Biochem. J. (2011) 436, 45–52 (Printed in Great Britain)
45
doi:10.1042/BJ20101684
Functional analysis of Dictyostelium IBARa reveals a conserved role of the
I-BAR domain in endocytosis
Douwe M. VELTMAN*, Giulio AUCIELLO†, Heather J. SPENCE*, Laura M. MACHESKY*, Joshua Z. RAPPOPORT† and
Robert H. INSALL*1
*Beatson Institute for Cancer Research, Switchback Road, Bearsden, Glasgow G61 1BD, U.K., and †School of Biosciences, University of Birmingham, Edgbaston,
Birmingham B15 2TT, U.K.
I-BAR (inverse-Bin/amphiphysin/Rvs)-domain-containing proteins such as IRSp53 (insulin receptor substrate of 53 kDa)
associate with outwardly curved membranes and connect them
to proteins involved in actin dynamics. Research on I-BAR
proteins has focussed on possible roles in filopod and lamellipod
formation, but their full physiological function remains unclear.
The social amoeba Dictyostelium encodes a single I-BAR/SH3
(where SH3 is Src homology 3) protein, called IBARa, along
with homologues of proteins that interact with IRSp53 family
proteins in mammalian cells, providing an excellent model to
study its cellular function. Disruption of the gene encoding
IBARa leads to a mild defect in development, but filopod and
pseudopod dynamics are unaffected. Furthermore, ectopically
expressed IBARa does not induce filopod formation and does
not localize to filopods. Instead, IBARa associates with clathrin
puncta immediately before they are endocytosed. This role
is conserved: human BAIAP2L2 (brain-specific angiogenesis
inhibitor 1-associated protein 2-like 2) also tightly co-localizes
with clathrin plaques, although its homologues IRSp53 and
IRTKS (insulin receptor tyrosine kinase substrate) associate with
other punctate structures. The results from the present study
suggest that I-BAR-containing proteins help generate the membrane curvature required for endocytosis and implies an
unexpected role for IRSp53 family proteins in vesicle trafficking.
INTRODUCTION
systems, in particular SCAR–WAVE [where WAVE is WASP
(Wiskott–Aldrich syndrome protein) verprolin homologous]
[7–9]. The presence of these proteins, in the absence of redundant
paralogues, makes Dictyostelium ideal to study the functional role
of I-BAR proteins.
The results of the present study reveal an unexpected conserved
role for I-BAR domains in clathrin-mediated endocytosis, and
thus that the specific curvature of the I-BAR domain does not
limit its physiological function to protrusion formation.
The organization of many cellular structures depends on the
coupling between membranes and the cytoskeleton. Adapter
proteins with membrane-binding domains play an important
role in this coupling. The BAR (Bin/amphiphysin/Rvs) domain
folds into an elongated α-helical structure and binds membranes
in vitro and in vivo [1]. BAR domains are unique in their
curvature. Classical BAR and F-BAR (FCH-BAR) domains have a
concave surface and preferentially bind to membranes with inward
(positive) curvature, such as the outside face of vesicles [2,3].
Accordingly, proteins with these domains, such as amphiphysin
and syndapin, are involved in vesicular processes such as clathrinmediated endocytosis and endosomal recycling [3,4].
Conversely, I-BAR (inverse-BAR) domains have a convex
surface and bind to outward (negative) membrane curvature [5,6].
Ectopical expression of I-BAR proteins in cultured cells generates
numerous amounts of filopodia and the purified I-BAR domain
is sufficient to tubulate membranes in vitro. The inside face
of a filopodium contains strong negative curvature and these
observations have led to the view that I-BAR proteins such as MIM
(missing in metastasis) and IRSp53 (insulin receptor substrate of
53 kDa) play a role in filopodia formation.
Dictyostelium encodes a single I-BAR-domain-containing
protein (Dictybase code DDB_G0274805). The Dictyostelium
genome also encodes homologues of various proteins that have
been reported to interact with I-BAR proteins in mammalian
Key words: brain-specific angiogenesis inhibitor 1-associated
protein 2-like 2 (BAIAP2L2), clathrin, endocytosis, inverseBin/amphiphysin/Rvs (I-BAR) domain, insulin receptor substrate
of 53 kDa (IRSp53), insulin receptor tyrosine kinase substrate (IRTKS).
