A Dictyostelium homologue of WASP is required for polarized F-actin
assembly during chemotaxis
Scott A. Myers1, Ji W. Han1, Yoonsung Lee1+, Richard A. Firtel2, and Chang Y. Chung12*
1Department of Pharmacology
Vanderbilt University Medical Center
Nashville TN 37232-6600
2Section of Cell and Developmental Biology
Division of Biological Sciences
Center for Molecular Genetics
University of California, San Diego
La Jolla CA 92093-0634
*Corresponding author: email@example.com
468 Robinson Research Building (MRB I)
1215 21st Ave. South @ Pierce
Nashville, TN 37232-6600
Phone: 615-322-4956, Fax: 615-343-6532
Key Words: WASP, Actin, Cytoskeleton, Cell Polarity, Cell Motility, PI3 kinase
Running title: WASP regulation of chemotaxis
+ Present address: Department of Cell Biology, Duke University Medical Center, Durham, NC
The actin cytoskeleton controls the overall structure of cells and is highly polarized in
chemotaxing cells, with F-actin assembled predominantly in the anterior leading edge and to a
lesser degree in the cell’s posterior. Wiscott-Aldrich syndrome protein (WASP) has emerged as
a central player in controlling actin polymerization. We have investigated WASP function and
its regulation in chemotaxing Dictyostelium cells and demonstrated the specific and essential role
of WASP in organizing polarized F-actin assembly in chemotaxing cells. Cells expressing very
low levels of WASP show reduced F-actin levels and significant defects in polarized F-actin
assembly, resulting in an inability to establish axial polarity during chemotaxis. GFP-WASP
preferentially localizes at the leading edge and uropod of chemotaxing cells and the B domain of
WASP is required for the localization of WASP. We demonstrated that the B domain binds to
PI(4,5)P2 and PI(3,4,5)P3 with similar affinities. The interaction between the B domain and
PI(3,4,5)P3 plays an important role for the localization of WASP to the leading edge in
chemotaxing cells. Our results suggest that the spatial and temporal control of WASP
localization and activation is essential for the regulation of directional motility.
Chemotaxis, cell movement up a chemical gradient, is central to a wide variety of
biological processes in eukaryotic cells, including migration of macrophages and neutrophils
during wound healing, homing of thymocytes, migration of neural crest cells, and aggregation of
Dictyostelium cells to form a multicellular organism (Aubry and Firtel, 1999; Downey, 1994;
Hosaka et al., 1979; Lahrtz et al., 1998; Locker et al., 1970). In Dictyostelium cells, neutrophils,
and macrophages, chemotaxis can be mediated through heterotrimeric G protein-coupled cell
surface receptors. The first step of chemotactic movement is a chemoattractant-mediated
increase in F-actin polymerization at the leading edge of the cell, which provides the motive
force for pseudopod extension and cell movement. An important biological question is how cells
regulate the formation of the leading edge in the direction of a chemoattractant source. Forward
extension of the leading edge of cells is driven by the protrusion of two F-actin-rich structures,
lamellipodia and filopodia. Dissecting the signaling mechanisms controlling F-actin
organization is a key step toward understanding directed cell movement.
The WASP (Wiskott-Aldrich Syndrome protein) family has emerged as important
regulatory molecules that connect multiple signaling pathways through Rac/Cdc42 to F-actin
polymerization. In mammals, this family includes WASP, N-WASP, and SCAR or WAVE
(Bear et al., 1998; Miki et al., 1996). The expression of WASP appears to be restricted to
hematopoietic cells (Ochs, 1998b), while the closely related N-WASP is more widely expressed
(Miki et al., 1996). Wiskott-Aldrich Syndrome (WAS) is a human X-linked immunodeficiency
characterized by recurrent infections, hematopoietic malignancies, eczema, and
thrombocytopenia that is caused by mutations in the gene encoding WASP (Aldrich et al., 1954;
Derry et al., 1994; Snapper et al., 1998). The size of hematopoietic cells, including platelets,
neutrophils, and lymphocytes, is significantly reduced in WAS patients, and the cell surfaces are
relatively smooth with a decrease in the number and size of microvilli, suggesting a defect in
cytoskeletal architecture (Remold-O'Donnell et al., 1997). Chemotaxis of macrophages and
monocytes from WAS patients is significantly defective.
