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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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Convergent elongation model for filopodia initiation. (1) Lamellipodial network is formed by Arp2/3-mediated dendritic nucleation. Elongation of some barbed ends in the network is terminated by capping protein, but other barbed ends acquire a privileged status by binding a complex of molecules (tip complex) that allows them to elongate continuously. Ena/VASP proteins are likely members of the tip complex mediating protection from capping. (2) Privileged barbed ends drift laterally during elongation and collide with each other. Tip complex mediates clustering of privileged barbed ends upon collision. (3) Converged filaments with linked barbed ends continue to elongate together. Other laterally drifting barbed ends encounter and join the initial cluster of tip complexes. Multiple collisions of barbed ends during elongation lead to gradual clustering of their barbed ends, multimerization of associated tip complexes, and convergence of filaments. (4) The multimeric filopodial tip complex initiates filament cross-linking by recruiting and/or activating fascin, which allows the bundling process to keep up with the elongation and guarantee efficient pushing. (5) In the nascent filopodium, the filopodial tip complex retains its functions of promoting coordinated filament elongation and bundling, as well as fusion with other filopodia.
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fig9: Convergent elongation model for filopodia initiation. (1) Lamellipodial network is formed by Arp2/3-mediated dendritic nucleation. Elongation of some barbed ends in the network is terminated by capping protein, but other barbed ends acquire a privileged status by binding a complex of molecules (tip complex) that allows them to elongate continuously. Ena/VASP proteins are likely members of the tip complex mediating protection from capping. (2) Privileged barbed ends drift laterally during elongation and collide with each other. Tip complex mediates clustering of privileged barbed ends upon collision. (3) Converged filaments with linked barbed ends continue to elongate together. Other laterally drifting barbed ends encounter and join the initial cluster of tip complexes. Multiple collisions of barbed ends during elongation lead to gradual clustering of their barbed ends, multimerization of associated tip complexes, and convergence of filaments. (4) The multimeric filopodial tip complex initiates filament cross-linking by recruiting and/or activating fascin, which allows the bundling process to keep up with the elongation and guarantee efficient pushing. (5) In the nascent filopodium, the filopodial tip complex retains its functions of promoting coordinated filament elongation and bundling, as well as fusion with other filopodia.

Mentions: Our kinetic and structural investigation of filopodial initiation in B16F1 melanoma cells demonstrated that filopodial bundles were formed by gradual reorganization of the lamellipodial dendritic network in a process that involved elongation of a subset of lamellipodial filaments, self-segregation of these filaments into filopodial precursors, and initiation of bundling at the tips of the precursors (Fig. 9). We propose that the mechanism of filopodia initiation is analogous to filopodial fusion, but that it begins at the level of individual filaments and gradually propagates to the fusion of thick bundles. We now discuss this hypothesis in detail.


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)

Convergent elongation model for filopodia initiation. (1) Lamellipodial network is formed by Arp2/3-mediated dendritic nucleation. Elongation of some barbed ends in the network is terminated by capping protein, but other barbed ends acquire a privileged status by binding a complex of molecules (tip complex) that allows them to elongate continuously. Ena/VASP proteins are likely members of the tip complex mediating protection from capping. (2) Privileged barbed ends drift laterally during elongation and collide with each other. Tip complex mediates clustering of privileged barbed ends upon collision. (3) Converged filaments with linked barbed ends continue to elongate together. Other laterally drifting barbed ends encounter and join the initial cluster of tip complexes. Multiple collisions of barbed ends during elongation lead to gradual clustering of their barbed ends, multimerization of associated tip complexes, and convergence of filaments. (4) The multimeric filopodial tip complex initiates filament cross-linking by recruiting and/or activating fascin, which allows the bundling process to keep up with the elongation and guarantee efficient pushing. (5) In the nascent filopodium, the filopodial tip complex retains its functions of promoting coordinated filament elongation and bundling, as well as fusion with other filopodia.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172658&req=5

fig9: Convergent elongation model for filopodia initiation. (1) Lamellipodial network is formed by Arp2/3-mediated dendritic nucleation. Elongation of some barbed ends in the network is terminated by capping protein, but other barbed ends acquire a privileged status by binding a complex of molecules (tip complex) that allows them to elongate continuously. Ena/VASP proteins are likely members of the tip complex mediating protection from capping. (2) Privileged barbed ends drift laterally during elongation and collide with each other. Tip complex mediates clustering of privileged barbed ends upon collision. (3) Converged filaments with linked barbed ends continue to elongate together. Other laterally drifting barbed ends encounter and join the initial cluster of tip complexes. Multiple collisions of barbed ends during elongation lead to gradual clustering of their barbed ends, multimerization of associated tip complexes, and convergence of filaments. (4) The multimeric filopodial tip complex initiates filament cross-linking by recruiting and/or activating fascin, which allows the bundling process to keep up with the elongation and guarantee efficient pushing. (5) In the nascent filopodium, the filopodial tip complex retains its functions of promoting coordinated filament elongation and bundling, as well as fusion with other filopodia.
Mentions: Our kinetic and structural investigation of filopodial initiation in B16F1 melanoma cells demonstrated that filopodial bundles were formed by gradual reorganization of the lamellipodial dendritic network in a process that involved elongation of a subset of lamellipodial filaments, self-segregation of these filaments into filopodial precursors, and initiation of bundling at the tips of the precursors (Fig. 9). We propose that the mechanism of filopodia initiation is analogous to filopodial fusion, but that it begins at the level of individual filaments and gradually propagates to the fusion of thick bundles. We now discuss this hypothesis in detail.

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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Related in: MedlinePlus