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Arp2/3 complex patches in filopodia are highly dynamic and co-localise with increases in RFP actin signal. Cells were spread for 60 minutes on fibronectin after co-transfection with GFP p21-arc and RFP actin. Solid arrows indicate Arp2/3 complex foci in filopodia and hollow arrows indicate the corresponding location in the RFP actin images. Images are taken from Movie 4. Scale bar 10 μm.
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Figure 4: Arp2/3 complex patches in filopodia are highly dynamic and co-localise with increases in RFP actin signal. Cells were spread for 60 minutes on fibronectin after co-transfection with GFP p21-arc and RFP actin. Solid arrows indicate Arp2/3 complex foci in filopodia and hollow arrows indicate the corresponding location in the RFP actin images. Images are taken from Movie 4. Scale bar 10 μm.

Mentions: Lamellipodia are protrusions of membrane driven by dendritic networks of actin, while filopodia are classically thought to be driven by parallel bundles of elongating actin filaments. We examined the dynamics of Arp2/3 complex in filopodia during cell spreading using p21-arc subunit of the Arp2/3 complex tagged with GFP. RFP actin was co-expressed to confirm the location of filopodia. The patches of Arp2/3 complex in filopodia were highly dynamic, changing size and intensity much in the same way as lamellipodial ruffles (Figure 4 and see Additional file 5 and 6). Arp2/3 complex puncta appeared and disappeared within filopodia, rather than seeming to be transported from within the cell or from lamellipodia.

Arp2/3 complex activity in filopodia of spreading cells

Johnston SA, Bramble JP, Yeung CL, Mendes PM, Machesky LM - BMC Cell Biol. (2008)

Bottom Line: Arp2/3 complex in filopodia co-localises with lamellipodial proteins such as capping protein and cortactin.We suggest that filopodia of spreading cells have regions of lamellipodial activity and that this activity affects the morphology and movement of filopodia.Our work has implications for how we understand the interplay between lamellipodia and filopodia and for how actin networks are generated spatially in cells.

Affiliation: University of Birmingham School of Biosciences, Edgbaston, Birmingham, UK. s.a.johnston@bham.ac.uk

ABSTRACT

Background: Cells use filopodia to explore their environment and to form new adhesion contacts for motility and spreading. The Arp2/3 complex has been implicated in lamellipodial actin assembly as a major nucleator of new actin filaments in branched networks. The interplay between filopodial and lamellipodial protrusions is an area of much interest as it is thought to be a key determinant of how cells make motility choices.

Results: We find that Arp2/3 complex localises to dynamic puncta in filopodia as well as lamellipodia of spreading cells. Arp2/3 complex spots do not appear to depend on local adhesion or on microtubules for their localisation but their inclusion in filopodia or lamellipodia depends on the activity of the small GTPase Rac1. Arp2/3 complex spots in filopodia are capable of incorporating monomeric actin, suggesting the presence of available filament barbed ends for polymerisation. Arp2/3 complex in filopodia co-localises with lamellipodial proteins such as capping protein and cortactin. The dynamics of Arp2/3 complex puncta suggests that they are moving bi-directionally along the length of filopodia and that they may be regions of lamellipodial activity within the filopodia.

Conclusion: We suggest that filopodia of spreading cells have regions of lamellipodial activity and that this activity affects the morphology and movement of filopodia. Our work has implications for how we understand the interplay between lamellipodia and filopodia and for how actin networks are generated spatially in cells.

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