WASP and N-WASP are complex "adaptor" proteins thought to link multiple signaling
inputs to regulate the actin cytoskeleton. Both WASP and N-WASP possess a WH1 (WASP
homology 1) domain, a basic (B) domain, a Cdc42/Rac binding (GBD) domain, a polyproline
(SH3-binding) domain, a G-actin binding verprolin homology (V) domain (sometimes referred
as WH2 domain), a central domain (C) and a C-terminal acidic (A) segment (Symons et al.,
1996; Zigmond, 2000). The GBD domain of WASP interacts with the GTP-bound form of
Cdc42 and Rac (Symons et al., 1996), key regulators of the actin cytoskeleton (Hall, 1998).
SCAR/WAVE family proteins possess a C-terminal VCA domain similar to WASP and N-
WASP, but have a long N-terminal domain lacking the WH1 and GBD domains (Bear et al.,
1998a; Marchand et al., 2001; Miki et al., 1996). WASP, N-WASP, and SCAR/WAVE have
been shown to activate the Arp2/3 complex, via C-terminal A segment, to stimulate actin
polymerization (Machesky and Insall, 1998; Machesky et al., 1999; Rohatgi et al., 1999).
Biochemical and structural data suggests that WASP is held in an inactive configuration through
the binding of an autoinhibitory domain that overlaps with the GBD to the C region of the VCA
domain (Prehoda et al., 2000). WASP is activated by signaling pathways through the binding of
Rac/Cdc42-GTP to the GBD, resulting in a conformational change, which allows the VCA
domain to activate the Arp2/3 complex(Kim et al., 2000). The presence of a GBD domain and
the interaction between WASP and Arp2/3 suggest that signaling pathways may regulate the
spatial and temporal control of WASP function, in part, through receptor activation of
Cdc42/Rac leading to actin cytoskeleton reorganization. Previous studies have suggested WASP
changes its conformation from auto-inhibited to active upon binding of activated Cdc42
(Cdc42GTP) and other ligands, such as PI(4,5)P2 (Higgs and Pollard, 2000; Rohatgi et al., 2000).
However, in order to better understand WASP function, we require a dynamic in vivo
system in which spatial and temporal regulation of WASP function in a biological response can
be dissected. In vivo studies suggest that WASP is involved in controlling directional motility
during chemotaxis and the spatial organization of F-actin in T-cell activation. Chemotaxis of
macrophages from WAS patients is significantly impaired, whereas the speed of random motility
of both cell types was found to be indistinguishable from that of control cells (Altman et al.,
1974; Ochs, 1998a; Zicha et al., 1998). Monocytes from WAS patients do not effectively
polarize and also have severely impaired migration in response to a variety of chemotactic agents
(Badolato et al., 1998; Zicha et al., 1998). To better understand the role WASP in directional
movement, we have examined spatial and temporal regulation of WASP function in
Dictyostelium. Through the analysis of WASP mutants, we demonstrated that WASP is required
for cellular polarity and motility during chemotaxis of Dictyostelium. We demonstrated that
WASP localization can be differentially regulated by the interaction of the B domain with
PI(4,5)P2 and PI(3,4,5)P3. Further, we provide evidence that WASP localization to the leading
edge requires the basic domain interacting with PI(3,4,5)P3 , the product of PI3Kinase. We
suggest that the spatial and temporal control of WASP localization and activation is essential for
the regulation of directional motility.
