Microtubules as Platforms for Assaying Actin Polymerization In Vivo
Citation: Oelkers JM, Vinzenz M, Nemethova M, Jacob S, Lai FPL, et al. (
Microtubules as Platforms for Assaying Actin Polymerization In Vivo
J. Margit Oelkers 0
Marlene Vinzenz 0
Maria Nemethova 0
Sonja Jacob 0
Frank P. L. Lai 0
Jennifer Block 0
Malgorzata Szczodrak 0
Eugen Kerkhoff 0
Steffen Backert 0
Kai Schlu ter 0
Theresia E. B. Stradal 0
J. Victor Small 0
Stefan A. Koestler 0
Klemens Rottner 0
Daniel J. Muller, Swiss Federal Institute of Technology Zurich, Switzerland
0 1 Helmholtz Centre for Infection Research , Braunschweig, Germany , 2 Institute of Molecular Biotechnology, Austrian Academy of Sciences , Vienna , Austria , 3 Department of Developmental and Regenerative Biology, Institute of Medical Biology, Immunos, Singapore, Singapore, 4 Molecular Cell Biology Laboratory, Department of Neurology, Bavarian Genome Research Network, University Hospital Regensburg , Regensburg, Germany , 5 School of Biomolecular and Biomedical Sciences, University College Dublin , Dublin, Ireland , 6 Institute for Molecular Cell Biology, University of Mu nster , M u nster, Germany , 7 Institute of Genetics, University of Bonn , Bonn , Germany
The actin cytoskeleton is continuously remodeled through cycles of actin filament assembly and disassembly. Filaments are born through nucleation and shaped into supramolecular structures with various essential functions. These range from contractile and protrusive assemblies in muscle and non-muscle cells to actin filament comets propelling vesicles or pathogens through the cytosol. Although nucleation has been extensively studied using purified proteins in vitro, dissection of the process in cells is complicated by the abundance and molecular complexity of actin filament arrays. We here describe the ectopic nucleation of actin filaments on the surface of microtubules, free of endogenous actin and interfering membrane or lipid. All major mechanisms of actin filament nucleation were recapitulated, including filament assembly induced by Arp2/3 complex, formin and Spir. This novel approach allows systematic dissection of actin nucleation in the cytosol of live cells, its genetic re-engineering as well as screening for new modifiers of the process.
Funding: This work was supported in part by grants from the Deutsche Forschungsgemeinschaft (to KR and TEBS). MS was supported through a Marie Curie
Early stage Training (contract MEST-2004-504990) of the European Communitys Sixth Framework Programme. JVS acknowledges grants from the Austrian
Science Foundation (projects FWF 1516-B09 and FWF P21292-B09). The funders had no role in study design, data collection and analysis, decision to publish, or
preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
. These authors contributed equally to this work.
The actin cytoskeleton in higher eukaryotes comprises
numerous sub-compartments, the molecular constituents and regulation
of which are just beginning to be elucidated. Examples include
protrusive organelles, such as lamellipodia and filopodia in
migrating cells, adhesive and invasive structures including focal
adhesions, the immunological synapse and invadosomes [1,2] and
also sensory structures, such as dendritic spines [3,4]. Through its
ability to form helical polar rods by polymerization, actin
constitutes a versatile building unit for both pushing and pulling
(in concert with myosin). The starting point for actin filament
assembly is the formation of a nucleus of three actin monomers,
which is considered to constitute the rate-limiting step in vitro and
in vivo. Once actin filaments are nucleated, other accessory proteins
are thought to take over and promote their elongation at their
fastgrowing ends. Whether this is accomplished by the nucleator or
another factor depends on the mechanism of nucleation (see also
below). Due to head to tail assembly of actin monomers, actin
filaments are intrinsically polar, harboring a fastgrowing, barbed
and a slowly growing, pointed end. The differential critical
concentrations of polymerization at the two ends can cause net
flow of actin monomers through the filament in a process known
as treadmilling [5,6], also operating in cellular structures like the
lamellipodium [7,8]. In addition, filament ends and sides are
subject to regulation by uncountable filament binding factors with
numerous activities. These include e.g. capping, stopping and
protecting ends from growth and depolymerization, respectively,
severing, generating filament ends presumably prone to
disassembly, or bundling, explicitly amplified in contractile structures or
finger-like protrusions such as filopodia and microvilli. It is
commonly agreed that a composite of all these activities drives the
turnover of a complex structure such as the lamellipodium.
Nevertheless, it is also clear that the essential prerequisite of
formation and maintenance of a given actin structure,
lamellipodium or smallish actin accumulation accompanying endocytosis,
constitutes the nucleation event, and an impressive progress has
recently been made in the discovery of mechanisms and factors
catalyzing this important process. Today, actin filament nucleators
are roughly divided into three classes : those that mimic a
nucleus, like Arp2/3 complex, those that stabilize spontaneously
formed intermediates, like formins, and those that recruit and
align actin monomers, like Spir. Arp2/3 complex contains two
actin-related proteins, Arp2 and Arp3 that, together with an actin
monomer, can assemble into a nucleus ready for elongation, either
individually or in association with a socalled mother filament .
In either case, Arp2/3 complex will stay attached to and protect
perhaps the pointed end from depolymerization, but the barbed
end will be free for monomer addition.
However, Arp2/3 complex is inactive in the absence of
nucleation promoting factors (NPFs), which can deliver actin
monomers and promote nucleation by inducing a conformational
change in the complex considered to bring Arp2 and Arp3 in close
proximity to each other . It is assumed today that the multiple
activities of the complex observed in vivo are mostly regulated by
the continuously growing group of NPFs, which now appear to
execute quite distinct and complementary functions determined
e.g. by differential localization [11,12,13]. NPFs are grouped into
two types. WASP family proteins constitute the type I NPFs like
the name-giving Wiskott-Aldrich-Syndrome protein (WASP),
NWASP or Scar/WAVE proteins, all of which share at their
Ctermini modules for binding actin and Arp2/3 complex . This
domain formerly known as WA comprises different numbers of
actin monomer-binding domains, also called V (for
verprolinhomology) or W (WASP homology 2) and a C-terminal CA
domain (connector and acidic) which activates Arp2/3 complex
. So in case of N-WASP, the C-terminus mediating Arp2/3
complex-dependent actin nucleation used in this study is
composed of two V-modules linked to the CA-domain (VVCA).
