The absence of a functional Arp2/3 complex in ARPC3^–/– fibroblasts results in an altered leading edge morphology with filopodia-like protrusions. Actin filaments were labelled using phalloidin (red), and nuclei labelled with DAPI (blue); green staining indicates fascin. Image courtesy of Dr Praveen Suraneni, Stowers Institute for Medical Research, Missouri, USA. 

Facilitated by nucleation-promoting factors, the Arp2/3 complex engineers dendritic actin networks that resemble lamellopodia actin organization; debranching and bundling can also mediate filopodia formation. Elongation of both structures is widely thought to promote cell protrusion, generating the force that initiates cell motility. But although several studies using RNA interference or dominant-negative constructs to disrupt Arp2/3 function support this model, another study reported no effect on lamellipodia extension or cell morphology and, perhaps surprisingly, a detailed analysis of the specific effect of inhibiting the Arp2/3 complex on cell motility in fibroblasts is lacking. By taking an alternative experimental approach to disrupt Arp2/3, however, Suraneni et al. have identified a role for this complex in lamellipodia extension, and demonstrate how these veil-like projections confer directional persistence on migrating fibroblasts.

The p21 subunit ARPC3 is one of seven subunits of the Arp2/3 complex. Although the authors were able to generate ARPC3^+/– adult heterozygous mice, ARPC3^–/– caused embryonic lethality, so Suraneni et al. derived embryonic stem (ES) cells from ARPC3^–/– blastocysts prior to lethality and subsequently differentiated them into ARPC3^–/– fibroblasts, on which they based their studies. Compared to isogenic wild-type ARPC3^+/+ fibroblasts, which displayed smooth, lamellipodia-like edges during spreading, fibroblasts lacking ARPC3 spread through spiky, filopodia-like protrusions that contained actin bundles and fascin; indeed, instead of Arp2/3, the tips of these bundles contained Diaphanous-related formins, which have been implicated in filopodia formation.

During wound healing, fibroblasts migrate towards the epithelial wound, but disruption of Arp2/3 in ARPC3^–/– fibroblasts considerably delayed closure of a wound made to a monolayer. Notably, ARPC3^–/– fibroblasts moved towards the wound with leading edges rich in filopodia-like protrusions that showed faster protrusion and retraction rates than those of lamellipodia in wild-type cells. A comparison of individual whole-cell motility behaviours showed that, although ARPC3^+/+ and ARPC3^–/– fibroblasts were similarly motile, the ARPC3 deficiency caused a lack of sustained migration directionality, which the authors propose might result from poor coordination of protrusion and retraction dynamics at the leading edge.

The ARPC3^–/– fibroblasts behaved similarly in response to the chemoattractant epidermal growth factor (EGF), with only 48% moving towards a source of EGF over a 12-hour period, compared with 85% of wild-type cells. Again, analysis of individual-cell behaviour showed that ARPC3^–/– fibroblasts lacked directional persistence.

So, although cells lacking ARPC3 could migrate, they did so in the absence of lamellipodia and persistent directional movement, indicating a requirement for the Arp2/3 complex in the generation of lamellipodia. Potentially, this is because Arp2/3 at the leading edge usually mediates nucleation of a new filament in the vicinity of another growing filament, thereby coordinating protrusion and retraction dynamics to promote lamellipodia extension and thus favouring directional persistence. As well as providing insights into the role of the Arp2/3 complex, this study has outlined the potential for studying cell motility in the diverse range of cell types that can be differentiated from ES cells. 

Author: Katrin Legg - Copyright © 2012 Cell Migration Consortium