EXPERIMENTAL
Bioinformatics
Novel I-BAR domain proteins were identified by searching GenBank® (http://blast.ncbi.nlm.nih.gov/Blast.cgi) using
BLASTP and TBLASTN with I-BAR domains of known family
members. Alignments of obtained sequences were performed with
ClustalX and phylogenetic trees were drawn with Treeview.
Strains and constructs
An ibrA knockout was generated in AX2 (sDM8) and in
AX3 (sDM7) as follows. The full-length ibrA gene amplified
from cDNA was cloned into pDONR221. A floxed blasticidinresistance cassette was ligated in between the unique MunI
Abbreviations used: BAIAP2L2, brain-specific angiogenesis inhibitor 1-associated protein 2-like 2; BAR, Bin/amphiphysin/Rvs; DIC, differential
interference contrast; GFP, green fluorescent protein; I-BAR, inverse-BAR; IRSp53, insulin receptor substrate of 53 kDa; IRTKS, insulin receptor tyrosine
kinase substrate; MIM, missing in metastasis; mRFP, monomeric red fluorescent protein; NA, numerical aperture; SH3, Src homology 3; TIRF, total internal
reflection; WASP, Wiskott–Aldrich syndrome protein; WAVE, WASP verprolin homologous.
1
To whom correspondence should be addressed (email [email protected]).
c The Authors Journal compilation c 2011 Biochemical Society
46
D. M. Veltman and others
and NdeI sites. A linear fragment was then obtained by PCR,
electroporated into cells and selected with 10 μg/ml blasticidin.
The full-length open reading frames of WASP and clathrin light
chain were obtained by PCR from cDNA and expressed using
Dictyostelium extrachromosomal vectors as described previously
[10].
Live cell assays
For the chemotaxis assays, vegetative cells were washed with
DB (10 mM sodium/potassium phosphate buffer, pH 6.5, 2 mM
MgCl2 and 1 mM CaCl2 ) and starved as a confluent monolayer
in a Petri dish under DB until the onset of streaming. Cells were
harvested and transferred to an Insall chemotaxis chamber [11].
Cells in this chamber reside on a 1 mm wide bridge that separates a
reservoir of DB from a reservoir of DB plus 1 μM cAMP. Movies
of chemotaxing cells were recorded at a position halfway over
the bridge. Quantification of the results was performed in ImageJ
with the MTrackJ plugin.
For quantification of the number of filopodia, vegetative cells
were placed on glass-bottomed dishes at low cell density and
movies of randomly moving cells were recorded using DIC
(differential interference contrast). TIRF (total internal reflection)
microscopy was performed on a Nikon Eclipse TE2000-U that
was fitted with a custom TIRF condenser and a Nikon 1.45 NA
(numerical aperture) 100× Plan Apo TIRF objective. MetaMorph
software was used to control the camera, shutters and light
sources.
HeLa cervical carcinoma cells (HPA) cultured in DMEM (Dulbecco’s modified Eagle’s medium; Invitrogen) containing 10 %
foetal bovine serum and penicillin (100 units/ml)/streptomycin
(100 μg/ml) were plated on to MatTek glass-bottomed 35 mm
dishes (MatTek corp) and transfected with LipofectamineTM
2000 (Invitrogen). Clathrin–dsRed was a gift from Professor
Tomas Kirchhausen (Harvard Medical School, Boston,
U.S.A.). Following fixation in 4 % paraformaldehyde (Electron
Microscopy Services), cells were imaged by TIRF microscopy
with a Nikon A1R through a 60×1.49 NA TIRF objective and an
Andor iXon 885 EMCCD (electron multiplying charge coupled
device). GFP (green fluorescent protein)-tagged proteins were
excited with a 488 nm laser, and clathrin–dsRed with a 568 nm
laser. Co-localization was quantified by the percentage of spots in
one channel co-incident with spots in the other.
RESULTS
Evolution of the I-BAR domain
The human genome encodes five proteins that contain I-BAR
domains. IRSp53, IRTKS (insulin receptor tyrosine kinase
substrate) and BAIAP2L2 [BAIAP2 (brain-specific angiogenesis
inhibitor 1-associated protein 2)-like 2] are collectively called
the IRSp53 family. MIM and ABBA (actin-bundling protein
with BAIAP2 homology) together comprise the MIM family
[12]. Homologues of these proteins are present throughout
the metazoa. The relatively simple eukaryote Dictyostelium
discoideum contains a single gene encoding a protein with an
I-BAR domain (dictyBase Gene ID DDB_G0274805). This gene
was first described in a review on IRSp53 [13]. The automated
gene model that was used in this paper predicted a protein
that contains a large N-terminal leucine-rich repeat domain.