Dictyostelium WASP was identified by yeast two-hybrid screen with constitutively active
HsCdc42 as bait as described in the previous study (Chung et al., 1998). The WASP ORF
encodes a protein of 399 amino acids (Figure 1A; GenBank accession no. AAG24442; locus
name: wasA). Direct pair-wise comparison with human WASP indicates that two proteins are
32% identical and 44% similar. Multiple sequence alignments with WASP from other species
indicates that WASP has similar domain architecture, as depicted in Figure 1B, to that of
mammalian WASP. Northern blot analysis shows that WASP expression is developmentally
regulated with peaks of expression during aggregation and multicellular stages, suggesting a role
for cellular motility during aggregation and morphogenesis in the multicellular stages of
development (Fig. 1C).
WASP deletion mutants
Southern blot analysis revealed two copies of genes encoding WASP in the Dictyostelium
genome (data not shown). To investigate WASP function, we attempted to generate a wasp null
mutant. We have not been able to obtain a null mutant (knockout of both copies of the gene)
using standard homologous recombination approaches, possibly because such a strain may
exhibit severe growth defects and thus be selected against using the standard protocol. The level
of WASP expression in cells stably expressing WASP antisense RNA is normally 20-40% of that
of wild-type cells and we have been unable to obtain stable antisense cell lines exhibiting less
WASP mRNA expression (data not shown). However, we were able to disrupt one of the copies
of the WASP gene by homologous recombination, and analysis of this hypomorphic strain
(WASPhypo) has provided us with direct evidence for involvement of WASP in the in vivo
regulation of F-actin assembly and cell motility during chemotaxis (see below). To acquire a
strain that has a very low level of WASP expression, we made a knockin construct in which the
level of WASP expression is regulated transcriptionally by a tetracycline (Tet)-regulated
promoter/transcription activator combination (Tet-Off TA; tTA)(Blaauw et al., 2000; Funamoto
et al., 2002). This construct was used to create a knock-in strain by replacing intact WASP gene
with WASP under the control of tTA in the single WASP gene null background (WASPTHY
starin). WASP knockin cells (WASPTK) express a very low level of WASP transcript in the
absence of the Tet-off TA transcription activator (Fig. 1D).
Aberrant F-actin organization and loss of polarity in cells expressing low levels of WASP
To examine whether reduced expression of WASP has an impact on the actin
cytoskeleton, we stained for actin filaments of aggregation-competent WASPhypo cells under the
cAMP gradient using rhodamine-phalloidin staining. The actin cytoskeleton controls the overall
structure of cells and is highly polarized in chemotaxing cells, with F-actin localized
predominantly in the anterior leading edge and to a lesser degree in the cell’s posterior (Firtel
and Chung, 2000; Gerisch et al., 1995; Parent and Devreotes, 1999; Westphal et al., 1997).
Wild-type cells are well polarized and show localized F-actin assembly at lamellipodia of the
leading edge and, to a lesser degree, at the posterior cortical region of the retracting cell body
(uropod) (Fig. 2A). WASPhypo cells appear elongated and polarized but show significantly less
F-actin at the leading edge. Moreover, F-actin at the uropod is almost not detectable. WASPTK
cells exhibited even stronger defects in F-actin organization. These cells exhibit neither a
prominent F-actin-enriched lamellipod nor cell polarity and were not elongated, presumably due
to the lack of polarized organization of F-actin. Some of WASPTK cells seem to maintain lower
but polarized distribution of F-actin. To determine if this polarized distribution is in the direction
of cAMP gradient, we measured fluorescence intensity by performing linescan of images in the
direction of the gradient and determine ratio of intensity of F-actin staining at the foremost part
(front) or rear end (back) of cells divided by the intensity of the center of cells. Wild type cells
showed 5 times higher F-actin staining at front whereas WASPTK cells showed virtually same
intensity of the center of cell, indicating the polarized distribution of F-actin, if any, is not in the
direction of cAMP gradient.
