Actin controls the I-BAR-MEMBRANE interaction

(1)

I-BAR

Extracellular

Extracellular Cytosol

Cytosol

BAR

Fig.6.: Fluorescence quenching experiments on I-BAR with acrilamide using different construction of lipisomes (no lipid (black square), 100 % PC (empty triangle), 100 % PS (black circle), the I-BAR concentration was 1

M).

The Inverse BAR (I-BAR) domain (Fig.1.) is the N-terminal 250 amino acid of the IRSp53 protein that induce negative membrane curvature both in vitro and in cells (Fig.2.). Generation of membrane curvature by I-BAR proteins (Fig.3.) often works together with actin dynamics. I-BAR shares its function between actin bundling and membrane binding but it is still obscured what molecular mechanisms are responsible for these functions. The aim of our project is to investigate the detailed membrane binding properties of the I-BAR of IRSp53 and its relations to the actin cytoskeleton. In vitro FRET experiments and fluorescence quenching studies were carried out between the I-BAR and liposomes made up from different lipid constructs. We have found that the I-BAR has preference to bind to the negatively charged lipids however it can also bind to the uncharged lipids. The fluorescence quenching studies reflected that the accessibility of the I-BAR surface was higher toward the negatively charged lipids than for the uncharged ones. TNS fluorescence assay reflected that the I-BAR domain binds to the surface of the micells rather than penetrating into its core. The I- BAR membrane interaction is controlled by the polimerizasion state of actin where filamentous actin stabilizes while the globular actin disrupts their interaction.

Kinga Futó ¹ , Laura M. Machesky ² , Balázs Visegrády ¹

1

Department of Biophysics, Faculty of Medicine, University of Pécs, Szigeti str. 12, Pécs H-7624, Hungary and

2

Beatson Institute for Cancer Research, Garscube Estate, Switchback Road, Bearsden, Glasgow G61 1BD, U.K.

e-mail: balazs.visegrady@aok.pte.hu

To dissect the mechanism by which I-BAR domain of IRSp53 contribute to the filopodia formation we investigate the interaction of I-BAR with the membrane and the actin-cytoskeleton using biochemical and biophysical approaches. We address the following questions:

,

Fig.3: The model of filopodia formation by BAR domains

Fig.1: The structure of IRSp53/MIM homology domain (I-BAR) dimer

Fig.4.: FRET efficiency was measured between IAEDANS-I-BAR (1M) and DiO-labelled liposomes made from different lipid components as the following: PC (balck triangle), PS/PC (70%/30%) (black square), PS (black circle).

Fig.5.: FRET efficiency was measured between IAEDANS-I-BAR (1M) and DiO-labelled liposomes made from different lipid components as the following: PC (balck triangle), PIP₂/PC (4%/96%) (empty square) and PIP₂/PC (15%/85%) (empty circle).

1. I-BAR HAS PREFERENCE TO THE NEGATIVELY CHARGED LIPIDS

LUVET composition IMD:lipid ratio K_d ( µM) Error

PS 1:50 1,58 0,26

PC 1:67 2,52 2,11

PS/PC (70/30%) 1:90 1,63 0,56

PIP2/PC ( 15/85%) 1:49 1,78 0,41

PIP2/PC ( 4/96%) 1:88 1,13 0,82

PS: phosphatidylserine PC: phosphatidilcholine

PIP₂: phosphatidylinositol-4,5-bisphosphate

Figure 8.: FRET efficiency was measured between IAEDANS-I-BAR (1M) and DiO- labelled PS-PC (70%/30%) liposomes in the presence of monomer (G) and filamentous (F) actin.We stabilize the different formes of actin with latrunculin-a or phalloidin treatment.

Figure 9.: FRET efficiency was measured between IAEDANS-I-BAR (1 μM) and IAF- F-actin with increasing concentration in case of different KCl concentration.

Figure 10.: Steady-state anisotropy of IAEDANS labeled G-actin (1 μM) as the function of I-BAR concentration.

Figure 11.: I-BAR at different concentrations was added to G- actin (2.5 μM, containing 2 % of pyrenyl labeled actin) simultaneously with the initiation of actin polymerisation (i.e.

adding 100 mM KCl and 2 mM MgCl₂). To study the kinetics of actin assembly the fluorescence intensity of the samples was measured as a function of time.

I-BAR domain influence the assembly of actin monomers to filaments in a concentration dependent manner.

2. I-BAR BINDS ACTIN MONOMERS AND FILAMENTS

Fig.7.: The effect of I-BAR on the structure of PIP2 micelles measured by TNS fluorescence.

