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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 tip complex. (A) Structure of tip complex in three filopodia seen by replica EM. Lower magnification, left; higher magnification, right. (B) Incubation of lysed cells in buffer causes dissociation of fascin (top) but not VASP (bottom) from filopodia. Phase contrast and GFP fluorescence images of cells transiently expressing indicated fusion proteins were taken immediately after cell lysis (“before incubation” phase images are not depicted) and after overnight incubation in phalloidin-containing buffer (“after incubation”). Acquisition and processing of fluorescence images was identical for each pair. (C) Filopodial bundle and tip complex after overnight incubation of the lysed cell in phalloidin-containing buffer. Replica EM. Filopodial tip complex keeps barbed ends of filopodial filaments together, whereas filaments within the bundle become loose. (D) Junctions between barbed ends of lamellipodial filaments (arrows) immediately after lysis or after incubation in buffer like in B or C (“after incubation”). Bars: 0.1 μm (A, C, and D), and 2 μm (B).
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fig8: Filopodial tip complex. (A) Structure of tip complex in three filopodia seen by replica EM. Lower magnification, left; higher magnification, right. (B) Incubation of lysed cells in buffer causes dissociation of fascin (top) but not VASP (bottom) from filopodia. Phase contrast and GFP fluorescence images of cells transiently expressing indicated fusion proteins were taken immediately after cell lysis (“before incubation” phase images are not depicted) and after overnight incubation in phalloidin-containing buffer (“after incubation”). Acquisition and processing of fluorescence images was identical for each pair. (C) Filopodial bundle and tip complex after overnight incubation of the lysed cell in phalloidin-containing buffer. Replica EM. Filopodial tip complex keeps barbed ends of filopodial filaments together, whereas filaments within the bundle become loose. (D) Junctions between barbed ends of lamellipodial filaments (arrows) immediately after lysis or after incubation in buffer like in B or C (“after incubation”). Bars: 0.1 μm (A, C, and D), and 2 μm (B).

Mentions: In EM images, many filopodial tips were associated with a distinct structure, which had a rough granular surface and variable shape and size (Fig. 8 A). To test whether this tip complex was involved in physical association of filopodial barbed ends with each other, we incubated lysed cells overnight in phalloidin-containing buffer. Phalloidin prevented depolymerization of actin filaments during incubation, whereas dissociation of other proteins was allowed. We monitored dissociation of fascin or VASP using cells expressing GFP-fusion proteins. Although lysis removed soluble and weakly bound proteins, lysed cells initially retained most of filopodia-associated fascin and VASP. However, fascin was completely gone after incubation, whereas VASP remained (Fig. 8 B). EM of incubated cells revealed that filopodial bundles became loose, consistent with the loss of fascin, but the tip complexes were mostly preserved, consistent with retention of VASP, and filament barbed ends remained associated with each other and with the filopodial tip complex (Fig. 8 C). These results suggest that the tip complex physically links barbed ends in filopodia independently of fascin. Occasionally, the tip complex detached partially or completely during incubation. In such cases, released filopodial filaments completely splayed apart.


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 tip complex. (A) Structure of tip complex in three filopodia seen by replica EM. Lower magnification, left; higher magnification, right. (B) Incubation of lysed cells in buffer causes dissociation of fascin (top) but not VASP (bottom) from filopodia. Phase contrast and GFP fluorescence images of cells transiently expressing indicated fusion proteins were taken immediately after cell lysis (“before incubation” phase images are not depicted) and after overnight incubation in phalloidin-containing buffer (“after incubation”). Acquisition and processing of fluorescence images was identical for each pair. (C) Filopodial bundle and tip complex after overnight incubation of the lysed cell in phalloidin-containing buffer. Replica EM. Filopodial tip complex keeps barbed ends of filopodial filaments together, whereas filaments within the bundle become loose. (D) Junctions between barbed ends of lamellipodial filaments (arrows) immediately after lysis or after incubation in buffer like in B or C (“after incubation”). Bars: 0.1 μm (A, C, and D), and 2 μm (B).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172658&req=5

fig8: Filopodial tip complex. (A) Structure of tip complex in three filopodia seen by replica EM. Lower magnification, left; higher magnification, right. (B) Incubation of lysed cells in buffer causes dissociation of fascin (top) but not VASP (bottom) from filopodia. Phase contrast and GFP fluorescence images of cells transiently expressing indicated fusion proteins were taken immediately after cell lysis (“before incubation” phase images are not depicted) and after overnight incubation in phalloidin-containing buffer (“after incubation”). Acquisition and processing of fluorescence images was identical for each pair. (C) Filopodial bundle and tip complex after overnight incubation of the lysed cell in phalloidin-containing buffer. Replica EM. Filopodial tip complex keeps barbed ends of filopodial filaments together, whereas filaments within the bundle become loose. (D) Junctions between barbed ends of lamellipodial filaments (arrows) immediately after lysis or after incubation in buffer like in B or C (“after incubation”). Bars: 0.1 μm (A, C, and D), and 2 μm (B).
Mentions: In EM images, many filopodial tips were associated with a distinct structure, which had a rough granular surface and variable shape and size (Fig. 8 A). To test whether this tip complex was involved in physical association of filopodial barbed ends with each other, we incubated lysed cells overnight in phalloidin-containing buffer. Phalloidin prevented depolymerization of actin filaments during incubation, whereas dissociation of other proteins was allowed. We monitored dissociation of fascin or VASP using cells expressing GFP-fusion proteins. Although lysis removed soluble and weakly bound proteins, lysed cells initially retained most of filopodia-associated fascin and VASP. However, fascin was completely gone after incubation, whereas VASP remained (Fig. 8 B). EM of incubated cells revealed that filopodial bundles became loose, consistent with the loss of fascin, but the tip complexes were mostly preserved, consistent with retention of VASP, and filament barbed ends remained associated with each other and with the filopodial tip complex (Fig. 8 C). These results suggest that the tip complex physically links barbed ends in filopodia independently of fascin. Occasionally, the tip complex detached partially or completely during incubation. In such cases, released filopodial filaments completely splayed apart.

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