The second group of Arp2/3 complex activators is known as type
II NPFs and comprises in mammals the Src-kinase substrate
cortactin and HS1 (haematopoietic-specific 1), the latter of which
is expressed in cells of the immune system. At variance to type I
NPFs, these proteins bind to actin filaments instead of monomers
via a central repeat domain and the Arp2/3 complex through an
N-terminal acidic region. Importantly, cortactin and HS1 appear
to activate Arp2/3 complex less potently in vitro than type I NPFs
As opposed to Arp2/3 complex, formin dimers can nucleate
and elongate actin filaments by surfing on their barbed ends in a
process also known as leaky capping . Formins are large
proteins, harboring numerous regulatory domains at their
Ntermini, and as business end the actin-binding FH2- (formin
homology 2-) domain, frequently aided by an FH1- (formin
homology 1-) module, thought to operate in delivering actin to
FH2 through profilin-actin recruitment [16,17]. The subgroup of
Diaphanous-related formins (DRFs) is activated in a
signaldependent fashion to promote actin assembly upon release of
autoinhibitory interactions between the DID- (Diaphanous
inhibitory-) and the DAD- (Diaphanous autoregulatory-) domain
at the very C-terminus. The class of proteins potentiating
nucleation by scaffolding actin monomers, such as Spir  or
Cobl  or Leiomodin in muscle  is still growing [21,22], and
considered to stay attached to the pointed end upon nucleation
while the barbed end continues to grow. These nucleators usually
employ three or four actin monomer-binding domains in one
protein, as in case of three and four WH2-domains in Cobl 
and Spir , respectively. However, additional studies e.g. on
Spir indicate more complex biochemical activities and potential
cooperations with other nucleators such as formins [23,24,25,26],
so precise mechanistic understanding of nucleation by these factors
and their subsequent fate will require future investigation.
Nevertheless, considerable knowledge has already been
obtained about the mode of function and regulation by accessory
proteins of all these factors, especially in vitro and in purified
conditions, but understanding their relevance in vivo is more
challenging for numerous reasons. These include the plethora of
unknown factors potentially interfering with straight forward
interpretation of results received upon inhibition of a given factor,
but also the fact that structures dependent on distinct nucleators
like to intermingle with each other (e.g. lamellipodia and filopodia)
within a given cellular compartment [2,27], complicating analyses
and faithful, objective interpretation. Finally, it is almost
impossible to directly compare the potencies of different nucleators
in physiologic actin structures, since cells perfectly tune the
engagement of nucleators in specific structures, both qualitatively
and quantitatively, so traditional functional interference with a
nucleator will provide information on its relevance for a given
structure, but not relative to another nucleator.
To circumvent this problem, and to directly examine the ability
of a given factor to drive actin filament nucleation in vivo, we
developed a novel assay in which the putative nucleator is targeted
to the sides of microtubules. Microtubules belong to those
subcellular structures that are essentially free of endogenous actin,
and they hardly associate with potentially interfering, cellular
membranes along their entire length. Moreover, microtubules
offer a homogenous topology and comparable stability, allowing
not only the detection of actin recruitment in fixed and live cells,
but also the analysis of actin turnover by FRAP (fluorescence
recovery after photobleaching) approaches. Finally, they are easily
relocated in the electron microscope and can thus potentially be
employed to study in vivo actin filament arrangements seeded by
different combinations of nucleators and/or accessory factors.
Targeting actin assembly to microtubules
To test if actin assembly can be targeted to microtubules, we
engineered a construct (pEGFP-MBD-VVCA, Figure 1A)
encoding EGFP followed by a microtubule-binding sequence (MBD),
and the VVCA-domain of murine N-WASP, which drives Arp2/3
complex-dependent actin nucleation . The MBD harbors two
independent microtubule-binding activities  located within the
C-terminus of human MAP4 comprising a proline-rich, a so-called
A4 domain and a tail region (see Methods). The EGFP-tagged
MBD-VVCA-fusion targeted to microtubules and induced the
assembly of actin filaments (F-actin), as evidenced by
counterstaining with phalloidin (Figure 1B). Actin accumulation on
microtubules was also detectable by live cell imaging (Figure S1A,
Movie S1), and coincided with recruitment of Arp2/3 complex,
visualized with mCherry-tagged p16B (also known as ArpC5B), a
ubiquitously expressed isoform  of the smallest out of seven
Arp2/3 complex subunits (Figure 1C, Movie S2). Among all
subunits, fusions to p16 proved most useful for following Arp2/3
complex dynamics in mammalian cells , due perhaps to its
peripheral location in the complex . Importantly,
MBDVVCA-labeled structures strongly overlapped with both
microtubules and actin, as evidenced by counter-staining of
EGFP-MBDVVCA and EBFP2-actin co-expressors with anti-tubulin
antibodies (Figure 1D). Moreover, actin and Arp2/3 complex
accumulation was specific for the presence of VVCA, since an identical
construct lacking this domain (MBD) failed to stimulate actin
assembly and Arp2/3 complex recruitment, in spite of its strong
accumulation on microtubules (Figure S1B, Movie S3 and Movie
S4). Conversely, EGFP-tagged VVCA alone failed to induce actin
assembly on microtubules, since in the absence of a targeting
domain it was unable to direct Arp2/3 complex activation to the
microtubule surface (Figure S1C). Finally, western blotting
confirmed expression of EGFP-tagged MBD or MBD-VVCA as
full-length proteins, since no degradation pattern was observed
using anti-GFP antibodies (Figure S1D).
