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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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Kinetics of marker proteins during filopodia initiation. Time-lapse sequences of GFP-Arp3 (A), GFP-fascin (B and C), or GFP-VASP (D–F). (A) Two flanking phase contrast frames (0 and 164 s) demonstrate formation of a new filopodium during the sequence. Positions of preexisting (arrow) and nascent (arrowhead) filopodia are indicated. No focal enrichment of Arp3 was seen during formation of the nascent filopodium. (B and C) Most nascent filopodia arise from bright GFP-fascin dots without obvious Λ-precursors. In some cases, a bright dot localizes to the tip of a faint Λ-shaped density, as in the inset in 16 s frame in C, which shows the region indicated by arrowhead in this frame after enlargement and adjustment of contrast to reveal weak fluorescence. Filopodia fusion occurs in C (arrows). (D–F) Bright GFP-VASP dots corresponding to nascent filopodia arise by gradual coalescence of the initially even line of leading-edge fluorescence. Brackets indicate regions of brighter GFP-VASP fluorescence that shrink into dots over time. In F, two smaller dots (24 s) are formed during coalescence of the shrinking region, which subsequently fuse with each other (32 s) and with the adjacent preexisting dot (40 s). Bars, 2 μm.
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fig3: Kinetics of marker proteins during filopodia initiation. Time-lapse sequences of GFP-Arp3 (A), GFP-fascin (B and C), or GFP-VASP (D–F). (A) Two flanking phase contrast frames (0 and 164 s) demonstrate formation of a new filopodium during the sequence. Positions of preexisting (arrow) and nascent (arrowhead) filopodia are indicated. No focal enrichment of Arp3 was seen during formation of the nascent filopodium. (B and C) Most nascent filopodia arise from bright GFP-fascin dots without obvious Λ-precursors. In some cases, a bright dot localizes to the tip of a faint Λ-shaped density, as in the inset in 16 s frame in C, which shows the region indicated by arrowhead in this frame after enlargement and adjustment of contrast to reveal weak fluorescence. Filopodia fusion occurs in C (arrows). (D–F) Bright GFP-VASP dots corresponding to nascent filopodia arise by gradual coalescence of the initially even line of leading-edge fluorescence. Brackets indicate regions of brighter GFP-VASP fluorescence that shrink into dots over time. In F, two smaller dots (24 s) are formed during coalescence of the shrinking region, which subsequently fuse with each other (32 s) and with the adjacent preexisting dot (40 s). Bars, 2 μm.

Mentions: The Arp2/3 complex is predicted to be enriched in the hypothetical filopodial nucleation center. Therefore, we performed kinetic analysis of GFP-Arp3–expressing cells (Fig. 3 A). Because filopodial bundles were invisible in GFP-Arp3 images, we acquired phase contrast images at the beginning and at the end of the sequence to detect nascent filopodia initiated during the sequence. Filopodia were observed to appear by phase microscopy and GFP-Arp3 was present throughout the lamellipodium, but no increase in GFP-Arp3 intensity was detected to spatially and temporally correlate with filopodial initiation. The essentially uniform distribution of the GFP-Arp3 signal does not support the hypothesis of an Arp2/3-based nucleation center for filopodial initiation.


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)

Kinetics of marker proteins during filopodia initiation. Time-lapse sequences of GFP-Arp3 (A), GFP-fascin (B and C), or GFP-VASP (D–F). (A) Two flanking phase contrast frames (0 and 164 s) demonstrate formation of a new filopodium during the sequence. Positions of preexisting (arrow) and nascent (arrowhead) filopodia are indicated. No focal enrichment of Arp3 was seen during formation of the nascent filopodium. (B and C) Most nascent filopodia arise from bright GFP-fascin dots without obvious Λ-precursors. In some cases, a bright dot localizes to the tip of a faint Λ-shaped density, as in the inset in 16 s frame in C, which shows the region indicated by arrowhead in this frame after enlargement and adjustment of contrast to reveal weak fluorescence. Filopodia fusion occurs in C (arrows). (D–F) Bright GFP-VASP dots corresponding to nascent filopodia arise by gradual coalescence of the initially even line of leading-edge fluorescence. Brackets indicate regions of brighter GFP-VASP fluorescence that shrink into dots over time. In F, two smaller dots (24 s) are formed during coalescence of the shrinking region, which subsequently fuse with each other (32 s) and with the adjacent preexisting dot (40 s). Bars, 2 μm.
© Copyright Policy
Related In: Results  -  Collection

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

fig3: Kinetics of marker proteins during filopodia initiation. Time-lapse sequences of GFP-Arp3 (A), GFP-fascin (B and C), or GFP-VASP (D–F). (A) Two flanking phase contrast frames (0 and 164 s) demonstrate formation of a new filopodium during the sequence. Positions of preexisting (arrow) and nascent (arrowhead) filopodia are indicated. No focal enrichment of Arp3 was seen during formation of the nascent filopodium. (B and C) Most nascent filopodia arise from bright GFP-fascin dots without obvious Λ-precursors. In some cases, a bright dot localizes to the tip of a faint Λ-shaped density, as in the inset in 16 s frame in C, which shows the region indicated by arrowhead in this frame after enlargement and adjustment of contrast to reveal weak fluorescence. Filopodia fusion occurs in C (arrows). (D–F) Bright GFP-VASP dots corresponding to nascent filopodia arise by gradual coalescence of the initially even line of leading-edge fluorescence. Brackets indicate regions of brighter GFP-VASP fluorescence that shrink into dots over time. In F, two smaller dots (24 s) are formed during coalescence of the shrinking region, which subsequently fuse with each other (32 s) and with the adjacent preexisting dot (40 s). Bars, 2 μm.
Mentions: The Arp2/3 complex is predicted to be enriched in the hypothetical filopodial nucleation center. Therefore, we performed kinetic analysis of GFP-Arp3–expressing cells (Fig. 3 A). Because filopodial bundles were invisible in GFP-Arp3 images, we acquired phase contrast images at the beginning and at the end of the sequence to detect nascent filopodia initiated during the sequence. Filopodia were observed to appear by phase microscopy and GFP-Arp3 was present throughout the lamellipodium, but no increase in GFP-Arp3 intensity was detected to spatially and temporally correlate with filopodial initiation. The essentially uniform distribution of the GFP-Arp3 signal does not support the hypothesis of an Arp2/3-based nucleation center for filopodial initiation.

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