However, we have manually inspected the gene model and
find that the leucine-rich repeat belongs to a separate protein.
The true start codon for DDB_G0274805 is positioned just
c The Authors Journal compilation c 2011 Biochemical Society
before the predicted I-BAR domain. This new gene model
is supported by the automated gene model of an apparently
orthologous protein that is found in Dictyostelium purpureum
(dictyBase Gene ID DPU_G0070382). A protein with an IBAR domain is also present in Polysphondylium pallidum
(http://sacgb.fli-leibniz.de; gene accession number 333206) and
Acytostelium subglobosum (http://acytodb.biol.tsukuba.ac.jp/cgibin/index.cgi?org=as; gene accession number ADB0000371).
BLAST searches with metazoan or protist I-BAR domains against
28 completed genomic sequences from the plant, chromalveolate
and excavate kingdoms did not yield any hits. This indicates that
the I-BAR domain evolved exclusively in the unikont kingdom
(amoebozoa, fungi and metazoa), after the split from plants and
other protozoa [14].
In order to determine the relationship between the I-BAR
proteins from different species, the sequences of their respective
I-BAR domains were aligned (see Supplementary Figure S1 at
http://www.BiochemJ.org/bj/436/bj4360045add.htm).Sequences
of prototypical members of other BAR subfamilies were
also included. A bootstrapped distance tree was calculated
from the results (Figure 1A). The I-BAR domains from protists
and metazoa are clearly separated from the other BAR families and
the indicated bootstrap value of 82 % gives good confidence
that the protist I-BAR domains are correctly classified. The
protist I-BAR domain is rooted close to the branch point of the
IRSp53 family and MIM family I-BAR domains, demonstrating
that it is equally similar to each. We have therefore renamed the
D. discoideum protein IBARa and the gene ibrA. All protist
proteins with an I-BAR domain have a C-terminal SH3 (Src
homology 3) domain, making them, if anything, closer to the
IRSp53-family proteins. A timeline of the most likely sequence
of events that has led to the current distribution of I-BARdomain-containing proteins across unikonts was constructed on
the basis of the phylogenetic analysis (Figure 1B).
Phenotype of IBARa mutants
In order to study the role of IBARa in Dictyostelium, ibrAknockout mutants were generated in two wild-type strains, AX2
and AX3, giving cells with similar phenotypes. The results shown
in the present study are from mutants in an AX3 background.
Given the suggested involvement of I-BAR proteins in
generation of protrusions, we tested motility and chemotaxis of
ibrA-null mutants. Aggregation-competent cells were placed in
a chemotaxis chamber that subjects the cells to a 2 % cAMP
difference between the front and the back of the cell at a
background concentration of 500 nM. This relatively shallow
gradient yields inaccurate chemotaxis which reveals even slight
defects [15]. Somewhat surprisingly, ibrA-null cells migrated
efficiently up the cAMP gradient in all experiments (Figure 2A).
In agreement with their effective chemotaxis, ibrA-null cells
aggregated normally. However, culminant fruiting bodies of ibrAnull cells were noticeably malformed. Stalks were irregular and
weak, and most fruiting bodies collapsed upon the slightest
disturbance, whereas fruiting bodies from wild-type cells were
sturdy and remained upright even when agitated (Figure 2B). Thus
IBARa is important for Dictyostelium biology, but apparently not
for migration or chemotaxis.
