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 renders the characterization of fibroblasts cells lacking functional the Arp2/3 complex. Characterization of the impact of the absence of functional Arp2/3 complex on these genetically matched cells included single cell spreading assays, wound healing assays, long-time single cell motility tracking, chemotaxis assays, fluorescence staining imaging with confocal or structured illumination microscopy [1.2]. ARPC3-/- fibroblasts maintained an ability to move but exhibited a strong defect in persistent directional migration in both wound healing and chemotaxis assays, while migrating at rates similar to wild-type cells.
Here, we will highlight our advances on determining the molecular-level organization of the leading edge actin networks, through an integrated approach that employs electron cryo-tomography of whole mammalian cells in conjunction with correlative light microscopy. We show by correlative fluorescence and cryo-tomography that the nanometer-scale actin-network organization of smooth lamellipodia in wild-type cells is replaced by massive, bifurcating actin-based protrusions with fractal geometry linked to self-organized criticality. Agent-based modeling shows that the Arp2/3 complex suppresses the formation of these protrusions by locally fine-tuning actin network morphology, providing the switch for directional movement.
This work is supported by NIGMS grant P01 GM098412.