First published online 6 May 2003
doi: 10.1242/jcs.00462
IRSp53 is colocalised with WAVE2 at the tips of protruding lamellipodia and filopodia independently of Mena
Hiroyuki Nakagawa1,2,
Hiroaki Miki3,
Motohiro Nozumi2,
Tadaomi Takenawa4,
Shigeaki Miyamoto2,
Jürgen Wehland5 and
J. Victor Small1,*
1 Department of Cell Biology, Institute of Molecular Biology, Austrian Academy
of Sciences, Billrothstraße 11, Salzburg A-5020, Austria
2 Department of Biochemical Engineering and Science, Kyushu Institute of
Technology, Iizuka, Fukuoka 820-8502, Japan
3 Division of Cancer Genomics, Institute of Medical Science, University of
Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan
4 Department of Biochemistry, Institute of Medical Science, University of Tokyo,
4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan
5 Gesellshaft für Biotechnologische Forschung (GBF), Mascheroder Weg 1,
38124 Braunschweig, Germany
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Fig. 7. Interaction between IRSp53, Mena and WAVE2. Mena is recruited to the
protruding tips of both lamellipodia and filopodia through the synergetic
function of its EVH1 and EVH2 domains. WAVE2 is recruited to the tips through
the WHD domain. IRSp53 binding to the proline-rich region of WAVE2 enhances
the activity of WAVE2 to stimulate the Arp2/3 complex. Because IRSp53 also
binds to the proline-rich region of Mena, IRSp53 may tie WAVE2 and Mena into a
complex. The shaded boxes show the proline-rich regions. Double-ended solid
arrows between molecules indicate the previously reported interactions. Grey
arrows show the tethering activity to the tips of both lamellipodia and
filopodia. Arp2/3, Arp2/3 complex; B, basic region; CRIB, Cdc42 and Rac
interactive binding motif; EVH, Ena/VASP homology; fil/ms,
filopodium/microspike; lam, lamellipodium; RCB, Rac binding region;
SH, Src homology domain; VCA, Verprolin-Cofilin homology and acidic domain;
WHD, WAVE/Scar homology domain.
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Fig. 1. Localisation of IRSp53 in B16 melanoma cells. (a-d) Cells transfected with
myc-IRSp53 were cultured on laminin and immunolabelled with monoclonal
anti-myc antibody 9E10 (a,c) and rhodamine-phalloidin (b,d). Arrows in (a,b)
indicate the lamellipodium extending from the cell moderately expressing
IRSp53. Arrowheads in (a,b) indicate an overexpressing cell with branched
filopodial extensions. Double-arrowheads point to the non-lamellipodium cell
membrane. Bars, 10 µm.
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Fig. 2. Dynamics of IRSp53 in B16 melanoma cells. (a-d) B16 melanoma cell
cotransfected with EGFP-IRSp53 and dominant active Rac1V12. The dynamics of
bracketed areas in (a,b) are represented as (c,d), respectively. In (c,d)
white lines indicate a region of protrusion (arrows) preceded by ruffling
(asterisks). Phase dense area corresponding to the lamellipodium region
extending parallel to the substrate is indicated by brackets in (c,d). (e,f)
B16 cell expressed EGFP-IRSp53 and stimulated by treatment with aluminium
fluoride 15 minutes before image acquisition. IRSp53 was not retained in
retraction fibres (arrowheads) and retracting ruffle (arrows). Time is given
in minutes and seconds. Bars, 10 µm.
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Fig. 6. Relative dynamics of IRSp53 and WAVE2 in lamellipodia and filopodia. Panels
show video frames of a B16 cell that was cotransfected with EGFP-IRSp53 and
WAVE2-DsRed. Lamellipodium formation was induced by microinjection of
constitutively active Rac1L61 a few minutes before imaging. (a) Fluorescence
channel for EGFP-IRSp53; (b) fluorescence channel for WAVE2-DsRed; (c) phase
contrast. Arrowheads indicate filopodium tip. Fluorescence intensity was
measured between white and black brackets. The profiles of fluorescence
intensity of the filopodium and lamellipodium are represented in the
corresponding image as fil and lam, respectively. Arrow in the profile
indicates the axis of fluorescence intensity. The fluorescence intensity of
EGFP-IRSp53 at the filopodium tip decreases before retraction and for
DsRed-WAVE2 only during retraction. Double arrowhead at time 1:24 indicates
the tip position at 0:49. (d) The specific fluorescence intensity (I)
at the tip was obtained by the subtraction of background fluorescence from the
tip fluorescence. IR corresponds to the ratio between fluorescence intensities
at n seconds (In) and that after i seconds (In+i). Time is in minutes and
seconds. Bar, 10 µm. See supplementary video.
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Fig. 3. Localisation of myc-tagged IRSp53 constructs in B16 cells. (a) Myc-tagged
IRSp53 constructs. RCB, CRIB and SH3 indicate Rac binding region, CRIB motif
and SH3 domain, respectively. Number indicates the position of corresponding
amino acid residue. (b-i) Cells transfected with myc- SH3, myc-SH3 or
myc-RCB, as indicated, and immunolabelled with monoclonal anti-myc antibody
9E10 (b,d,f,h) and rhodamine-phalloidin (c,e,g,i). Arrows and arrowheads
indicate the tips of filopodia and lamellipodia, respectively. Bars, 10
µm.
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Fig. 4. Localisation of IRSp53 in MVD7 at the tips of lamellipodia and
filopodia of Ena/VASP family deficient cells. The cells were cotransfected
with EGFP-IRSp53 and Rac1V12 were cultured on laminin and visualised in
fluorescence (a,c) and phase contrast (b,d). Bars, 5 µm.
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Fig. 5. Localisation of WAVE2 in B16 cells plated on laminin. (a,b) A cell
immunolabelled with an anti-WAVE antibody that recognises all three WAVE
isoforms (a) and with rhodamine-phalloidin (b). (c,d) Sequential video frames
taken in fluorescence (c) and phase contrast (d) of a cell that was
transfected with WAVE2- DsRed. For living cell observation, lamellipodia
protrusion was stimulated by treatment with aluminium fluoride 15 minutes
before image acquisition. White line indicates region of retraction followed
by lamellipodium protrusion; arrows and arrowheads indicate filopodia. Time is
in seconds. Bars, 10 µm.
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© The Company of Biologists Ltd 2003