Filopodia and pseudopodia in ibrA mutants
Ectopic overexpression of the I-BAR domain in mammalian cells
leads to a dramatic increase of spiky protrusions [12,16]. We
quantified the number of actively protruding filopodia in ibrAknockout Dictyostelium and in wild-type cells overexpressing
Conserved role for I-BAR proteins in endocytosis
Figure 1
47
Evolution of the I-BAR domain
(A) I-BAR-domain-containing proteins were identified from GenBank® . The domain topology of these proteins is shown in the rectangular boxes. The I-BAR domains of these proteins and of
prototypical classical BAR domains were aligned in ClustalX and a distance tree was calculated. Different groups are indicated by different coloured areas. The bootstrap value is given for the branch
that separates I-BAR domains from classical BAR domains (1000 trials). Species abbreviations: Hs, Homo sapiens ; Mm, Mus musculus ; Gg, Gallus gallus ; Dr, Danio rerio ; Sp, Strongylocentrotus
purpuratus ; Dm, Drosophila melanogaster ; Ta, Trichoplax adhaerens ; Dd, D. discoideum ; Dp, D. purpureum ; and Pp, Polysphondylium pallidum . (B) Timeline of the most likely sequence of events
in the evolution of I-BAR proteins, derived from the phylogenetic analysis in (A).
IBARa (Figure 2C). Unexpectedly, the number of filopodia
was, if anything, greater in the knockout and lower in
the overexpressing cell line, although the difference was
not significant in a t test (P > 0.1). Thus IBARa, and
I-BAR domains in general, are not required for filopodia to
form.
Another way I-BAR proteins can regulate protrusions is via
their SH3 domains. It has been proposed that vertebrate IRSp53
c The Authors Journal compilation c 2011 Biochemical Society
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Figure 2
D. M. Veltman and others
Characterization of ibr A-null cells
(A) Aggregation-competent wild-type and ibrA -null cells were subjected to a stable gradient of 1 μM cAMP/mm. A time-lapse movie was recorded and individual cells were tracked using ImageJ.
Tracks of seven cells are shown for each strain. Numbers are the average of 20 cells from two movies. (B) Monolayers of wild-type and ibrA -null cells were developed on non-nutrient agar and
imaged after 36 h. (C) DIC images of vegetative cells of the indicated strains. Arrows indicate the position of actively protruding filopodia. (D) The average number of filopodia for wild-type and
ibr A-null cells was determined from short movies, which allowed discrimination between retraction fibres and true filopodia. Error bars indicate the S.E.M. (E) TIRF and brightfield images of the
GFP-tagged SCAR–WAVE complex member HSPC300 in wild-type and ibr A-null cells. (F) Kymograph of GFP-tagged SCAR–WAVE complex recorded along the white line drawn in (E).
is an adapter that couples Rac to the SCAR–WAVE complex [17].
Both Rac and all members of the SCAR–WAVE complex are
tightly evolutionarily conserved in Dictyostelium [18,19]. In wildtype cells, SCAR accumulates in a thin contour at the leading edge
of pseudopodia (Figure 2E). This association is very dynamic;
pseudopodia extend as long as SCAR is associated with the edge,
and pseudopod growth immediately halts when SCAR dissipates.
Figure 2(F) shows that these dynamics are unchanged in ibrAnull cells. Pseudopod frequency and pseudopod extension speed
also did not show any obvious differences between wild-type
and ibrA-null cells (results not shown). Thus in Dictyostelium,
I-BAR domain proteins are not needed to couple activated Rac
to SCAR–WAVE, activation of the Arp2/3 complex and actin
polymerization or to drive pseudopodia.
c The Authors Journal compilation c 2011 Biochemical Society
Localization of IBARa to endosomal clathrin pits
To assess the physiological role of Dictyostelium IBARa, we
observed the GFP-tagged protein in live cells. Consistent with
the findings above, IBARa was not observed at the leading
edge of the cell or in filopodia. Instead, transient puncta were
visible at the basal membrane. These were strongly reminiscent
of clathrin in endocytic coated pits, but shorter lived. To
test whether the IBARa localization represents a subset of
clathrin-coated endosomal pits, cells were co-transfected with
mRFP (monomeric red fluorescent protein)-tagged clathrin light
chain and observed by TIRF microscopy (Figure 3A). Due
to the short duration of the IBARa spots compared with
clathrin, IBARa appears absent from most clathrin spots in
Conserved role for I-BAR proteins in endocytosis
Figure 4
49
Time frame of recruitment of IBARa, WASP and Arp2/3
(A–D) The proteins indicated on the right-hand side were fluorescently tagged (one with GFP,
the other with mRFP), co-expressed in wild-type cells, and observed using TIRF microscopy.