WASPTK cells were tested for in vivo actin polymerization responses to cAMP
stimulation. Wild-type cells show a rapid and transient increase of F-actin assembly (~70-90%
increase) by 10 seconds after cAMP stimulation (Fig. 2B), as previously described (Hall, 1989;
Zigmond et al., 1997). WASPTK cells have a moderately lower level of F-actin assembly in
unstimulated cells. Upon chemoattractant stimulation, these cells show a significantly reduced
level of actin assembly, which is presumably due to the lower expression of WASP.
To investigate spatial regulation of F-actin organization in living cells, we used the green
fluorescent protein-actin-binding domain (GFP-ABD) probe (Pang et al., 1998) that is a fusion of
EGFP and the actin-binding domain from ABP-120, the major F-actin cross-linking protein in
Dictyostelium cells (Bresnick et al., 1990). The fusion protein binds specifically and dynamically
to the same F-actin structures that phalloidin recognizes in fixed cells. In chemotaxing wild-type
cells, prominent accumulation of GFP-ABD was observed at the leading edge and uropod (Fig.
2C). In addition, there was a moderate localization of GFP-ABD along the entire cell cortex.
However, in WASPTK cells, GFP-ABD is more diffusely distributed, and no polar distribution is
observed, consistent with the results of the phalloidin staining shown in Fig., 2A. More
importantly, as shown in Fig. 2D, WASPTK cells show a greatly decreased number of free barbed
ends relative to wild-type cells, as might be expected due to the lack of WASP. In aggregation-
competent wild-type cells, the distribution of free barbed ends was very polarized. In WASPTK
cells, the labeling of free barbed ends is greatly reduced and is not localized. This observation is
consistent with our findings in phalloidin staining and in agreement with models suggesting that
WASP is required for polarized F-actin assembly in migrating cells.
Localized myosin II assembly is key in maintaining cell polarity and cortical tension and
retracting the posterior cell body (Clow and McNally, 1999; Egelhoff et al., 1996; Sanchez-
Madrid and del Pozo, 1999; Stites et al., 1998). PAKa plays important roles for the regulation of
cell polarity, chemotaxis, and cytokinesis in Dictyostelium by controlling myosin II assembly
(Chung and Firtel, 1999). Our previous study demonstrated that the N-terminal domain of PAKa
is necessary and sufficient for PAKa’s localization to the posterior cortex of polarized,
chemotaxing cells (Chung and Firtel, 1999; Chung et al., 2001). We used a GFP fusion of N-
PAKa (N-PAKa-GFP) as a reporter to examine axial polarity of cytoskeleton. Wild-type cells
showed biased distribution of N-PAKa-GFP at the uropod in chemotaxing cells, as expected
(Fig. 2E). When this polarity is disrupted by globally stimulating cells (bathing the cells in
cAMP to simultaneously activate all of the cell-surface cAMP receptors), N-PAKa-GFP
becomes uniformly distributed around the cells’ periphery. As the rounded morphology of
WASPTK might predict, N-PAKa-GFP is distributed more uniformly along the cortex of these
cells compared to wild-type cells, indicating that establishment of cytoskeleton polarity is
significantly impaired in WASPTK cells.
Chemotaxis of WASP mutants
To test whether the changes in the actin cytoskeleton described above alter
chemoattractant-induced cell migration, we employed a chemotaxis assay combined with time-
lapse video microscopy (Fig. 3). Wild-type cells are usually well polarized, move quickly and
linearly toward the cAMP source, extend pseudopodia predominantly in the direction of cAMP
gradient, and produce very few random lateral or rear pseudopodia. Cells of WASPhypo strain
exhibit less vigorous pseudopodia extension and do not appear to retract the uropod as efficiently
as wild-type cells (Fig. 3A). Pseudopod extension and uropod retraction of wild-type cells are
well coordinated so that the length of cell body of migrating cells is relatively constant (Fig. 3C).
WASPhypo cells appear defective in this process, which is consistent with their lack of F-actin
assembly at the uropod (Fig. 2A). The rear cell body of the WASPhypo cells does not retract
actively whereas pseudopod extension is not largely impaired. This results in a very elongated
cell body and greater variations in cell body length as depicted in Figure 3B. Mechanical tension
caused by extension of pseudopod forced the posterior of WASPhypo cells to become detached
from the substratum and contracts, resulting in abrupt shortening of the length of cell body (Fig.