It is thinkable that actin filaments are not nucleated on
microtubules, but recruited instead as pre-nucleated, small
filaments. Interestingly, the annealing of small actin oligomers
has recently been proposed to contribute to actin assembly in yeast
cells . In addition, the two consecutive WH2-domains (VV) of
N-WASP-VVCA were also demonstrated recently to interact with
the barbed ends of actin filaments . However, the following
observations argue against oligomer recruitment to significantly
contribute to actin assembly in our system. First, a truncated
VVCA-domain lacking the C-terminus but comprising the two
WH2-domains (VV) failed to recruit actin and Arp2/3 complex
(Figure S2 and Figure S10, Movie S5 and Movie S6). The lack of
Arp2/3 recruitment under these conditions emphasizes the need
for physical interaction between the CA fragment and the Arp2/3
complex (see also below). Furthermore, to test whether an actin
filament binding protein is capable of actin recruitment to
microtubules, we produced chimeras of fascin and the
actinbinding domain of a-actinin with MBD (Figure 2). Fascin is a
prominent actin filament bundling protein, most famous for its
strong association with microspikes and filopodia , which it
serves to stabilize . a-actinins are a family of dimeric
actinbinding proteins with essential functions e.g. in signaling and
stabilization of the contractile apparatus in muscle and analogous
structures in non-muscle cells . The fusion of EGFP-tagged
MBD with fascin displayed a dual specificity pattern in vivo
(Figure 2A): the MBD mediated microtubule localization, while
fascin also targeted the fusion protein to microspike bundles at the
cell periphery, as expected [35,37]. Importantly, only the latter
structures co-localized with actin, whereas MBD-fascin on
Figure 2. Actin filament binding is not sufficient to target actin to microtubules. Epifluorescence images of live cells co-expressing
mCherry-actin with (A) EGFP-MBD-fascin or (B) EGFP-MBD-ABD (ABD: actin-binding domain of a-actinin). Arrowheads mark accumulation of
MBDfascin and MBD-ABD in microspikes embedded into the lamellipodium, as expected. Arrow points to a former microspike that became integrated into
the lamella, as evidenced by video microscopy (data not shown). Merges (right) corresponding to boxed insets (left) reveal the absence of
colocalization of these MBD-constructs with actin on microtubules. Bar, 5 mm.
microtubules did not show any sign of actin recruitment. Since
MBD-fascin was expressed as a full-length protein (Figure S3B)
capable of targeting to microspikes, the lack of actin accumulation
on microtubules is unlikely due to non-functional protein folding.
We also cloned the actin-binding domain (ABD) of a-actinin,
which when tagged to EGFP alone labeled the actin cytoskeleton
(Figure S3A). In contrast, a chimera of MBD and ABD of
aactinin (Figure 2B) again targeted to microspike bundles at the cell
periphery and to microtubules, but there was no actin
accumulation on the latter. Thus, we conclude that targeting of an
actinbinding activity to microtubules potentially capable of recruiting
small filaments or oligomers is not sufficient to drive actin
assembly in this assay. Together with the lack of actin
accumulation induced by N-WASP-VV, these data strongly
suggest that VVCA-induced actin assembly on microtubules is
mediated by de novo nucleation of actin filaments through the
Topology of actin filaments assembled on microtubules
To assess the arrangement of Arp2/3 complex-induced actin
filaments associated with microtubules, we performed correlative
light microscopy and negative stain electron tomography. Control,
non-transfected cells showed no specific association of actin
filaments with microtubules, as expected (for representative
microtubule see Figure 3E). Co-expression of mCherry-actin and
EGFP-MBD-VVCA allowed confirmation of actin assembly on
microtubules by observation in the light microscope (Figure3A, B),
and subsequent processing of the same cells by negative staining
and electron microscopy. Individual microtubules labeled for
MBD-VVCA and actin filaments in the light microscope were
relocated in the electron microscope (Figure 3C). As shown, short
actin filaments were found arranged as a disorganized cloud
concentrated around microtubules, consistent with stochastic
nucleation of actin filaments at the microtubule surface
(Figure 3D). Individual actin filaments or filament stubs in physical
contact with the microtubule surface could also be discerned
(Figure 3D), indicative of at least transient direct interactions of
nucleated actin filaments with microtubules, and also consistent
with results obtained by FRAP (see below). These data confirm
that microtubules can be exploited as platforms for actin
nucleation in vivo, and can generate actin assemblies analyzable
in detail by different types of light and electron microscopy.
Arp2/3 dependent actin nucleation on microtubules is
instantaneous and continuous
Although the induction of actin filament assembly on
microtubules on average reduced microtubule dynamics as compared to
cells expressing MBD alone (compare e.g. Movie S1 and Movie
S3), it was still possible to explore actin dynamics during growth or
shrinkage of individual microtubules. Interestingly, actin
nucleation on growing microtubules did not lag behind the
MBDVVCA-binding to their growing tips, neither for the frame rate
shown here (0.1 Hertz) (Figure 4A; Movie S7) nor at higher image
acquisition frequencies (1 Hertz, not shown). We assume that the
moderate average growth rates of microtubules of roughly
2.2 mm/min in these cells were not fast enough to detectably
escape potent actin nucleation at the microtubule tip. These data
indicate that MBD-VVCA binding can instantly drive actin
filament nucleation at these sites. Furthermore, the shrinkage of
microtubules again visualized with MBD-VVCA coincided with
abrupt depolymerization or dissipation of actin filaments at
microtubule tips (Figure S4; Movie S8), indicating that actin
filaments on microtubules undergo rapid turnover, comparable to
other Arp2/3 complex-dependent structures such as the
lamellipodium . To compare the dynamics of MBD-VVCA and actin
at the surface of microtubules, we performed fluorescence recovery
after photobleaching (FRAP) experiments (Figure 4BE).
Interestingly, MBD-VVCA bound along the length of microtubules
continuously exchanged with the cytosolic pool with an average
half time of 7.7 seconds (Figure 4B, D), similar to the turnover of
the Arp2/3 complex activator WAVE at the lamellipodium tip .
