Mechanism of filopodia initiation by reorganization of a dendritic network

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 Λ-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 Λ-precursors, whereas Arp2/3 was not. Subsequent recruitment of fascin to the clustered barbed ends of Λ-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.

Microvilli appear to represent the first step in actin bundle formation inDrosophilabristles

“…We show that the formation of microvilli in situ occurs by filament elongation from small densities located on the plasma membrane, not by bundling of filaments in the cell cortex as recently suggested for microspike formation in motile cells (Svitkina et al, 2003). Although descriptive, a format viewed by some as passé, this study is paramount because it answers in-depth questions furthering our understanding of the formation of bristles and pattern development in this model system.…”

Nucleation geometry governs ordered actin networks structures

“…3a, see Supplementary Fig. S4 and Video S4), reminiscent of that present in vivo 6 . This interaction forced the filaments to bend, so bundle formation depended on filaments' ability to change their growing direction.…”

“…Interestingly, this propagative coalescence of actin filaments initiated by a precursor, such as the -precursors corresponding to the splayed filopodial roots observed in vivo 6 , accounts for the emergence of the parallel bundles from the dense surrounding network in cells. Moreover, this propagative process explains the presence of short actin filaments within bundles 25 , consistent with the high barbedend capping activity present at the leading edge of lamellipodia 26 .…”

“…Assembled into bundles in filopodia or in stress fibres, they play a pivotal role in eukaryotes during cell morphogenesis, adhesion and motility. The bundle emergence has been extensively related to specific actin regulators 1-3 in vivo [4][5][6][7] . Such dynamic modulation was also highlighted by biochemical reconstitution of the actin-network assembly, in bulk solution or with biomimetic devices [8][9][10][11][12][13][14][15][16][17][18] .…”

Dual Actin-bundling and Protein Kinase C-binding Activities of Fascin Regulate Carcinoma Cell Migration Downstream of Rac and Contribute to Metastasis

“…In vitro, fascin cross-links filamentous actin (Factin) into tightly packed, parallel bundles in cooperation with Arp2/3 complex and Wiskott-Aldrich syndrome protein Haviv et al, 2006). In intact cells, fascin-and-actin bundles support cortical cell protrusions and growth cone filopodia Adams et al, 1999;Cohan et al, 2001, Adams andAnilkumar et al, 2003;Svitkina et al, 2003;Vignjevic et al, 2006). Fascin contains N-and C-terminal actin-binding sites and the actin cross-linking activity of fascin is negatively regulated by a protein kinase C (PKC) phosphorylation site (serine-39 in human fascin) that lies within the amino-terminal actin-binding site (Ono et al, 1997).…”