3C). WASPTK cells have even greater defects in chemotaxis. They are very flattened and round
as they are unable to establish polarity (Fig. 3A). Some of WASPTK cells contain more than one
nucleus (not shown), indicating growth defects. In the presence of chemoattractant, small
portion of WASPTK cells (30%) appear to develop a pseudopod slowly in the direction of the
chemoattractant gradient but the remainder of the cell body does not polarize (Fig. 3A), resulting
in very slow movement with a higher angular deviation due to frequent change of the direction of
movement. They move at a speed of 2-3 µm/minute, four times slower than wild-type cells (8-
10 µm/min) as shown in Fig. 3D. WASPTK cells showed significantly lower chemotaxis index
than wild type cells, suggesting that their defects are due to the lack of persistence. The speed
and angular deviations were recovered by the expression of tTA, a chimeric tetracycline-
controlled transcriptional activator protein that results in higher expression of WASP. These
cells also exhibited recovery of cellular polarity (Fig. 3A and D), indicating the necessity of
WASP for cell motility and polarity. Inability of WASPTK cells to chemotax efficiently was
clearly manifested in the traces of cells migrating toward cAMP source (Fig. 3B). Wild type
cells chemotax in a directed manner, showing straight paths of movement whereas WASPTK cells
show unbiased movement indicated by higher angular deviation. To determine if the chemotaxis
defects are due to a general lack of responsiveness to the chemoattractant, we examined the
expression of cAR1, the major cAMP chemoattractant receptor that regulates aggregation. The
expression of cAR1 was normal (not shown). To ensure that other signaling pathways are not
altered, we examined the translocation of AktPH-GFP to the membrane upon cAMP stimulation
and the kinetics of translocation in WASPTK was the same as in wild type cells (Fig. 3E),
indicating that pathways activating PI3 kinase is intact. Combined with normal cAR1
expression, this result suggests that other signaling pathways are not altered.
Motility defects of cells expressing low level of WASP during multicellular development
plated on non-nutrient agar and development was examined (Fig. 4). WASPhypo cells exhibit a
To examine the effect of disrupting WASP on multicellular development, cells were
very significant motility defect during morphogenesis, remaining at the mound stage three times
longer than wild-type cells. At the mound stage, the prestalk cells sort from the prespore cells by
chemotaxis and differential cell adhesion, forming at tip that elongates to form the migrating
slug(Firtel and Meili, 2000; Weijer, 1999). We expect the delay at the mound stage to be due to
motility defects of WASPhypo cells. Similar morphogenesis defects have been demonstrated for
other mutants affecting the actin-myosin cytoskeleton. The delay of development probably
results from the abnormal regulation of the cytoskeletal organization, which would affect cell
movement during chemotaxis and late development. At the mound stage cells differentiate into
different cell types and sort themselves out to specific area of the mound due to differential
adhesion and motility. The motility defect of WASPhypo cells probably delays this cell sorting,
which presumably prolongs the mound period. When mixed with wild-type cells, the WASPhypo
cells initially distribute randomly in the mound. WASPhypo cells do not actively migrate into the
stream of cells rotating in the mound, resulted in the exclusion of WASPhypo cells to the mound
periphery (Fig. 4B). The motility defects are more significant in WASPTK cells as they do not
aggregate when starved on non-nutrient agar plates. Defects of WASPTK cells can be rescued by
the ectopic expression of GFP-WASP as shown in Fig. 4A. WASPTK cells do not effectively
participate in the aggregation when they are mixed with wild-type cells and are excluded from
the mound as morphogenesis ensues (Fig. 4C). Wild type cells in the mound are well elongated
and polarized, but majority of WASPTK cells are neither present in the mound nor well polarized,
also indicating that WASPTK cells do not actively chemotaxing. As the expression of cAMP
receptor is normal and other signaling pathways are intact, we conclude that the failure to
aggregate results from defects in chemotactic motility, not from an inability to respond to the
Subcellular localization of WASP in migrating cells
To determine if there is a dynamic subcellular localization of WASP in response to
chemoattractant stimulation, we fused GFP to the N-terminus of full-length WASP, separated by
a flexible linker Gly-Ser-Gly-Ser-Gly. We examined cells stably expressing moderate levels of
GFP-WASP (GFP-WASP/WASPhypo) and found that GFP-WASP is transiently concentrated at
the leading edge and, to a lesser extent, at the uropod of Dictyostelium cells migrating toward a
chemoattractant gradient (Fig. 5A). This subcellular localization is consistent with the
organization of the actin cytoskeleton indicated by phalloidin staining in migrating cells. The
enrichment of GFP-WASP at the leading edge of chemotaxing cells suggests that cAMP-
activated signaling pathways may regulate WASP translocation to the leading edge that, in turn,
stimulates F-actin assembly.