This observation might explain the efficient activation of Arp2/3
complex on microtubules, since the residence times of a given
Arp2/3 complex activator at a specific subcellular location could
well be a limiting factor for activation efficiency. The turnover of
actin filaments on microtubules was significantly slower than
MBD-VVCA (t1/2 = 16 sec, Figure 4C, E), again reminiscent of
turnover rates observed for actin and Arp2/3 complex in the
lamellipodium which are longer than the NPF at the tip, due to the
treadmilling of the network . These data indicate that the
physical association of MBD-VVCA molecules with microtubules
is sufficiently long to allow nucleation and elongation of individual
actin filaments (see Figure 3). Turnover of the actin network is
delayed however, because nucleation may not occur as instantly as
MBD binding or because fluorescence in the network will have to
recover by treadmilling or both. The turnover data of both
MBDVVCA and actin on microtubules best fitted bi-exponential
models (Figure 4D, E), presumably due to the multitude of
parameters potentially contributing to average exchange. More
specifically, actin on microtubules may be lost by dissociation of
entire filaments or oligomers and not just by depolymerization,
and the residence times of individual MBD-VVCA molecules may
vary depending on active engagement in Arp2/3 activation for
instance or on steric restraints. Whatever the case, the data
unequivocally show that the actin filament turnover observed in
physiological, Arp2/3 complex-dependent structures such as the
lamellipodium [8,38] is largely recapitulated in this assay.
Cortactin cannot activate Arp2/3 complex on
The experiments described above demonstrate the
development of an assay that allows asking whether a given
actinbinding protein can recruit proteins or protein machineries
driving actin filament assembly. The type II NPF cortactin has
been implicated in the regulation of multiple Arp2/3-dependent
structures and co-localizes with Arp2/3 complex in lamellipodia
and at sites of clathrin endocytosis [39,40]. However, its precise
functions in vivo are elusive, especially since genetic deletion in
fibroblasts did not abolish lamellipodia formation or actin
assembly accompanying endocytosis [38,41]. Although these
data indicated cortactin to be dispensable for Arp2/3 complex
activation in these structures, they did not address directly
whether cortactin is able in principle to activate Arp2/3
mediated actin assembly in vivo.
To test this, full-length cortactin was fused to MBD in analogy
to N-WASP-VVCA and co-expressed again with mCherry-tagged
actin or p16B for visualization of Arp2/3 complex. Interestingly,
the cortactin molecule was functional, since it mediated additional
targeting of the EGFP-tagged fusion protein to its physiological
localization in lamellipodia and microspikes (Figure 5A), and was
expressed as a full-length protein as confirmed by western blotting
(Figure S5). Although the MBD-cortactin chimera was mainly
targeted to microtubules, this was not accompanied by
accumulation of actin or Arp2/3 complex at these sites (Figure 5A; Movie
S9 and Movie S10). Activation of Arp2/3 complex by cortactin in
vitro requires interaction domains for both Arp2/3 complex and
actin filaments , both of which were present in the chimera,
however, the full-length molecule is potentially regulated by a
complex network of additional interactions in vivo , which may
influence the outcome of our assay. Thus, we also asked whether
the isolated Arp2/3 binding surface of cortactin (N-terminal
residues 184 , Figure 5B) might be able at least to recruit
Arp2/3 complex. However, neither Arp2/3 complex nor actin
(used as negative control) were targeted to microtubules in this
case (Figure 5B; Movie S11 and Movie S12), indicating that
binding of the N-terminus to Arp2/3 complex is not sufficient to
ectopically target Arp2/3 complex in vivo. Based on these and our
previous results obtained with genetic deletion of cortactin in
fibroblasts , we conclude that the comparably weak Arp2/3
complex activation of cortactin observed in vitro does not suffice to
potently activate Arp2/3 complex in vivo. The interaction of
cortactin with Arp2/3 complex might indeed serve distinct
functions, as suggested for instance by the observation of
competitive binding with Arp2/3 complex between type I NPFs
such as N-WASP and cortactin . Finally, the intimate
connection between cortactin and Arp2/3 complex function was
Figure 4. Turnover of MBD-VVCA and associated actin filaments on microtubules. (A) Selected frames derived from time-lapse movie of
B16-F1 cell expressing EGFP-MBD-VVCA and mCherry-actin. Arrowheads point to the growing tip of a MBD-VVCA-labeled microtubule instantly
recruiting actin. Time is in seconds; bar, 2 mm. (B, C) Representative frames taken from FRAP movies of cells transfected either with EGFP-tagged
MBD-VVCA (B) or mCherry-MBD-VVCA (false-colored green in [C]) and EGFP-actin (false-colored red in [C] for clarity). Simultaneous imaging of
MBDVVCA was used to ensure bleaching of actin structures co-localizing with MBD-labeled microtubules. Circles in (B, C) indicate areas of bleaching and
dashed lines enclose those regions used for measurements of fluorescence intensity changes over time. Time is in seconds; bars, 2 mm. (D, E)
Fluorescence recovery curves obtained for EGFP-MBD-VVCA (D) or EGFP-actin (E). Data are means and standard errors of means (error bars) measured
for each time point and construct as indicated. n values correspond to number of movies analyzed. Half times of fluorescence recovery (t1/2) were
calculated from best exponential fits (green curves).
underscored by the observation that full-length cortactin was
corecruited to microtubules with actin filaments induced by
MBDVVCA and Arp2/3 complex but not other nucleators (see also
below), indicating that subcellular cortactin positioning in vivo
occurs subsequently and not prior to Arp2/3 complex activation
Figure 5. Cortactin does not drive actin assembly or Arp2/3 complex accumulation on microtubules. Epifluorescence images of live cells
co-transfected with (A) EGFP-MBD-cortactin (MBD-Cttn) or (B) the EGFP-MBD-tagged N-terminus of Cortactin (MBD-Cttn 184) with mCherry-actin or
mCherry-p16B as indicated. In neither case, a co-localization of the respective MBD-construct with actin or Arp2/3 complex was discernible. Merged
images correspond to enlarged insets on the left. Arrowheads indicate co-localization of MBD-Cttn with microspikes. Bar, 10 mm.
