Cells employ protrusive leading edges to navigate and promote their migration in diverse physiological environments. Classical models of leading edge protrusion rely on a treadmilling dendritic actin network that undergoes continuous assembly nucleated by the Arp2/3 complex, forming ruffling lamellipodia. Although the dendritic nucleation model has been rigorously evaluated in several computational studies. Experimental evidence demonstrating a critical role for Arp2/3 in the generation of protrusive actin structures and cell motility has been far from clear. Most components of the pathway have been probed for their relevance by RNA interference or dominant-negative constructs. However, given that the Arp2/3 complex nucleates actin at nanomolar concentrations, even a dramatic knockdown could still leave behind a level sufficient to fully or partially support Arp2/3-complex dependent functions.
Our recent work characterizes newly developed fibroblasts cell lines completely lacking functional Arp2/3 complex. We tested the impact Arp2/3-complex function on these genetically matched cells with and without Arp2/3 complex using single cell spreading assays, wound healing assays, long-time single cell motility tracking, chemotaxis assays, and fluorescence imaging with confocal or structured illumination microscopy [1,2]. In the absence of functional Arp2/3 complex, the fibroblasts were unable to extend lamellipodia but generated dynamic leading edges composed primarily of filopodia-like protrusions (FLPs), with formin proteins (mDia1 and mDia2) concentrated near their tips. These mutant fibroblasts maintained an ability to move but exhibited a strong defect in persistent directional migration in both wound healing and chemotaxis assays [1,2]. Here, we will highlight our advances in determining the molecular-level organization of the actin networks of these cells through an integrated approach that employs electron cryo-tomography of whole mammalian cells in conjunction with correlative light microscopy.
References:
[1] Suraneni P, Rubinstein B, Unruh JR, Durnin M, Hanein, D, and Li R (2012). The Arp2/3 complex is required for lamellipodia extension and directional fibroblast cell migration. J Cell Biol. 197, 239.
[2] Suraneni P, Fogelson B, Rubinstein B, Noguera P, Volkmann N, Hanein D, Mogilner A, Li R (2015). A mechanism of leading edge protrusion in the absence of Arp2/3 complex. Mol Biol Cell. 26:901-912.
[3] This work is supported by NIGMS grants P01 GM066311 and P01 GM098412.