Conditions: (a) 100 uM TNS alone; (b) TNS with 10 M PIP₂; (c) TNS with 1 M IMD and 10 M PIP₂; (d) 10 mM MgCl₂ with TNS.

3. I-BAR INFLUENCE THE ASSEMBLY OF ACTIN

MONOMERS TO FILAMENTS IN A CONCENTRATION DEPENDENT MANNER

Table 1.: The binding stochiometry and the dissoiation constants of I-BAR-lipid interaction were calculated.

0 5 10 15 20

0,0 0,1 0,2 0,3 0,4 0,5 0,6 0,7

K

d

= 9,9 ± 1,2



M K

d

= 17,3 ± 4,1



M

FRET ef fi ciency

Actin concentration (  M)

25 mM KCl 100 mM KCl

0 4 8 12 16

0,12 0,14 0,16 0,18

K

d

= 12,5 ± 3,4 (



M)

Steady-state an isotropy

I-BAR concentration (  M)

2,5 μM actin +[I-BAR], μM

,

The IRSp53-I-BAR was cloned into a pGEX-4T2 expression vector and was expressed in BL21 cell line.

Steady state Förster Type Resonancy Energy Transfer (FRET) can be measured between fluorescence I-BAR (labeled with IAEDANS on cystein as donor) and DiO-labeled liposomes (as acceptor), and the transfer efficiency can be calculated, similarly as we used before (5).

Fluorescence quenching experiments were carried out with acrylamide quencher on the IAEDANS fluorophore. The IAEDANS bound to the I-BAR and the experiments were made in the presence or absence of the liposomes.

TNS assay were used to detect changes in protein conformation and can also detect changes in the structure of lipid assemblies (6).

(1) Yamagishi, A., Masuda, M., Ohki, T., Onishi, H., and Mochizuki, N. (2004) A novel actin bundling/filopodium-forming domain conserved in insulin receptor tyrosine kinase substrate p53 and missing in metastasis protein. J Biol Chem 279, 14929-36.

(2) Lee, S. H., Kerff, F., Chereau, D., Ferron, F., Klug, A., and Dominguez, R. (2007) Structural basis for the actin-binding function of missing-in-metastasis. Structure 15, 145-55.

(3) Frost, A., De Camilli, P., and Unger, V. M. (2007) F-BAR proteins join the BAR family fold. Structure 15, 751-3.

(4) Millard, T. H., Bompard, G., Heung, M. Y., Dafforn, T. R., Scott, D. J., Machesky, L. M., and Futterer, K. (2005) Structural basis of filopodia formation induced by the IRSp53/MIM homology domain of human IRSp53. Embo J 24, 240-50.

(5) Visegrady, B., Lorinczy, D., Hild, G., Somogyi, B., and Nyitrai, M. (2004) The effect of phalloidin and jasplakinolide on the flexibility and thermal stability of actin filaments. /FEBS Lett/ /565/, 163-6.

(6) Langner, M., D. Cafiso, S. Marcelja, and S. McLaughlin. 1990. Electrostatics of phosphoinositide bilayer membranes. Theoretical and experimental results. Biophys J 57:335-349.

0 1000 2000 3000 4000 5000 0,0

0,2 0,4 0,6 0,8 1,0 1,2

Py reny l fluores ce nc e inten sity, a.u.

Time (s)

The I-BAR binds actin monomers at low salt concentration (K

_d

= 12,5 ± 3,4 µM).

The I-BAR-F-actin interaction does not depend on the KCl concentration.

The I-BAR membrane interaction is controlled by the polimerizasion state of actin where filamentous actin stabilizes while the globular actin disrupts their

interaction.

TNS assay reflects that I-BAR do not have any effect on the fluorescence mixtures of PIP

₂

micelles and 2-p- toluidinyl-napthylene-6-sulfonate (TNS).

IRSp53-I-BAR:

• The FRET experiments show that the I-BAR is able to bind to all the different lipid constructs.

• The protein binds stonger to liposomes containing more negative charges.

•The I-BAR binds to PIP

₂

head groups on the surface of the micelles, rather than penetrating the core of the micelles.

•The I-BAR is simultaneously connected with the membrane and the actin where filamentous actin stabilizes while the globular actin disrupts their interaction.

• I-BAR domain promotes the assembly of actin monomers to filaments in high concentration and inhibits it in low concentration.

Our in vitro result suggest that actin controls the

IRSp53-I-BAR-membrane interaction. These proteins may play a role in the regulation of assembly and disassembly of filopodia.

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b c

Fig.2: Cos7 cells were transfected with mCherry-I- BAR and labeled with Alexa 488-Phalloidin