Actin assembly on microtubules can be induced by
distinct nucleation mechanisms
As described above, our assay allows the induction of Arp2/3
complex-dependent actin assembly at ectopic sites in vivo. Next we
asked whether actin polymerization could also be induced by
Arp2/3 complex-independent nucleation mechanisms, e.g. by
formins or Spir. Interestingly, fusion of the EGFP-MBD targeting
unit with Drf3 lacking DAD (Drf3DDAD), an active variant of the
formin Drf3 (also known as mDia2), which was previously
concluded to potently stimulate filopodia formation in vivo through
actin filament nucleation  was also capable of inducing actin
assembly on microtubules (Figure 6A). Moreover, an N-terminal
fragment of human Spir-1 (Spir-NT), which comprises all four
WH2-domains, also mediated strong actin polymerization on
microtubules (Figure 6B). Virtually identical results were obtained
with the full-length variant of Spir-1 (data not shown). With both
Drf3 and Spir-NT, actin assembly could be scored both in fixed
cells stained with phalloidin to prove the presence of actin
filaments (Figure 6A, B), and in live cells co-transfected with
mCherry-actin (Figure S7A, B). The latter approach is more
sensitive and easier to interpret. To confirm the specificity of each
actin nucleation pathway, cells expressing EGFP-MBD-tagged
Spir-NT or Drf3DDAD were counterstained with an antibody
specific for the Arp2/3 complex subunit p16A (Figure S8AC).
Although the antibody strongly labeled lamellipodia and ruffles as
well as vesicular structures in non-transfected cells as expected
(Figure S8A), no co-localization was observed with ectopic Spir- or
formin-induced actin filaments on microtubules (Figure S8B, C),
indicating Arp2/3 independent actin assembly.
Collectively, these data demonstrate the general applicability of
the microtubule platform to assay actin nucleation induced by
different mechanisms in vivo.
Employing the assay: probing the minimal requirements
of Arp2/3 dependent actin assembly in vivo
There is consensus that the WH2-domains (VV) in
N-WASPVVCA bind actin monomers and the acidic domain Arp2/3
complex, whereas the connector can interact with both . All
these interactions are considered essential for Arp2/3 dependent
actin assembly . The A-region harbors a tryptophan residue,
the mutation of which to serine was sufficient to eliminate
detectable Arp2/3 complex binding of WASP-VCA . Notably,
the same domain harboring the WH2 and connector regions had
previously been observed to induce weak actin nucleation .
However, this was subsequently interpreted as an artifact of
GSTinduced dimerization . A more recent study described strongly
reduced but not abolished affinity of VVC for Arp2/3 complex,
and actin assembly induced by this domain on synthetic vesicles in
vitro . Whether the acidic region might also be dispensable for
Arp2/3 dependent actin assembly in vivo has remained unclear.
We employed the MBD-assay to compare effects of microtubule
targeting of VVCA (wildtype) with VVC and VV. For expression
of all variants see Figure S9. MBD-VV failed to induce actin and
Arp2/3 complex recruitment (Figure S2, see above). However,
expression of MBD-VVC induced significant actin and Arp2/3
complex accumulation on microtubules (Figure 7A, B), although
less robustly especially in case of Arp2/3 complex than usually
observed for VVCA. This conclusion is based on the fact that
accumulation on microtubules was more difficult to distinguish
from the cytosolic fraction than observed in VVCA-expressors
(compare Figure 1 and Figure 7). Furthermore, when counting the
number of cells capable in principle of actin accumulation for each
construct, it became evident that the frequency of actin
colocalization with microtubules (97.2% for VVCA-expressors
[n = 252]) was significantly reduced to 43.2% (n = 264) in case of
VVC (p,0.001), whereas no single co-localization was scored in
case of VV (n = 278) (Figure S10). The accumulation of Arp2/3
complex followed a similar trend, although the detection
frequency on microtubules was generally reduced for all constructs
(Figure S10). Nevertheless, Arp2/3 complex enrichment could
clearly be detected also with VVC (10.7%; n = 190), albeit much
less robustly than with VVCA (69.4%; n = 193). Again, no
recruitment could be scored with VV alone (n = 271) (Figure
S10). These data strongly suggest that the acidic domain
contributes to, but is not essential for Arp2/3 complex-dependent
actin assembly in vivo. The physiologic relevance of this
observation was confirmed by comparing N-WASP-dependent
actin tail formation induced by intracellular Shigella in N-WASP
null cells reconstituted either with EGFP-tagged full length
NWASP or N-WASP lacking the acidic domain (N-WASPDA)
(Figure 7CE). Interestingly, both N-WASP variants were capable
of driving actin tail formation, although reduced efficiency of
NWASPDA was reflected e.g. by increased expression levels
required for inducing prominent actin tails (not shown). These
results demonstrate for the first time the dispensability of the acidic
Figure 7. The acidic domain of N-WASP is dispensable for recruitment and activation of Arp2/3 complex. Epifluorescence images of
cells transfected with EGFP-MBD-VVC and stained with phalloidin (A) or co-expressing fluorescently labeled p16B (B). Merged images and arrows
indicate co-localization of MBD-VVC with actin filaments (A) and Arp2/3 complex (B). Bar, 10 mm. (CE) Epifluorescence images of N-WASP-deficient
cells transfected with EGFP (C), EGFP-N-WASP full length (D) or EGFP-N-WASPDA (E) and infected with S. flexneri (Shigella). No actin tails are visible in
cells transfected with EGFP alone (C), as expected , whereas both ectopically expressed N-WASP full length and N-WASPDA bind to the surfaces of
Shigella, and induce actin tail formation in N-WASPdeficient cells (see insets). Bar, 10 mm.
domain in an N-WASP-dependent process in vivo, and call for
revision of our views on the functional relevance of this domain in
both type I and II NPFs.
