Review
I-BAR domains, IRSp53 and filopodium formation

https://doi.org/10.1016/j.semcdb.2009.11.008Get rights and content

Abstract

Filopodia and lamellipodia are dynamic actin-based structures that determine cell shape and migration. Filopodia are thought to sense the environment and direct processes such as axon guidance and neurite outgrowth. Cdc42 is a small GTP-binding protein and member of the RhoGTPase family. Cdc42 and its effector IRSp53 (insulin receptor phosphotyrosine 53 kDa substrate) have been shown to be strong inducers of filopodium formation. IRSp53 consists of an I-BAR (inverse-Bin-Amphiphysin-Rvs) domain, a Cdc42-binding domain and an SH3 domain. The I-BAR domain of IRSp53 induces membrane tubulation of vesicles and dynamic membrane protrusions lacking actin in cells. The IRSp53 SH3 domain interacts with proteins that regulate actin filament formation e.g. Mena, N-WASP, mDia1 and Eps8. In this review we suggest that the mechanism for Cdc42-driven filopodium formation involves coupling I-BAR domain-induced membrane protrusion with SH3 domain-mediated actin dynamics through IRSp53.

Introduction

Cells form the fundamental building blocks of all living matter [1]. Thus understanding the form and function of cells will help to reveal the complex biology of tissues, and ultimately whole organisms. Cardinal features of cells are their shape or morphology, and their ability to migrate. Disease states such as neurodegeneration and cancer can be linked to defects in cell morphology and migration. Two cellular compartments, the membrane and the cytoskeleton, play pivotal roles in regulating cell shape and migration.

The actin-based structures at the leading edge – lamellipodia and filopodia – determine cell shape and ability to migrate. Motile cells put forward thin, sheet-like protrusive structures at their leading edge as they crawl across the substratum. The region closest to the leading edge is referred to as the lamellipodium. It is made up of highly branched dendritic microfilaments assembled by the Arp2/3 complex [2].

Filopodia are membrane-based actin-rich finger-like protrusions that are highly dynamic. Filopodia extend and retract rapidly from the cell surface as the cell explores its environment, seeking biological cues. Their movements are not limited to extension and retraction in the horizontal plane; filopodia are also able to swing laterally, as well as lift up away from and down towards the substratum in the vertical plane. Ultimately they form adhesions with the matrix, facilitating lamellae to fill gaps between them and move the cell forward. Filopodia are thought to play important roles in a number of cellular and developmental processes, including (i) neuritogenesis [3], [4], (ii) axon guidance in neuronal growth cones [5], [6], [7], (iii) receptor–ligand endocytosis [8], [9], (iv) dengue virus uptake [10], (v) detection of pathogen targets for phagocytosis [11] and (vi) dorsal closure in Drosophila embryos [12].

Here, we show how studies of the I-BAR and SH3 domain-containing protein IRSp53 have begun to reveal a mechanism of filopodium formation. Essential to this model for filopodium formation is that the Cdc42–IRSp53 effector complex allows the coupling of membrane protrusion (driven by the I-BAR domain) to actin dynamics (mediated by the SH3 domain).

Section snippets

Filopodia: diversity, form and composition

In mammalian cells, each individual filopodium is made up of a cylindrical plasma membrane extension enclosing a tight bundle of 15–20 linear actin filaments all oriented in parallel, with their barbed ends distal from the cell body [13]. In addition to actin filaments, a number of proteins are associated with filopodia. The formin Dia2 (Diaphanous 2) nucleates actin filaments and has been found in both mammalian and Dictyostelium filopodia, including the tips of these structures [14], [15].

Definition of mammalian filopodia

At the outset of this review it is important to define the structure and dynamics of filopodia so that results from different laboratories can be compared and their discrete features investigated. Mammalian filopodia can be followed in cell culture using time-lapse microscopy. Widefield dual channel fluorescence microscopy using sensitive CCD (charged-coupled device) cameras are an ideal set-up to follow filopodia. Individual frames can be acquired in the range of 100 ms each and at a rate of

Small GTPases as regulators of filopodium formation

Several members of the Ras superfamily of small GTPases have been linked to filopodium formation, with strongest evidence having emerged for Cdc42 [17], Rif (Rho in filopodia) [18] and Rab35 (Ras-like protein in brain 35) [19]. Apart from these, other small GTPases implicated in filopodium formation include RalA (Ras-like A) [20], TC10, Wrch-1 (Wnt-1 responsive Cdc42 homologue-1) and Wrch-2 (Wnt-1 responsive Cdc42 homologue-2) [17].

Domain organisation and binding partners

Also known as BAIAP2 (brain-specific angiogenesis inhibitor-1 associated protein 2), IRSp53 is composed of three main domains – an N-terminal I-BAR domain, followed by a partial CRIB domain and an SH3 domain. At the extreme C-terminal there exists a PDZ (post-synaptic density 95, disc large, zonula occludens-1) and/or a WH2 (WASP homology 2) domain. The isoforms and tissue distribution of IRSp53 have been reviewed by Scita et al. [26].

IRSp53 was found to bind Cdc42 [23], Rac1 [27] and the

I-BAR domains and membrane protrusion

The BAR (Bin-Amphiphysin-Rvs) domain is a highly conserved protein domain involved in remodelling of cellular membranes. BAR domains form dimers with natural curvature and can induce curvature of membranes that they bind to. There are three distinct families of BAR domain-containing proteins: classical BAR, F-BAR (Fer/CIP4-homology-BAR) and I-BAR. Each BAR domain dimer can induce distinct degrees of membrane curvature depending on its shape. For example, the F-BAR domains induce curvature

Actin polymerisation in filopodium formation

Lamellipodia have been postulated to be prerequisite for filopodium formation, as filopodia were observed to arise from them [42]. This led to the ‘convergent elongation model’ of filopodium formation, whereby filopodial tip complex proteins such as Ena/VASP and Dia2 bring together and protect the barbed ends of selected Arp2/3-nucleated lamellipodial microfilaments from capping proteins. These microfilaments are thus able to continue elongating to form long, unbranched filaments that are

A mechanism for filopodium formation: coupling membrane protrusion and actin polymerisation

We now integrate the components that have been discussed during the course of this review – Cdc42, IRSp53, membrane protrusion (induced by I-BAR domain) and actin polymerisation (driven by SH3 domain binding partners) – into a model for filopodium formation. The process starts with the activation of Cdc42 via membrane-bound receptors and exchange factors. Active Cdc42 forms ‘hotspots’ on the plasma membrane for recruitment of proteins that are necessary to build filopodia. Cdc42 recruits

Filopodial dynamics

Actin assembly at tips and retrograde flow can be used to describe filopodial dynamics. Mallavarapu and Mitchison used caged rhodamine-labelled actin and GFP-actin to examine filopodial growth and retraction in the growth cones of NG108 cells. Using these probes they measured rates of actin assembly and retrograde flow throughout the lifetimes of filopodia. In the majority of filopodia examined assembly rates at the tip were dominant in determining filopodial extension or retraction, with

Conclusions

The discovery of the I-BAR domain in IRSp53 and its ability to induce membrane protrusion has begun a new chapter in understanding filopodium formation. The Cdc42–IRSp53 effector complex initiates filopodium formation. It is likely that I-BAR domain oligomerisation is important for membrane protrusion and the recruitment of SH3 domain partners for actin filament formation. Once formed, filopodia can either become stabilised or disassemble. Filopodium stabilisation is connected with the

Acknowledgements

We would like to thank members of the SA lab for their contribution over the years in the IRSp53 project. We would also like to thank A*STAR for supporting this research.

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