Filopodial protrusion driven by density-dependent Ena-TOCA-1 interactions

Abstract

Filopodia are narrow actin-rich protrusions with important roles in neuronal development. The neuronally-enriched TOCA-1/CIP4 family of F-BAR and SH3 domain adaptor proteins have emerged as upstream regulators that link membrane interactions to actin binding proteins in lamellipodia and filopodia, including WAVE and N-WASP nucleation promoting factors and formins. Here, we demonstrate a direct interaction between TOCA-1 and Ena/VASP actin filament elongators that is mediated by clustered SH3 domain interactions. Using Xenopus retinal gangion cell axonal growth cones, where Ena/VASP proteins have a native role in filopodia extension, we show that TOCA-1 localises to filopodia and lamellipodia with a retrograde flow of puncta and correlates with filopodial protrusion. Two-colour single molecule localization microscopy of TOCA-1 and Ena supports their nanoscale association. TOCA-1 clusters coalesce at advancing lamellipodia and filopodia and operate synergistically with Ena to promote filopodial protrusion dependent on a functional SH3 domain. In analogous yet distinct ways to lamellipodin and IRSp53, we propose that transient TOCA-1 clusters recruit and promote Ena activity to orchestrate filopodial protrusion.

Filopodia In Vitro and In Vivo

“…Surprisingly, BAR proteins associated with positive curvature, including ARHGAP44, FCHSD2, and TOCA-1, can also generate filopodia and recruit actin filament-elongating proteins (Blake et al 2023, Bu et al 2009, Galic et al 2014, Zhai et al 2022. BAR proteins binding to a positively curved membrane could stabilize stochastic membrane fluctuations, working with I-BAR proteins, reducing the energetic barrier to the highly curved membrane, and ensuring robust membrane responsiveness to actin bundles (Mancinelli et al 2021).…”

“…Ena and VASP coalesce into larger clusters before initiation (Cheng & Mullins 2020, Disanza et al 2013, with abundance peaking at the moment of filopodial initiation (Urbančič et al 2017). When they extend filopodia, they work with multiple membrane adaptors, including lamellipodin, IRSp53, and TOCA-1 (Blake et al 2023, Cheng & Mullins 2020, Disanza et al 2013. Ena and VASP are required for cortical neurite initiation and are important in axonal growth cone filopodia (Dent et al 2007) and for 2D migration and microspike formation within melanoma cells (Damiano-Guercio et al 2020).…”

“…Other BAR proteins associated with positively curved membrane are implicated in filopodial formation, due either to positively curved membrane present during filopodial formation or protrusion or alternative binding modes (Frost et al 2008, Galic et al 2014, Mancinelli et al 2021, Shimada et al 2007). F-BAR protein TOCA-1 localizes to neuronal filopodial tips, where it coincides with Ena during filopodial protrusion (Blake et al 2023), and the related proteins FBP17 and CIP4 are associated with either endocytic tubules or membrane protrusions in neurons, depending on the isoform expressed (Taylor et al 2019). PX-BAR protein SNX9 is present in a subset of filopodia, where it may also be involved in the budding of extracellular vesicles (EVs), similar to what has been reported for the missing in metastasis (MIM) protein ( Jarsch et al 2020, T. Nishimura et al 2021.…”