Here, we investigate the features of WAVE complex organization that may instruct the generation and dynamics of lamellipodia. The WAVE complex is required for the formation of lamellipodia and efficient chemotaxis in a range of immune cells ( Weiner et al., 2006 Leithner et al., 2016 Graziano et al., 2019 Park et al., 2008 Evans et al., 2013 Kheir et al., 2005). In neutrophils and other motile cells, the dynamics of the WAVE complex closely correspond to the leading edgeвЂ™s morphology and pattern of advance ( Weiner et al., 2007 Steffen et al., 2004 Davidson et al., 2018 Hahne et al., 2001 Huang et al., 2013 Zallen et al., 2002 Leithner et al., 2016 Fritz-Laylin et al., 2017a). As a pentameric heterocomplex, the WAVE complex contains the subunits WAVE/Scar, Abi1/2, HSPC300, Sra1/Cyfip1, and Nap1/Hem1 ( Weiner et al., 2006 Eden et al., 2002 Innocenti et al., 2004). The WAVE complex is required for proper cell migration and regulation of cell shape across eukaryotes including mammals, amoeba, and plants ( Pollard and Borisy, 2003 Weiner et al., 2007 Leithner et al., 2016 Fritz-Laylin et al., 2017a Ibarra et al., 2006 Szymanski, 2005 Rakeman and Anderson, 2006 Kunda et al., 2003). The rules for instructing filopodial versus lamellipodial cell morphologies are not understood. Though these are their typical actin structures, WASP can compensate for the lack of Scar/WAVE in building lamellipodia in some cell types like Dictyostelium discoideum ( Veltman et al., 2012 Zhu et al., 2016), but not in neutrophils or dendritic cells ( Graziano et al., 2019 Leithner et al., 2016). Despite these similarities, the morphological structures they build are distinct: N-WASP typically participates in spiky filopodial protrusions, invadopodia, and endocytic vesicles ( Takenawa and Suetsugu, 2007 Miki et al., 1998 Bu et al., 2009 Naqvi et al., 1998 Yamaguchi et al., 2005 Yu and Machesky, 2012 Yu et al., 2012 Benesch et al., 2005 Taylor et al., 2011 Veltman et al., 2012), whereas WAVE participates in broad, sheet-like lamellipodial protrusions ( Hall, 1998 Weiner et al., 2006 Leithner et al., 2016 Graziano et al., 2019). 1 A Takenawa and Suetsugu, 2007 Pollard and Borisy, 2003 Rohatgi et al., 1999 Oikawa et al., 2004 Weiner et al., 2007 Ho et al., 2004 Koronakis et al., 2011 Suetsugu et al., 2006 Abou-Kheir et al., 2008 Etienne-Manneville and Hall, 2002). Both are stimulated by phosphoinositides, Rho GTPases, and curvature-sensitive Bin-amphiphysin-Rvs (BAR) domain proteins both activate the Arp2/3 complex both are recycled in an actin-dependent fashion and both show evidence of oligomerization at the membrane ( Fig. N-WASP and WAVE are nested in similar signaling topologies. Our work defines organizing principles of the WAVE complex lamellipodial template and suggests how feedback between cell shape and actin regulators instructs cell morphogenesis. Therefore, the WAVE complex has additional roles in cell morphogenesis beyond Arp2/3 complex activation. Although the WAVE complex stimulates actin nucleation via the Arp2/3 complex, sheet-like protrusions are still observed in ARP2-null, but not WAVE complex-null, cells.

The WAVE complex recruits IRSp53 to sites of saddle curvature but does not depend on IRSp53 for its own localization. This pattern of enrichment could explain several emergent cell behaviors, such as expanding and self-straightening lamellipodia and the ability of endothelial cells to recognize and seal transcellular holes. Using super-resolution microscopy, we find that the WAVE complex forms actin-independent 230-nm-wide rings that localize to regions of saddle membrane curvature. Here we investigate how the WAVE complex organizes sheet-like lamellipodia. How local interactions of actin regulators yield large-scale organization of cell shape and movement is not well understood.

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