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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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Filopodial filaments originate from the surrounding dendritic network. Platinum replica EM. (A) Filopodium contains a tight bundle of actin filaments that splays apart at its root and becomes an integral part of the surrounding network. Filaments in the roots are long compared with the branching network of the adjacent lamellipodium (inset). (B) Recently fused filopodium consists of two sub-bundles, each of which has a splayed root; the boxed region at the root of the right sub-bundle is enlarged in C and shows many branches (encircled) at which filopodial filaments originate. Rough background outside the cell edge is due to laminin coating of the glass coverslip. Bars, 0.2 μm.
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fig5: Filopodial filaments originate from the surrounding dendritic network. Platinum replica EM. (A) Filopodium contains a tight bundle of actin filaments that splays apart at its root and becomes an integral part of the surrounding network. Filaments in the roots are long compared with the branching network of the adjacent lamellipodium (inset). (B) Recently fused filopodium consists of two sub-bundles, each of which has a splayed root; the boxed region at the root of the right sub-bundle is enlarged in C and shows many branches (encircled) at which filopodial filaments originate. Rough background outside the cell edge is due to laminin coating of the glass coverslip. Bars, 0.2 μm.

Mentions: The majority of apparently young filopodial bundles were splayed apart at their roots into smaller bundles or individual filaments (Fig. 5), suggesting that the bundles were formed by convergence of the composing elements. Filopodial roots consisting of two or more smaller bundles are consistent with an event of filopodial fusion in the recent history of that filopodium (Fig. 5 B). More importantly, we observed many examples of filopodial bundles whose roots suggested the convergence of individual filaments originating from distant places in the surrounding lamellipodial network and entering the bundle at different levels. In some cases, it was possible to track filaments back from the bundle toward their origin as a branch on another filament in the surrounding network (Fig. 5 C). These findings suggest that filaments comprising filopodial bundles were asynchronously recruited from the dendritic network. Remarkably, filaments entering filopodial bundles were long compared with the branched network near the leading edge (Fig. 5, inset in A). Older filopodia, which could be recognized by their length and thickness, either had their actin bundles rooted deeply in the cytoplasm, which impeded visualization, or had tapered (not splayed) roots. This is consistent with depolymerization from the pointed ends of the composing filaments causing progressive elimination of the original splayed roots.


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)

Filopodial filaments originate from the surrounding dendritic network. Platinum replica EM. (A) Filopodium contains a tight bundle of actin filaments that splays apart at its root and becomes an integral part of the surrounding network. Filaments in the roots are long compared with the branching network of the adjacent lamellipodium (inset). (B) Recently fused filopodium consists of two sub-bundles, each of which has a splayed root; the boxed region at the root of the right sub-bundle is enlarged in C and shows many branches (encircled) at which filopodial filaments originate. Rough background outside the cell edge is due to laminin coating of the glass coverslip. Bars, 0.2 μm.
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Related In: Results  -  Collection

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fig5: Filopodial filaments originate from the surrounding dendritic network. Platinum replica EM. (A) Filopodium contains a tight bundle of actin filaments that splays apart at its root and becomes an integral part of the surrounding network. Filaments in the roots are long compared with the branching network of the adjacent lamellipodium (inset). (B) Recently fused filopodium consists of two sub-bundles, each of which has a splayed root; the boxed region at the root of the right sub-bundle is enlarged in C and shows many branches (encircled) at which filopodial filaments originate. Rough background outside the cell edge is due to laminin coating of the glass coverslip. Bars, 0.2 μm.
Mentions: The majority of apparently young filopodial bundles were splayed apart at their roots into smaller bundles or individual filaments (Fig. 5), suggesting that the bundles were formed by convergence of the composing elements. Filopodial roots consisting of two or more smaller bundles are consistent with an event of filopodial fusion in the recent history of that filopodium (Fig. 5 B). More importantly, we observed many examples of filopodial bundles whose roots suggested the convergence of individual filaments originating from distant places in the surrounding lamellipodial network and entering the bundle at different levels. In some cases, it was possible to track filaments back from the bundle toward their origin as a branch on another filament in the surrounding network (Fig. 5 C). These findings suggest that filaments comprising filopodial bundles were asynchronously recruited from the dendritic network. Remarkably, filaments entering filopodial bundles were long compared with the branched network near the leading edge (Fig. 5, inset in A). Older filopodia, which could be recognized by their length and thickness, either had their actin bundles rooted deeply in the cytoplasm, which impeded visualization, or had tapered (not splayed) roots. This is consistent with depolymerization from the pointed ends of the composing filaments causing progressive elimination of the original splayed roots.

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