Membrane binding properties of IRSp53-missing in metastasis domain (IMD) protein

https://doi.org/10.1016/j.bbalip.2013.07.006Get rights and content

Highlights

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    IRSp53–IMD stabilizes the filopodia formation in live cells binding to the lipid membrane.

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    We determined the binding properties of the IMD to different phospholipids by in vitro fluorescence assays.

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    IMD has a stronger binding interaction with the negatively charged phospholipids.

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    The polymerisation state of the actin interferes with IMD–lipid binding.

Abstract

The 53-kDa insulin receptor substrate protein (IRSp53) organizes the actin cytoskeleton in response to stimulation of small GTPases, promoting the formation of cell protrusions such as filopodia and lamellipodia. IMD is the N-terminal 250 amino acid domain (IRSp53/MIM Homology Domain) of IRSp53 (also called I-BAR), which can bind to negatively charged lipid molecules. Overexpression of IMD induces filopodia formation in cells and purified IMD assembles finger-like protrusions in reconstituted lipid membranes. IMD was shown by several groups to bundle actin filaments, but other groups showed that it also binds to membranes. IMD binds to negatively charged lipid molecules with preference to clusters of PI(4,5)P2. Here, we performed a range of different in vitro fluorescence experiments to determine the binding properties of the IMD to phospholipids. We used different constructs of large unilamellar vesicles (LUVETs), containing neutral or negatively charged phospholipids. We found that IMD has a stronger binding interaction with negatively charged PI(4,5)P2 or PS lipids than PS/PC or neutral PC lipids. The equilibrium dissociation constant for the IMD–lipid interaction falls into the 78–170 μM range for all the lipids tested. The solvent accessibility of the fluorescence labels on the IMD during its binding to lipids is also reduced as the lipids become more negatively charged. Actin affects the IMD–lipid interaction, depending on its polymerization state. Monomeric actin partially disrupts the binding, while filamentous actin can further stabilize the IMD–lipid interaction.

Introduction

The 53-kDa insulin receptor substrate protein (IRSp53) is part of a regulatory network that organizes the actin cytoskeleton in response to stimulation by small GTPases, promoting formation of actin-rich cell protrusions such as filopodia and lamellipodia [1], [2], [3]. Overexpression or microinjection of wild-type IRSp53 drives cytoskeletal rearrangement [4], resulting in increased filopod formation. IRSp53 interacts with Rho GTPases through its Rac Binding Domain (RCB), while its SH3 domain binds to WAVE2 [5], [6], Mena/VASP, Eps8 [7], [8], mDia [9], espin [10], DRLPA [11], Shank-1 [11], [12] and synaptopodin [13]. IRSp53 also interacts with the Rac GEF Tiam1 and Eps8, which forms a Rac GEF complex with other members [7], [8].

An important discovery in understanding IRSp53 was the characterization of the IMD (IRSp53/MIM Homology Domain) [14]. The IMD comprises a 250 amino acid domain at the N terminus of both IRSp53 and MIM-B [15]. IMD can induce filopodia formation in cells, both when expressed in isolation, and in the context of full length IRSp53. Purified recombinant IMD bundles actin filaments in vitro, and it was postulated that the actin bundling property was critical to IMD function [14]. IMD shows structural similarity to BAR domains, which bind to membranes of a specific curvature and can be curvature inducing [16], [17], [18], [19]. Positively charged lysines are concentrated at the ends of the IMD [5], [15], [19], [20] and are important for interaction between the IMD and the membrane. These results suggest that the IMD can enhance filopodia formation by simply bundling actin filaments at the cell periphery. However, more recent work has suggested that the IMD can bind to and deform membrane phospholipids and questioned the relevance of the actin interaction [5], [20].

The role of IRSp53 in Rac-dependent activation of Scar/WAVE2 was studied using liposomes made with PI(3,4,5)P3 [5]. The same group found that IMD interacts by nonspecific electrostatic binding with a range of phospholipids including PS, PI(3)P, PI(3,5)P2, PI(4,5)P2 and PI(3,4,5)P3. In another recent paper, a similar method was undertaken to understand MIM and IRSp53–IMD interactions with membranes [20]. Initially two different splice variants of MIM–IMD (long and short) were found, with four additional amino acids at the distal ends of the dimer in the IMD-L. MIM–IMD-L showed interaction with PI(3,4)P2 and PI(4,5)P2 which is different from what Suetsugu and colleagues observed for the IRSp53–IMD [5]. They also found that IMD has specificity for interaction with PI(4,5)P2 that was clustered in model membranes.

The primary aim of this work was to quantify the binding properties of the IMD to model membranes. We have prepared large unilamellar vesicles (with 200 nm diameter) containing different combination of PS, PC and PI(4,5)P2 lipids investigating the charge-dependency of the binding. The interaction between IMD and the lipid vesicles were followed by in vitro fluorescence-based assays like fluorescence resonance energy transfer, fluorescence quenching or TNS fluorescence assays. In our current work we present these assays as a suitable tool to investigate the binding interactions between IMD and lipids. Since actin filaments are important in filopodia formation, we also explored how the polymerization state of actin affects the lipid binding of the IMD.

Section snippets

Chemicals

KCl, MgCl2, CaCl2, NaOH, Mops, Tris, N-(((iodoacetyl)amino)ethyl)-5-naphthylamine-1-sulfonate (IAEDANS), acrylamide and phalloidin were obtained from Sigma Chem. Co. (St. Louis, MO, USA). ATP and 2-mercaptoethanol were obtained from MERCK (Darmstadt, Germany) and NaN3 was purchased from Fluka (Switzerland). 2-p-toluidinyl-naphtylene-6-sulphonate (TNS) and DiO was purchased from Invitrogen (Carlsbad, CA, USA).

Protein and lipid preparation

Plasmids pGEX4T2 containing the coding sequence of the IMD domain of IRSp53 were

Lipid binding by IMD

Previously we have expressed and purified the IMD and labeled it with IAEDANS fluorescent probe on its surface cysteines [19]. In the present work LUVETs were prepared from different lipid constructs and labeled with DiO fluorescence dye that penetrates into the lipid bilayer. IAEDANS-labeled IMD and DiO-labeled LUVETs were used in in vitro FRET assays to study their binding interactions. IAEDANS served as donor and DiO as acceptor. Samples of IMD and lipids in various composition and

Conclusions

Earlier studies provided evidence that the IMD interacts with lipid membranes. IMD was shown to bind to negatively charged phospholipids, preferably binding to the PIP2 containing vesicles and driving finger-like invaginations in vitro [20]. However, different studies interpret the function of the IMD differently and importantly, precise measurements of dissociation binding constants between the IMD and membranes have not yet been made. We have used a FRET method to follow the binding

Acknowledgments

This work was supported by grants from the Hungarian Scientific Research Fund (OTKA grants K 77840 M.Ny. and NN107776), and by the Operational Programmes of New Hungary Development Plan (TÁMOP-4.2.1. B-10/2/KONV-2010-0002 and TÁMOP 4.2.4.A/2-11-1-2012-0001).

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