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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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Structural organization of filopodia with known history. Correlative live imaging and EM. (A) Overview of the cell lamellipodium at different stages of sample processing. GFP-actin fluorescence images taken just before (live, 16 s) and immediately after (lysed, 35 s) cell lysis were merged (live + lysed) in green and red channels, respectively; cell advance during the 19 s between images appears as red strip at the leading edge. Low magnification EM image of the same region (EM) is overlaid with actin fluorescence image of the lysed cell (EM + actin). (B) Boxed region in A is enlarged in B to show correlation between light and EM in more detail. Brighter areas in fluorescence image correspond to denser actin arrays in EM. (C) Detailed history of the boxed region in A. Time in seconds. The 35 s frame is taken from the lysed cell. Arrows of different color indicate position of individual nascent filopodia. (D) Enlarged EM of the boxed region in B showing the structure of nascent filopodia, whose history is presented in C; individual nascent filopodia are outlined in colors corresponding to colors of arrows in C. Some filaments converging into the bundle of the “blue” filopodium are highlighted in shades of blue. Boxes 1 and 2 are further enlarged in corresponding insets and show organization of the filopodial tip (1) and of the root of “green” filopodium (2); branching filaments are highlighted in green in the inset (2). See detail in text. Bars, 2 μm (A) and 0.2 μm (D).
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fig7: Structural organization of filopodia with known history. Correlative live imaging and EM. (A) Overview of the cell lamellipodium at different stages of sample processing. GFP-actin fluorescence images taken just before (live, 16 s) and immediately after (lysed, 35 s) cell lysis were merged (live + lysed) in green and red channels, respectively; cell advance during the 19 s between images appears as red strip at the leading edge. Low magnification EM image of the same region (EM) is overlaid with actin fluorescence image of the lysed cell (EM + actin). (B) Boxed region in A is enlarged in B to show correlation between light and EM in more detail. Brighter areas in fluorescence image correspond to denser actin arrays in EM. (C) Detailed history of the boxed region in A. Time in seconds. The 35 s frame is taken from the lysed cell. Arrows of different color indicate position of individual nascent filopodia. (D) Enlarged EM of the boxed region in B showing the structure of nascent filopodia, whose history is presented in C; individual nascent filopodia are outlined in colors corresponding to colors of arrows in C. Some filaments converging into the bundle of the “blue” filopodium are highlighted in shades of blue. Boxes 1 and 2 are further enlarged in corresponding insets and show organization of the filopodial tip (1) and of the root of “green” filopodium (2); branching filaments are highlighted in green in the inset (2). See detail in text. Bars, 2 μm (A) and 0.2 μm (D).

Mentions: Because not every Λ-precursor produced a filopodium in kinetic studies, we performed correlative EM for cells with known history. For this purpose, we acquired time-lapse sequences of GFP-actin–expressing cells. After extraction and fixation, we prepared those cells for EM and analyzed filopodia formed in the course of the sequence (Fig. 7). Fig. 7 A illustrates the correlation between the last live image of one such cell, the image of the lysed cell, and the EM image of the same cell taken at low magnification comparable with that of light microscopy. During the 19-s interval between the last live image and the image of the lysed cell, the lamellipodium protruded ∼0.9 μm, which is evident in the superimposed image. The subsequent processing for EM did not introduce significant distortions into the structure of the lamellipodium because extracted light and low power EM images could be almost perfectly overlapped. Coincidence of light and EM features could also be seen at higher magnification, where brighter areas in fluorescence corresponded to denser actin arrays in EM (Fig. 7 B).


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)

Structural organization of filopodia with known history. Correlative live imaging and EM. (A) Overview of the cell lamellipodium at different stages of sample processing. GFP-actin fluorescence images taken just before (live, 16 s) and immediately after (lysed, 35 s) cell lysis were merged (live + lysed) in green and red channels, respectively; cell advance during the 19 s between images appears as red strip at the leading edge. Low magnification EM image of the same region (EM) is overlaid with actin fluorescence image of the lysed cell (EM + actin). (B) Boxed region in A is enlarged in B to show correlation between light and EM in more detail. Brighter areas in fluorescence image correspond to denser actin arrays in EM. (C) Detailed history of the boxed region in A. Time in seconds. The 35 s frame is taken from the lysed cell. Arrows of different color indicate position of individual nascent filopodia. (D) Enlarged EM of the boxed region in B showing the structure of nascent filopodia, whose history is presented in C; individual nascent filopodia are outlined in colors corresponding to colors of arrows in C. Some filaments converging into the bundle of the “blue” filopodium are highlighted in shades of blue. Boxes 1 and 2 are further enlarged in corresponding insets and show organization of the filopodial tip (1) and of the root of “green” filopodium (2); branching filaments are highlighted in green in the inset (2). See detail in text. Bars, 2 μm (A) and 0.2 μm (D).
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fig7: Structural organization of filopodia with known history. Correlative live imaging and EM. (A) Overview of the cell lamellipodium at different stages of sample processing. GFP-actin fluorescence images taken just before (live, 16 s) and immediately after (lysed, 35 s) cell lysis were merged (live + lysed) in green and red channels, respectively; cell advance during the 19 s between images appears as red strip at the leading edge. Low magnification EM image of the same region (EM) is overlaid with actin fluorescence image of the lysed cell (EM + actin). (B) Boxed region in A is enlarged in B to show correlation between light and EM in more detail. Brighter areas in fluorescence image correspond to denser actin arrays in EM. (C) Detailed history of the boxed region in A. Time in seconds. The 35 s frame is taken from the lysed cell. Arrows of different color indicate position of individual nascent filopodia. (D) Enlarged EM of the boxed region in B showing the structure of nascent filopodia, whose history is presented in C; individual nascent filopodia are outlined in colors corresponding to colors of arrows in C. Some filaments converging into the bundle of the “blue” filopodium are highlighted in shades of blue. Boxes 1 and 2 are further enlarged in corresponding insets and show organization of the filopodial tip (1) and of the root of “green” filopodium (2); branching filaments are highlighted in green in the inset (2). See detail in text. Bars, 2 μm (A) and 0.2 μm (D).
Mentions: Because not every Λ-precursor produced a filopodium in kinetic studies, we performed correlative EM for cells with known history. For this purpose, we acquired time-lapse sequences of GFP-actin–expressing cells. After extraction and fixation, we prepared those cells for EM and analyzed filopodia formed in the course of the sequence (Fig. 7). Fig. 7 A illustrates the correlation between the last live image of one such cell, the image of the lysed cell, and the EM image of the same cell taken at low magnification comparable with that of light microscopy. During the 19-s interval between the last live image and the image of the lysed cell, the lamellipodium protruded ∼0.9 μm, which is evident in the superimposed image. The subsequent processing for EM did not introduce significant distortions into the structure of the lamellipodium because extracted light and low power EM images could be almost perfectly overlapped. Coincidence of light and EM features could also be seen at higher magnification, where brighter areas in fluorescence corresponded to denser actin arrays in EM (Fig. 7 B).

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