Eps8, Arp2/3 and IRSp53 in TNT formation

Tunneling nanotubes (TNTs) connect distant cells and mediate cargo transfer for intercellular communication in physiological and pathological contexts. How the cell controls a common pool of proteins to generate these actin-mediated protrusions spanning length scales beyond those attainable by canonical filopodia remains unknown. Through a combination of surface micropatterning, microscopy and optical tweezer-based approaches, we found that Arp2/3-dependent pathways attenuate the extent with which actin polymerizes in nanotubes, limiting the formation and attainable lengths of TNTs. Upon Arp2/3 inhibition Epidermal growth factor receptor kinase substrate 8 (Eps8) exhibited heightened interactions with the inverted Bin/Amphiphysin/Rvs (I-BAR) domain protein IRSp53 resulting in increased TNTs. In these conditions, Eps8 interaction with proteins enhancing filament turnover and depolymerization were reduced. Our data reveals how common players in protrusions (Eps8 and IRSp53) drive outward linear actin extension to form TNTs, and that their interaction is enhanced when competing pathways overutilizing actin in branched Arp2/3 networks are inhibited, suggesting a shift in the equilibrium (and proteins usage) between branched and linear actin polymerization to form different cell protrusions.