The nucleation of actin filaments is key to numerous processes
regulating cell division, morphogenesis, migration, signaling and
host-pathogen interaction. A constantly increasing number of
molecules can influence actin nucleation activity in vitro, but how
and where exactly they function in vivo is often unknown. Whether
or not actin assembly mediated by a given factor in vitro translates
into bona fide actin nucleation activity in the cytoplasm frequently
remains unclear. Here we introduce a novel and robust assay to
analyze actin nucleation ectopically targeted to the surface of
microtubules. Ectopic actin assembly had previously been induced
on mitochondria [50,51,52] or late endosomes in Dictyostelium .
The use of endosomes is complicated by the fact that these
structures do assemble actin filaments by themselves to counteract
their fusion . As opposed to this, it is quite safe to assume that
a construct engineered to induce actin assembly on the surface of
microtubules will find essentially no endogenous actin and thus
other actin binding proteins that might aid the ectopic actin
assembly process. The conclusion that Arp2/3 complex activation
in live cells is feasible in the absence of the acidic domain of the
NWASP C-terminus would not have been legitimate on subcellular
structures suspected to recruit endogenous actin. Mitochondria
recruit little actin , but are highly dynamic structures
undergoing fusion and fission events within seconds , thus
compromising the in-depth analysis of actin assembly by live-cell
imaging. This is most critical for turnover studies such as those
using FRAP, since the relatively slow turnover rates observed for
actin and actin regulators can only reliably be determined on
comparably rigid, and thus stable structures. Likewise, the
topology and comparably firm surface of microtubules will both
be advantageous for successful relocation and for detailed analyses
of the ultrastructure of actin networks generated by distinct actin
Actin polymerization could be induced irrespective of the
molecular mechanism of nucleation, demonstrating the versatility
of the assay. In principle this approach is applicable to any
transfectable cell line, irrespective of model organism or cell type.
In our hands, transfection with MBD-VVCA of murine NIH 3T3
or fish CAR fibroblasts also caused robust actin assembly on
microtubules (not shown). However, we recommend analyses best
be completed by 2448 hours after transfection. It should be
pointed out that the strongly-induced reorganization of the actin
cytoskeleton might affect additional, actin-dependent processes, so
analyses of MBD-construct expressors should be restricted to actin
assembly events on the surface of microtubules. Nevertheless,
spindle formation and cytokinesis could still be observed in cells
expressing MBD-VVCA (not shown), indicating that these
processes are not generally blocked, but the treatment could
interfere with efficiency or frequency of their occurrence.
Moreover, MBD-VVCA-induced reprograming of Arp2/3
complex activation onto the microtubule surface abolished
lamellipodia formation, which is not surprising given that Arp2/3 complex
is considered to be essential for the formation of these protrusions
 and that simple sequestration of Arp2/3 complex in the
cytosol also interfered with lamellipodia formation . In
contrast, cells expressing MBD-Spir-NT appeared more likely
to form lamellipodia (not shown), consistent with the absence
of a reported function for Spir in the formation of these structures
The assay is ideally suited for the analysis of co-recruitment of
additional regulators (exemplified here by cortactin), and can be
extended to tuning output actin assembly by RNAi-mediated
suppression of specific components. Furthermore, assaying actin
assembly induced on the same structure by distinct nucleators
upon knockdown or knockout of a given factor will allow to
directly uncover its potential differential functions in distinct actin
Combination of the simple actin accumulation readout with
high content screening should facilitate identification of novel
activators or inhibitors of specific actin nucleation mechanisms in
vivo. This way the assay will help to increase our understanding of
the complex interplay of different actin assembly mechanisms in
normal and diseased cells.
EGFP tagged human b-actin was purchased from Clontech
(Mountain View, CA, USA), and the following constructs were
described previously: mCherry-actin , EGFP-N-WASP and
pEGFP-C3-VVCA . EBFP2-actin was obtained by fusing
human b-actin into pEBFP2-C1, kindly provided by Dr. Robert E.
Campbell . mCherry-p16B was obtained by exchanging
EGFP in EGFP-p16B  for mCherry. For generation of
microtubule targeting constructs, the MBD encoded by residues
6881151 of human MAP4, (isoform 3; genebank accession:
U19727) was amplified with pMEP-MAP4 as template , kindly
provided by Dr. Martin Gullberg (Umea, Sweden) and using
primers 59-gacatgtacaccccaccgaac-39 (forward) and
59-gtcatctgtacatgcttgtctcc-39 (reverse), thereby introducing BsrGI sites. For the
control vector, the MBD-fragment was fused into pEGFP-C1
(Clontech) using BsrGI digestion. For EGFP-MBD-VVCA, the
fragment was fused in frame into pEGFP-C3-VVCA . Fusion
with other cDNAs of interest was routinely done in an analogous
fashion based on constructs harboring the respective N-terminally
EGFP-tagged sequences. These included EGFP-Drf3DDAD ,
EGFP-fascin  or EGFP-cortactin. For generation of the latter,
murine cortactin (genebank accession: NM_007803) was cloned
into pEGFP-C1 vector. mCherry-cortactin, mCherry-VVCA and
mCherry-Spir-NT were generated by exchanging EGFP in
respective vectors described above for mCherry.
MBD-Spir-NT was generated by fusing Spir-NT (see below)
into pEGFP-MBD-C1. Spir-NT (the N-terminal KIND and four
WH2 domains) corresponded to residues 2402 of human Spir-1
(isoform 2). Cttn 184 was made by amplification of the
Nterminal 84 amino acids of murine cortactin using primers
59-gagagaattcatgtggaaagcctctgc-39 (forward) and
59-gagagtcgacatagccgtgggaagcctt-39 (reverse) and cloning into EGFP-MBD-C2.