Kymographs show one clathrin-mediated endocytosis event for each strain. (E) Timing of IBARa,
Arp2/3 and WASP recruitment relative to the disappearance of clathrin, based on the averages
of at least five kymographs for each pair.
Figure 3
Co-localization of IBARa and clathrin
(A) TIRF microscopy image of a wild-type Dictyostelium cell expressing GFP-tagged IBARa and
mRFP-tagged clathrin (light chain). (B) Time series of the same cells as in (A) showing two
disappearing clathrin puncta in high magnification. The IBARa channel is shown in the lower
panel. (C) At each IBARa spot visible in the GFP channel it was recorded if a clathrin spot was
disappearing at the same time in the RFP channel. Vice versa, for each disappearing clathrin
spot it was recorded if IBARa was present. A total of 40 spots were counted from three different
cells.
still pictures, but the correlation between clathrin and IBARa
is very strong, which is particularly clear in Supplementary
Movie S1 (http://www.BiochemJ.org/bj/436/bj4360045add.htm).
Accumulation of IBARa occurs just before the clathrin signal
disappears (Figure 3B). Essentially all clathrin puncta accumulate
a substantial amount of IBARa, and IBARa puncta are only visible
at places where clathrin is present but about to disappear from the
TIRF illumination field (Figure 3C).
Disappearance of clathrin puncta has been linked to endocytosis
of the vesicle. In yeast and mammalian cells, this is assisted
by Arp2/3-mediated actin polymerization [20]. The arrival of
WASP at the coated pit is thus an effective marker for incipient
endocytosis. Additional co-localization experiments (Figures 4A–
4D, and schematically in Figure 4E) show that the timing of
recruitment of IBARa coincides with the recruitment of WASP
and Arp2/3. The timing of the recruitment is consistent, which
strongly indicates that IBARa is involved in F-actin-driven vesicle
invagination. However, disruption of ibrA did not affect the
recruitment of WASP or Arp2/3 to sites of clathrin-mediated
endocytosis. The half-life of fluorescence intensity of clathrin
during endocytosis was also not significantly different in wildtype cells compared with ibrA-null cells (results not shown).
Although this does not rule out that IBARa is involved in vesicle
budding, it indicates that its function is not essential for this.
The recruitment of I-BAR proteins to sites of clathrin-mediated
endocytosis, not actin protrusion, was a surprise, so we examined
its evolutionary conservation. The human genome encodes three
proteins with analogous domain topologies to Dictyostelium
IBARa. These proteins are the most likely candidates to have
conserved the function of the ancestral IBARa. IRSp53 is
relatively well characterized, whereas the function of IRTKS
and BAIAP2L2 is much less clear and it is not known what the
functional differences between these proteins are. We expressed
GFP fusions of each protein in HeLa cells, together with clathrin–
dsRed. In mammalian cells, clathrin is organized in at least two
distinct structures. Apart from the canonical clathrin-coated pits,
larger and less dynamic structures called ‘coated plaques’ are
also observed at the basal membrane in many isolated cell lines
[21]. In mammalian cells, it is the coated plaques, rather than
the coated pits, that associate with F-actin [22]. Figure 5(A)
shows that all three GFP-fusion proteins are present in punctate
structures at the basal membrane. Quantification of these images
reveals that these puncta do not co-localize with the small
clathrin puncta that represent the canonical clathrin-coated pits.
However, BAIAP2L2 co-localizes consistently and accurately
with coated plaques, whereas IRSp53 and IRTKS do not
(Figure 5B). These results could be reproduced in MDA MB231
cells, confirming that the recruitment of BAIAP2L2 to coated
plaques is not cell-type specific (see Supplementary Figure S2A at
http://www.BiochemJ.org/bj/436/bj4360045add.htm). Moreover,
c The Authors Journal compilation c 2011 Biochemical Society
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Figure 5
D. M. Veltman and others
Localization of IRSp53 family proteins in HeLa cells
(A) Co-expression of fluorescently tagged clathrin with IRSp53, IRTKS and BAIAP2L2 in HeLa cells. (B) For each clathrin spot in the respective strains, it was determined if the co-expressed protein
was also present at that position and vice versa. (C) Three-dimensional representation of a clathrin-coated vesicle in the process of endocytosis. Cross-sections in the three planes are shown below
(XZ and YZ planes are identical). Areas of positive curvature are highlighted in green and negative curvature in red.
c The Authors Journal compilation c 2011 Biochemical Society
Conserved role for I-BAR proteins in endocytosis
in BSC1 cells, in which coated plaques are absent, the number
of BAIAP2L2 spots is substantially reduced and the remaining
spots do not co-localize with clathrin (see Supplementary
Figure S2B).
