markers, Arp2/3 complex (C), cortactin (D), and capping protein (E), are excluded from filopodia and are partially depleted from Λ-precursors. (F) α-Actinin localizes to proximal parts of lamellipodia and filopodia. Bars, 2 μm.

Mentions: Our kinetic analysis identified Λ-precursors as intermediates in filopodial initiation. Next, we examined whether molecular markers that discriminate between lamellipodia and filopodia are present in Λ-precursors (Fig. 4). Expression of GFP-actin or staining with labeled phalloidin was used to visualize actin. Putative Λ-precursors were identified in the actin channel based on their characteristic shape, and slightly increased actin density. Immunostaining or expression of GFP-tagged proteins was used to localize the second protein.


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)

Localization of filopodial and lamellipodial markers in Λ-precursors. Left column; actin revealed by Texas Red-phalloidin (A, B, and F) or by GFP-actin expression (C–E). Central column; actin-binding proteins (as indicated) revealed by expression of GFP-fusion proteins (A, B, and F) or by immunostaining (C–E). Right column; merged images. Λ-precursors are indicated by arrowheads. (A) Fascin is strongly enriched in established filopodia and localizes to the distal section of some Λ-precursors (wide arrowheads), but not others (narrow arrowheads). (B) VASP forms bright dots at the tips of filopodia and Λ-precursors. Additional dots can be seen along the leading edge (arrows), which apparently do not correspond to Λ-precursors. (C–E) Lamellipodial markers, Arp2/3 complex (C), cortactin (D), and capping protein (E), are excluded from filopodia and are partially depleted from Λ-precursors. (F) α-Actinin localizes to proximal parts of lamellipodia and filopodia. Bars, 2 μm.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172658&req=5

fig4: Localization of filopodial and lamellipodial markers in Λ-precursors. Left column; actin revealed by Texas Red-phalloidin (A, B, and F) or by GFP-actin expression (C–E). Central column; actin-binding proteins (as indicated) revealed by expression of GFP-fusion proteins (A, B, and F) or by immunostaining (C–E). Right column; merged images. Λ-precursors are indicated by arrowheads. (A) Fascin is strongly enriched in established filopodia and localizes to the distal section of some Λ-precursors (wide arrowheads), but not others (narrow arrowheads). (B) VASP forms bright dots at the tips of filopodia and Λ-precursors. Additional dots can be seen along the leading edge (arrows), which apparently do not correspond to Λ-precursors. (C–E) Lamellipodial markers, Arp2/3 complex (C), cortactin (D), and capping protein (E), are excluded from filopodia and are partially depleted from Λ-precursors. (F) α-Actinin localizes to proximal parts of lamellipodia and filopodia. Bars, 2 μm.
Mentions: Our kinetic analysis identified Λ-precursors as intermediates in filopodial initiation. Next, we examined whether molecular markers that discriminate between lamellipodia and filopodia are present in Λ-precursors (Fig. 4). Expression of GFP-actin or staining with labeled phalloidin was used to visualize actin. Putative Λ-precursors were identified in the actin channel based on their characteristic shape, and slightly increased actin density. Immunostaining or expression of GFP-tagged proteins was used to localize the second protein.

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