Initiation and disassembly of filopodia tip complexes containing VASP and lamellipodin

Abstract

The shape of many eukaryotic cells depends on the actin cytoskeleton, and changes in actin assembly dynamics underlie many changes in cell shape. Ena/VASP-family actin polymerases, for example, modulate cell shape by locally accelerating actin filament assembly and slowing filament capping. When concentrated into discrete foci at the leading edge, VASP promotes filopodia assembly, and forms part of a poorly understood molecular complex that remains associated with growing filopodia tips. Here we identify precursors of this filopodia tip complex in migrating B16F1 cells: small leading-edge clusters of the adaptor protein lamellipodin (Lpd) which subsequently recruit VASP and initiate filopodia formation. Dimerization, membrane association, and VASP binding are all required for lamellipodin to incorporate into filopodia tip complexes, and over-expression of monomeric, membrane-targeted lamellipodin mutants disrupts tip complex assembly. Once formed, tip complexes containing VASP and lamellipodin grow by fusing with each other, but their growth is limited by a size-dependent, dynamic instability. Our results demonstrate that assembly and disassembly dynamics of filopodia tip complexes are determined, in part, by a network of multivalent interactions between Ena/VASP proteins, EVH1 ligands, and actin filaments. [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text]

Stochastic combinations of actin regulatory proteins are sufficient to drive filopodia formation

“…While I-BAR protein IRSp53 localizes to the supported lipid bilayer, it does not enrich at FLS tip complexes ( Fig. S1, H–J ), which agrees with the shaft rather than tip localization observed for endogenous IRSp53 in filopodia in B16F1 cells ( Cheng and Mullins, 2020 ). We optimized the signal-to-noise-ratio while keeping within reasonable range of the endogenous protein levels when adding labeled protein and monitored the appearance and accumulation of actin regulators at the FLS tip complex using highly inclined and laminated optical sheet (HILO) illumination at the membrane, with spinning disk confocal microscopy of the same samples in the actin channel in the volume above the coverslip ( Fig.…”

Multiple roles for actin in secretory and endocytic pathways

“…In addition, endophilin can interact with N-WASP 58 . Lamellipodin interacts with Ena/VASP proteins, which control actin filament elongation in other structures 59,60 . Indeed, the Ena/VASP protein Mena appears to act in FEME 61 .…”

A Diaphanous and Enabled dependent asymmetric actin cable array repositions nuclei during Drosophila oogenesis

“…Ena localization during stage 10B (Spracklen et al, 2014b), and is required for nurse cell dumping (Tootle and Spradling, 2008). Another candidate, Lamellipodin (Lpn, Drosophila Pico), is important for the localization of Ena to lamellipodia in mammalian cells (Carmona et al, 2016;Cheng and Mullins, 2020;Hansen and Mullins, 2015;Michael et al, 2010), and also contributes to nurse cell dumping (Spracklen et al, 2019). Loss of Abelson (Abl), a tyrosine kinase targeting Ena, results in a dumping defect, premature cable assembly, and some abnormal Ena localization (Gates et al, 2009).…”