DISCUSSION
Adapter proteins play a critical role in the assembly of many
multi-protein complexes by recruiting and binding individual
components, but their exact physiological role is often difficult
to pinpoint. The discovery of the specific curvature of BAR
domains has been a welcome guide to establish the function of
their respective proteins. The outwardly curved I-BAR domain is
able to tubulate membranes in vitro and ectopic expression of the
I-BAR domain induces numerous spiky protrusions in cultured
cells [6]. These results quickly established a physiological role
for IRSp53 in filopodia formation. However, this view has been
challenged as other researchers have found that the protrusions
induced by IRSp53 overexpression lack many characteristics of
endogenous filopodia [23]. The present study now puts the relation
between the I-BAR domain and filopodia formation into an
evolutionary perspective. We find that the I-BAR domain evolved
specifically in the unikont kingdom, whereas organisms that can
form filopodia are found throughout the eukaryotic tree. We also
find no evidence for a role of the ancestral I-BAR adapter protein
IBARa in Dictyostelium in filopodia formation. These results do
not exclude a role for I-BAR domain proteins such as IRSp53 in
filopodia formation in mammalian cells, but indicate that I-BAR
domains in principle are not essential for filopodia formation and
that they first evolved as part of a different pathway.
The list of processes that involve I-BAR domain proteins
has been steadily expanding and the intuitive structure–function
relationship does not always appear to hold. Formation of
lamellipodia, actin pedestals and tight junctions have now all been
found to involve I-BAR domain proteins [24–26]. A study using
primary cultured hippocampal neurons also found that IRSp53
localizes to the post-synaptic density together with PSD-95 [27].
Post-synaptic densities have very active membrane dynamics
and vesicle trafficking, but do not form filopodia. Our evidence
from the present study that Dictyostelium IBARa is involved in
clathrin-mediated endocytosis further underscores the wide range
of processes that involve I-BAR domains. Adapter proteins with
positively curved BAR domains already have a well-established
role in this process, and at a first glance the opposite curvature of
the I-BAR domain does not appear compatible with endocytosis.
However, the narrowest point in the neck of a budding clathrincoated vesicle is a mathematical saddle point, which is curved
in opposite directions when viewed either parallel to the cell
membrane or perpendicular to it (Figure 5C). In this respect,
I-BAR proteins could very well be recruited to the same site as
BAR proteins. The proposition that the curvature of the BAR
domain does not necessarily impose protein function is also
signified by the observation that filopodia-like protrusions are
generated by the overexpression of syndapin, which contains an
inwardly curved BAR domain [28].
The lack of an obvious phenotype in the IBARa-knockout cells
prevents absolute confirmation of a role in endocytosis, but it does
not rule it out. In a milestone knockout study of 60 Saccharomyces
proteins that are known to function in endocytic internalization
or have a localization that is consistent with involvement in this
process, only 14 showed abnormalities in localization or temporal
dynamics of markers of either F-actin or the clathrin coat [29]. It
appears that internalization is so robust that loss of any one gene
usually has little effect on the overall process. The subtle defect
51
of the Dictyostelium IBARa knockout is consistent with that of
Dictyostelium clathrin-light-chain-null cells, which are viable, but
form aberrant fruiting bodies [30] and that of the mouse knockout,
which appears healthy [31]. Expression of mammalian IBARa is
particularly high in the hippocampus, but the knockout mouse also
does not show any obvious defects in neurons in this region [31].
Paralogues often acquire different specialized roles and this is
no different for the three paralogous I-BAR/SH3 adapter proteins
found in vertebrates. IRSp53 has been proposed to couple Rac
proteins to the SCAR–WAVE complex. Both SCAR and WASP
are evolutionarily more ancient than IBARa. The putative partial
CRIB [Cdc42 (cell-division cycle 42)/Rac-interacting binding]
motif of IRSp53 that is thought to mediate Rac binding to SCAR
emerged particularly late (it is absent in Dictyostelium IBARa,
vertebrate IRTKS and BAIAP2L2.) This suggests that ancestral
SCAR did not require I-BAR proteins as an adapter, either as
part of its complex or for activation by small GTPases. Instead
of being a principal activator, I-BAR proteins may fine-tune the
functions of SCAR. This is consistent with our observations in
Dictyostelium and with earlier studies that find that IBARa only
optimizes human SCAR–WAVE activity [32].
