Structural basis of filopodia formation induced by the IRSp53/MIM homology domain of human IRSp53

Abstract

The scaffolding protein insulin receptor tyrosine kinase substrate p53 (IRSp53), a ubiquitous regulator of the actin cytoskeleton, mediates filopodia formation under the control of Rho-family GTPases. IRSp53 comprises a central SH3 domain, which binds to proline-rich regions of a wide range of actin regulators, and a conserved N-terminal IRSp53/MIM homology domain (IMD) that harbours F-actin-bundling activity. Here, we present the crystal structure of this novel actin-bundling domain revealing a coiled-coil domain that self-associates into a 180 Å-long zeppelin-shaped dimer. Sedimentation velocity experiments confirm the presence of a single molecular species of twice the molecular weight of the monomer in solution. Mutagenesis of conserved basic residues at the extreme ends of the dimer abrogated actin bundling in vitro and filopodia formation in vivo, demonstrating that IMDmediated actin bundling is required for IRSp53-induced filopodia formation. This study promotes an expanded view of IRSp53 as an actin regulator that integrates scaffolding and effector functions.

Filopodia: Complex models for simple rods

“…Overexpression of full length IRSp53 or of isolated IMDs induces filopodium-like membrane protrusions, the majority of which, however, differs significantly from canonical filopodia (Mattila et al, 2007, Millard et al, 2005, Suetsugu et al, 2006. First, ectopically overexpressed IRSp53/IMD localises to the entire plasma membrane enveloping these filopodialike structures (Figure 3), which is different from the localisation pattern of low expressors where IRSp53 is detected in tip complexes of both filopodia (Figure 3) and lamellipodia (compare Disanza et al, 2006, Millard et al, 2005, Nakagawa et al, 2003, Suetsugu et al, 2006.…”

“…First, ectopically overexpressed IRSp53/IMD localises to the entire plasma membrane enveloping these filopodialike structures (Figure 3), which is different from the localisation pattern of low expressors where IRSp53 is detected in tip complexes of both filopodia (Figure 3) and lamellipodia (compare Disanza et al, 2006, Millard et al, 2005, Nakagawa et al, 2003, Suetsugu et al, 2006. Notably, a convincing localisation of endogenous IRSp53 family porteins in protrusions of motile cells is still lacking, which would be instrumental to resolve the functional relevance of IMD-induced structures.…”

The Insulin Receptor Substrate of 53 kDa (IRSp53) Limits Hippocampal Synaptic Plasticity

“…GTPase binding to IRSp53 enables interactions of its SH3 domain with downstream effectors WAVE2, Mena, Eps8, or N-WASP, all of which are known regulators of actin dynamics (3)(4)(5)(6). In addition, the N-terminal IRSp53/missing in metastasis homology domain of IRSp53 assists in generating cellular protrusions by bundling actin filaments (5,7,8) and promoting membrane curvature (9,10). Expression of IRSp53 is particularly high in the brain, and consequently IRSp53 contributes to the formation of dendritic spines in the cultured hippocampal neuron model (11).…”

Hyaluronan Synthesis Induces Microvillus-like Cell Surface Protrusions

“…In selected environments, most of the components in the actin polymerization apparatus, when overexpressed or experimentally activated, have been reported to increase the growth of filopodia or microvilli (2)(3)(4)(5)(6)(7). This suggests that there is a strong intrinsic potential in cells to display these extensions, ready for implementation by factors even outside the effector and signaling chains directly related to actin.…”

“…Hyaluronan synthases (HAS1, HAS2, HAS3) 3 are a family of vertebrate plasma membrane enzymes synthesizing hyaluronan. They are integral membrane proteins that catalyze the alternate addition of glucuronic acid and N-acetylglucosamine from their UDP derivatives to a growing hyaluronan polymer, continuously translocated through the plasma membrane into extracellular matrix (22) and eventually reaching a molecular mass up to 10 7 Da (23).…”