The sequence encoding the actin-binding domain of a-actinin
comprising both calponin homology domains  was amplified
from EGFP-a-actinin , using the following primers:
59gagagaattcatggaccattatgattctc-39 (forward) and
59-gagagtcgactgctgtctccgccttctgg-39 (reverse). The PCR fragment was fused into
EGFP-MBD-C2 or EGFP-C2 as control. MBD-VV (amino acids
392460 of murine N-WASP) was amplified from EGFP-VVCA
 using primers 59-gagagaattccatcaagttccagctcct-39 (forward)
and 59-gagagtcgacagtgggtgcgggtgttgg-39 (reverse), and MBD-VVC
(amino acids 392483 of murine N-WASP) using primers
59gagagaattccatcaagttccagc-39 (forward) and
59-tgtcgtcgacttattcatctgagga-39 (reverse). Both fragments were ligated into
EGFP-MBDC2. To obtain N-WASPDA, amino acid residues 1483 of murine
N-WASP  were amplified with primers
59-gagagaattcatgagctcgggccagcag-39 (forward) and
59-gagagtcgacttattcatctgaggaatga-39 (reverse) and were cloned into EGFP-C2. All PCR
fragments were sequenced to ensure correct amplification.
Cells, transfections and western blotting
Mouse melanoma cells (B16-F1) were purchased from
American Type Culture Collection (ATCC CRL-6323) and were
cultured in DMEM (Invitrogen, Germany) with 10% FCS (PAA
Laboratories, Austria) and 2 mM glutamine (Invitrogen) at 37uC
in the presence of 7.5% CO2. Cells were transfected using
Superfect (Quiagen) according to manufacturers instructions. One
day after transfection, B16-F1 cells were seeded onto coverslips
coated with 25 mg/ml laminin (Sigma-Aldrich) and either
examined by video microscopy or fixed and processed for
immunolabeling or electron microscopy. Ectopic expression of
EGFP-tagged proteins was analyzed by western blotting using
standard protocols. Monoclonal anti-GFP antibody (clone 101G4)
is available from Synaptic Systems (Gottingen, Germany).
NWASP-deficient cells  were maintained and transfected as
Shigella actin tail formation
Infections with S. flexneri were performed as described .
Briefly, N-WASP-deficient cells were seeded onto
fibronectincoated coverslips, transfected and infected with Shigella (M90T
wild-type invasive strain serotype 5) two days after plating.
Bacteria were slowly centrifuged to bring them in close proximity
to the host cell layer, and allowed to infect for 1.5 h before
extracellular bacteria were killed using 50 mg/ml gentamicin
(Sigma). Cells were fixed for 20 min with 4% paraformaldehyde
(PFA) and 0.1%Triton X100 in phosphate-buffered saline (PBS),
and subjected to immunolabeling.
Light and video microscopy
Phalloidin stainings and immunolabeling experiments were
performed as described . Transfected cells were seeded onto
glass coverslips coated with laminin (25 mg/ml) or fibronectin
(25 mg/ml, for N-WASP2/2 cells), and fixed with 4%
paraformaldehyde (PFA) in PBS (37uC) for 20 min followed by extraction with
0.1% Triton X100 for 1 min. Subsequently, samples were stained
with Alexa594- or Alexa350-labelled phalloidin to detect actin
filaments, anti-a-tubulin (clone 3A2, Synaptic Systems), monoclonal
anti-p16A antibody (clone 323H3 ) for staining of the Arp2/3
complex or polyclonal anti-Shigella antibody (Abcam), followed by
secondary, Alexa594-labelled goat anti mouse antibody or
Alexa350-labelled goat anti rabbit antibody (both Invitrogen).
Light microscopy was performed on an inverted microscope
(Axiovert 100TV; Carl Zeiss, Jena, Germany) using standard
epifluorescence illumination (light source HXP120, Zeiss) and
636/NA1.4 or 1006/NA1.4 plan-apochromatic objectives.
Images were acquired with a back-illuminated, cooled
chargecoupled-device camera (CoolSNAP HQ2, Photometrics, Tucson,
AZ, USA) driven by Metamorph software (Molecular Devices
Corp., Downingtown, PA, USA).
For video microscopy and FRAP experiments, cells were mounted
in an open, heated chamber (Warner Instruments, Reading, United
Kingdom) at 37uC. Actin assembly and Arp2/3 complex
accumulation induced by VVCA, VVC versus VV were quantified using live
cell imaging, scoring cells as detailed in the legend to Figure S10.
Statistical analyses were done using OriginPro 8.5 (OriginLab
Corporation, Northampton, USA). FRAP experiments were
performed with minor modifications as described before  using a
double-scan-headed confocal microscope (Fluoview1000, Olympus,
Hamburg, Germany) equipped with a 1006/1.45 PlanApo TIRF
objective (Olympus). Circular regions drawn around individual
microtubules were bleached with a 405 nm diode using tornado
mode. Movies were acquired at a scanning rate of 1.644 s per frame.
Average fluorescence intensities of microtubules or background were
measured with Metamorph software (Molecular Devices Corp.)
before and after bleaching. The fluorescence intensity of the last
frame before bleaching was defined as maximum and normalized to
1. Background fluorescence was subtracted and data were analyzed
using SigmaPlot 11.0 and Microsoft Excel 2000. Exponential curves
in Figure 4 corresponded to best fits of means. Fitted data followed
equation y = a(12exp(2bx))+c(12exp(2dx)), with a = 0.5628,
b = 0.1583, c = 0.3708 and d = 0.0253 for MBD-VVCA and
a = 0.2743, b = 0.3601, c = 0.6246 and d = 0.0164 for actin. Half
times of recovery (t1/2) were calculated by solving the corresponding
equations at 50% of the maximal recovery value derived from each
Correlated live cell imaging and electron tomography was
performed essentially as described in . Briefly, B16-F1
melanoma cells were co-transfected with EGFP-MBD-VVCA and
mCherry-actin and plated onto formvar-coated coverslips. Cells
expressing both constructs were located by fluorescence microscopy
and imaged before and after fixation in a mixture of 0.5% Triton
X100 and 0.25% glutaraldehyde in cytoskeleton buffer (10 mM
MES, 150 mMNaCl, 5 mM EGTA, 5 mM glucose and 5 mM
MgCl2, at pH 6.1). After an initial fixation of 1 min the cells were
post-fixed in 2% glutaraldehyde containing 10 mg/ml phalloidin
and stored in the same mixture at 4uC. The film was subsequently
peeled from the coverslip, an EM grid positioned over the area
containing the cell of interest and the grid negatively-stained with
6% sodium silicotungstate containing 10 mg/ml phalloidin and a
10 nm gold sol. The cell was relocated in the electron microscope
(FEI Tecnai F30 Polara operating at 300 kV) and tomographic
series recorded around two orthogonal axes. Re-projections from
the tilt series were generated using IMOD software from the
Boulder Laboratory for 3D Electron Microscopy of Cells, using the
gold particles as fiducials for alignment .