Of the three paralogous IBAR/SH3 adapter proteins in
vertebrates, only BAIAP2L2 is involved in clathrin-mediated
endocytosis and thus forms the link with ancestral IBARa. Until
now, BAIAP2L2 has remained poorly characterized and to our
knowledge no functions of BAIAP2L2 have been proposed.
Its specific recruitment to clathrin-coated plaques and not to
coated pits shows that plaques and pits are differentially regulated
processes. It has been described that clathrin-coated vesicles bud
from the edges of the coated plaques [33]. In this scenario,
BAIAP2L2 may stabilize the transition state between the flat
clathrin lattice of the core of the plaque and the curved neck that
must be present on the budding vesicle.
In conclusion, the specific curvature of the I-BAR domain does
not restrict its function to protrusion formation, but I-BAR domain
proteins are involved in endocytosis and perhaps a range of other
related cellular processes.
AUTHOR CONTRIBUTION
Douwe Veltman and Robert Insall planned and performed the
Dictyostelium work, and wrote the paper. Heather Spence and
Laura Machesky identified the human IRSp53 homologues
and made the expression clones. Giulio Auciello and Joshua
Rappoport performed the transfection and microscopy of human
cells. All authors contributed to and commented on the final text.
ACKNOWLEDGEMENTS
We are grateful to Dr John Dawson for helpful discussions. dictyBase was, as usual, an
essential source of sequence data and analysis. We also thank the Acytostelium genome
consortium for the use of their online genome BLASTing service.
FUNDING
This work was funded by an institute Cancer Research UK grant (to R.H.I.) and
a Biotechnology and Biological Sciences Research Council grant [grant number
BB/H002308/1 (to J.Z.R.)]. G.A. is funded by a Medical Research Council Doctoral
Studentship. The TIRF microscope used in the present study was obtained through
Birmingham Science City Translational Medicine Clinical Research and Infrastructure
Trials Platform, with support from Advantage West Midlands.
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doi:10.1042/BJ20101684
SUPPLEMENTARY ONLINE DATA
Functional analysis of Dictyostelium IBARa reveals a conserved role of the
I-BAR domain in endocytosis
Douwe M. VELTMAN*, Giulio AUCIELLO†, Heather J. SPENCE*, Laura M. MACHESKY*, Joshua Z. RAPPOPORT† and
Robert H. INSALL*1
*Beatson Institute for Cancer Research, Switchback Road, Bearsden, Glasgow G61 1BD, U.K., and †School of Biosciences, University of Birmingham, Edgbaston,
Birmingham B15 2TT, U.K.
Figure S1
Alignment of the I-BAR domain
I-BAR domains from the indicated organisms were aligned in ClustalX. Residues are coloured according to the standard ClustalX colour scheme. The second half of the domain is shown below the
first. Species abbreviations are as follows: Hs, Homo sapiens ; Mm, Mus musculus ; Gg, Gallus gallus ; Dr, Danio rerio ; Tr, Takifugu rubripes ; Sp, Strongylocentrotus purpuratus ; Dm, Drosophila
melanogaster ; Ta, Trichoplax adhaerens ; Dd, Dictyostelium discoideum ; Dp, Dictyostelium purpureum ; and Pp, Polysphondylium pallidum .
1
To whom correspondence should be addressed (email [email protected]).
c The Authors Journal compilation c 2011 Biochemical Society
D. M. Veltman and others
Figure S2
Co-localization between clathrin and BAIAP2L2
(A) Co-expression of fluorescently tagged clathrin with BAIAP2L2 in MDA MB231 cells and BSC1 cells. (B) For each clathrin spot in the respective strains, it was determined if BAIAP2L2 was also
present at that position and vice versa.
Received 14 October 2010/28 February 2011; accepted 14 March 2011
Published as BJ Immediate Publication 14 March 2011, doi:10.1042/BJ20101684
c The Authors Journal compilation c 2011 Biochemical Society