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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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Interconversion between microspikes, filopodia, and retraction fibers. Time-lapse sequences of untransfected (A, phase contrast) or GFP-actin expressing (B and C, fluorescence) cells. Time in seconds. (A) Lamellipodium containing several microspikes (triple arrow and arrowhead in first frame) retracts leaving microspikes in the form of retraction fibers (240 s), some of which continue to protrude (240–400 s, arrow). At ∼400 s, lamellipodium resumes protrusion and absorbs retraction fibers, some of which disappear, one becoming a filopodium (560 s, arrow), and another becoming a microspike (560 s, arrowhead). (B) Transition of a microspike (0 and 20 s) to filopodium (20–60 s) to retraction fiber (60–140 s). Actin bundle displayed continuous elongation, whereas surrounding lamellipodium initially kept up with the bundle (0–20 s), paused (40 s), and then withdrew (60–140 s). (C) Transition of microspike (0–30 s) to filopodium (45–75 s) and back to microspike (90–105 s) as a result of uncoordinated protrusive behavior of the bundle and the lamellipodium. Bars, 2 μm.
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fig1: Interconversion between microspikes, filopodia, and retraction fibers. Time-lapse sequences of untransfected (A, phase contrast) or GFP-actin expressing (B and C, fluorescence) cells. Time in seconds. (A) Lamellipodium containing several microspikes (triple arrow and arrowhead in first frame) retracts leaving microspikes in the form of retraction fibers (240 s), some of which continue to protrude (240–400 s, arrow). At ∼400 s, lamellipodium resumes protrusion and absorbs retraction fibers, some of which disappear, one becoming a filopodium (560 s, arrow), and another becoming a microspike (560 s, arrowhead). (B) Transition of a microspike (0 and 20 s) to filopodium (20–60 s) to retraction fiber (60–140 s). Actin bundle displayed continuous elongation, whereas surrounding lamellipodium initially kept up with the bundle (0–20 s), paused (40 s), and then withdrew (60–140 s). (C) Transition of microspike (0–30 s) to filopodium (45–75 s) and back to microspike (90–105 s) as a result of uncoordinated protrusive behavior of the bundle and the lamellipodium. Bars, 2 μm.

Mentions: We followed the kinetics of peripheral actin bundles by phase contrast or fluorescence microscopy in untransfected or GFP-actin–expressing cells, respectively. We observed many examples of transition between filopodia, microspikes, and retraction fibers (Fig. 1). The predominant order of transitions was from microspike to filopodium to retraction fiber. Transitions in the opposite direction were also observed. For each type of structure, the filament bundle was able to protrude, suggesting that the actin polymerizing machinery was functional in each morphological state. The protrusive activity of the surrounding lamellipodium seemed to be an important factor determining the transitions between these organelles. Depending on whether the lamellipodium advanced as fast as or slower than an actin bundle elongated, the bundle appeared as a microspike or a filopodium. If the lamellipodium withdrew while the actin bundle remained stable or elongated, the bundle appeared as a retraction fiber. Increased net protrusion of an actin bundle also contributed to the transition from microspikes to filopodia, especially after fusion of two microspikes. Thus, filopodia, microspikes, and retraction fibers are interconvertible organelles, which may transform one into another because of a disparity in the protrusion velocity of the bundles themselves and of the surrounding lamellipodium. Therefore, in this paper, we will consider these types of peripheral actin bundles together and will refer to them collectively as “filopodia,” because this is the most commonly used term.


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)

Interconversion between microspikes, filopodia, and retraction fibers. Time-lapse sequences of untransfected (A, phase contrast) or GFP-actin expressing (B and C, fluorescence) cells. Time in seconds. (A) Lamellipodium containing several microspikes (triple arrow and arrowhead in first frame) retracts leaving microspikes in the form of retraction fibers (240 s), some of which continue to protrude (240–400 s, arrow). At ∼400 s, lamellipodium resumes protrusion and absorbs retraction fibers, some of which disappear, one becoming a filopodium (560 s, arrow), and another becoming a microspike (560 s, arrowhead). (B) Transition of a microspike (0 and 20 s) to filopodium (20–60 s) to retraction fiber (60–140 s). Actin bundle displayed continuous elongation, whereas surrounding lamellipodium initially kept up with the bundle (0–20 s), paused (40 s), and then withdrew (60–140 s). (C) Transition of microspike (0–30 s) to filopodium (45–75 s) and back to microspike (90–105 s) as a result of uncoordinated protrusive behavior of the bundle and the lamellipodium. Bars, 2 μm.
© Copyright Policy
Related In: Results  -  Collection

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

fig1: Interconversion between microspikes, filopodia, and retraction fibers. Time-lapse sequences of untransfected (A, phase contrast) or GFP-actin expressing (B and C, fluorescence) cells. Time in seconds. (A) Lamellipodium containing several microspikes (triple arrow and arrowhead in first frame) retracts leaving microspikes in the form of retraction fibers (240 s), some of which continue to protrude (240–400 s, arrow). At ∼400 s, lamellipodium resumes protrusion and absorbs retraction fibers, some of which disappear, one becoming a filopodium (560 s, arrow), and another becoming a microspike (560 s, arrowhead). (B) Transition of a microspike (0 and 20 s) to filopodium (20–60 s) to retraction fiber (60–140 s). Actin bundle displayed continuous elongation, whereas surrounding lamellipodium initially kept up with the bundle (0–20 s), paused (40 s), and then withdrew (60–140 s). (C) Transition of microspike (0–30 s) to filopodium (45–75 s) and back to microspike (90–105 s) as a result of uncoordinated protrusive behavior of the bundle and the lamellipodium. Bars, 2 μm.
Mentions: We followed the kinetics of peripheral actin bundles by phase contrast or fluorescence microscopy in untransfected or GFP-actin–expressing cells, respectively. We observed many examples of transition between filopodia, microspikes, and retraction fibers (Fig. 1). The predominant order of transitions was from microspike to filopodium to retraction fiber. Transitions in the opposite direction were also observed. For each type of structure, the filament bundle was able to protrude, suggesting that the actin polymerizing machinery was functional in each morphological state. The protrusive activity of the surrounding lamellipodium seemed to be an important factor determining the transitions between these organelles. Depending on whether the lamellipodium advanced as fast as or slower than an actin bundle elongated, the bundle appeared as a microspike or a filopodium. If the lamellipodium withdrew while the actin bundle remained stable or elongated, the bundle appeared as a retraction fiber. Increased net protrusion of an actin bundle also contributed to the transition from microspikes to filopodia, especially after fusion of two microspikes. Thus, filopodia, microspikes, and retraction fibers are interconvertible organelles, which may transform one into another because of a disparity in the protrusion velocity of the bundles themselves and of the surrounding lamellipodium. Therefore, in this paper, we will consider these types of peripheral actin bundles together and will refer to them collectively as “filopodia,” because this is the most commonly used term.

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