Figure S1 MBD or VVCA alone do not target actin to
microtubules. Epifluorescence images of cells co-expressing (A)
EGFP-tagged MBD-VVCA and mCherry-actin as control or (B)
EGFP-tagged MBD and mCherry-actin or mCherry-p16B as
indicated. Note that MBD-VVCA and actin co-localize, whereas
no overlap of MBD with actin or MBD with p16B is visible. Bar,
10 mm. (C) Phalloidin staining (blue in merge) and
immunolabeling with anti-a-tubulin antibodies (red in merge) of a cell
transfected with EGFP-VVCA (green in merge). Merge
corresponds to boxed regions in left panels. Since VVCA does not
target to microtubules, they are completely devoid of actin
filaments. Bar, 10 mm. (D) Immunoblot confirming expression of
EGFP-tagged MBD and MBD-VVCA at appropriate molecular
Figure S2 The WH2-domains of N-WASP cannot
nucleate actin filaments on microtubules. Selected frames from
time-lapse movie of B16-F1 cell co-expressing EGFP-tagged
MBDVV and mCherry-actin (upper panel) or mCherry-p16B (lower
panel). Insets on the left are magnified in merged images on the
right, revealing the absence of co-localization of MBD-VV (green in
merge) with actin or Arp2/3 complex (red in merges). Bar, 10 mm.
Figure S3 Subcellular localization of the actin-binding
domain of a-actinin (ABD) and its expression compared
to MBD-tagged ABD and fascin. (A) Phalloidin staining of a
B16-F1 cell ectopically expressing EGFP-ABD revealing that the
ABD of a-actinin robustly associates with actin networks and
bundles located in the lamellipodium and the lamella behind, as
expected. Bar, 5 mm. (B) Verification of correct expression of
EGFP-tagged MBD-fascin, ABD and MBD-ABD as indicated.
Figure S4 MBD-VVCA and polymerized actin dissociate
instantly from shrinking microtubules. Selected frames
from time-lapse movie of B16-F1 cell co-expressing EGFP-tagged
MBD-VVCA and mCherry-actin as indicated. Arrowheads point
to a shrinking microtubule. Time is in seconds; bar, 2 mm.
Figure S6 Cortactin is recruited to Arp2/3 but not
SpirNT-induced actin assemblies. Epifluorescence images of live
cells co-transfected with (A) mCherry-MBD-VVCA and
EGFPcortactin (false-colored in merge for clarity) or (B) EGFP-Spir-NT
and mCherry-cortactin. Merged image and arrows in (A) show
significant accumulation of cortactin at MBD-VVCA-stimulated
actin structures. In contrast, no targeting to microtubules
decorated with Spire-NT was discernible. Bar, 5 mm.
Figure S7 Actin polymerization on microtubules by
MBD-Drf3DDAD and MBD-Spir-NT. Live cell imaging of
B16-F1 cells co-expressing mCherry-actin and (A) EGFP-tagged
MBD-Drf3DDAD or (B) MBD-Spir-NT. Merged images and
arrows indicate robust co-localization of respective
MBD-construct with actin. Bar, 5 mm.
Figure S8 MBD-Drf3DDAD and MBD-Spir-NT nucleate
actin filaments on microtubules independently of Arp2/
3 complex. Immunolabeling experiments showing Arp2/3
complex localization (p16A) in (A) non-transfected control cell or
in cells expressing EGFP-tagged (B) MBD-Drf3DDAD or (C)
MBD-Spir-NT. Bar, 5 mm.
Figure S9 Immunoblot showing expression of
EGFPtagged MBD-VV and MBD-VVC compared to MBD and
MBD-VVCA. Asterisk marks an additional, truncated product
detected by anti-GFP antibodies (MBD-VVC lane) with an
approximate size of 50 kDa, thus unable presumably to interfere
with microtubule targeting and thus actin assembly induced by the
full length fragment (Figure 1A).
Figure S10 MBD-VVCA co-localizes more frequently
with actin and p16B compared to MBD-VVC and
VV. Cells were transfected with MBD-VVCA, MBD-VVC or
MBD-VV and additionally with either actin or p16B. Living cells
were classified into categories: actin (A) or p16B (B) co-localizing
or not co-localizing with the respective MBD-construct on
microtubules, as indicated. Cells in which actin or p16B
accumulation on microtubules could not be determined due to
overexpression of either construct were classified ambiguous.
Data are means and standard errors of means (error bars) and n
values correspond to number of cells analyzed. The differences
between MBD-VVCA- and MBD-VVC-expressors co-localizing
with actin or p16B were confirmed to be statistically significant by
two-sided two-sample t test.
actin assembly on
by Arp2/3 complex
We thank Dr. Martin Gullberg for MAP4 cDNA, Dr. Robert E. Campbell
for EBFP2 expression plasmid, Brigitte Denker for excellent technical
assistance and Jan Faix for discussion.
Conceived and designed the experiments: JVS SAK KR. Performed the
experiments: JMO MN MV SAK SJ. Analyzed the data: JMO MN MV SJ
KR. Contributed reagents/materials/analysis tools: FPLL JB MS EK SB
KS TEBS. Wrote the paper: JMO SAK JVS KR. Made figures: JMO
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