The basic domain of WASP might be a major determinant for WASP localization
The biased localization of WASP at the leading edge and uropod raises two major
questions. First, is spatial localization of WASP directly correlated with the stimulation of F-
actin nucleation? We attempted to answer this question by simultaneously monitoring WASP
localization and F-actin assembly in live cells. We fused spectrally distinct variants of the green
fluorescent protein (GFP), YFP and CFP, to WASP and to coronin respectively, and co-
expressed them in WASPhypo cells. By using a high-speed excitation filter wheel, we acquired
differential images of the two reporters with time-lapse microscopy. Coronin is a conserved
component of the actin cytoskeleton found in all eukaryotes examined from yeast to mammals
and binds specifically to F-actin and bundles of actin filaments and localizes to sites of dynamic
F-actin assembly (Asano et al., 2001; de Hostos, 1999). Coronin has been used as a reporter for
F-actin distribution in live cells (Gerisch et al., 1995). As shown in Figure 5C, newly formed
pseudopodia are initially enriched with YFP-WASP and preceded the localization of CFP-
coronin, consistent with F-actin assembly at the leading edge being downstream from the
localization and activation of WASP. The second question is: what are the determinants for
WASP localization? Studies suggested that the activity of PI3 Kinase is essential for the
polarized F-actin organization in migrating cells (Devreotes and Janetopoulos, 2003; Merlot and
Firtel, 2003; Rickert et al., 2000). The WH1 and B (basic) domains of N-WASP have been
shown to mediate binding phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) (Imai et al., 1999;
Miki et al., 1996), suggesting that these domains might be responsible for targeting WASP to the
membrane via the interaction with the B domain. GFP-WASP lacking the WH1 and B domain
displays a diffuse distribution in chemotaxing cells, suggesting that these domains are required
for the dynamic regulation of WASP localization in moving cells (Fig. 5A). We then expressed
the GFP-fused WH1 domain and B domain linked to the GBD (GTPase binding domain) (B-
GBD) individually in wild-type cells to determine whether they have similar distribution to
WASP. GFP-WH1 distributes uniformly in the cell, whereas GFP-B-GBD shows polarized
distribution to the leading edge and uropod, indicating that the B domain is a major determinant
for WASP localization. The presence of GBD domain might be required, but not sufficient for
the localization of WASP. Interestingly, GFP-B-GBD appeared to be associated with vesicle-
like structures as revealed by punctate pattern of GFP signal at the leading edge and uropod,
which is different from more diffuse distribution of GFP-WASP. To test if more diffuse
distribution of GFP-WASP is due to higher expression level, we expressed GFP-WASP in
WASPTK cells. GFP-WASP still shows biased distribution at the leading edge and uropod, but
GFP-WASP indeed showed punctate vesicle localization at the leading edge and uropod (Fig.
5B). Higher expression of GFP-WASP in WASPTK cells showed more diffuse distribution
presumably because binding sites on vesicles are saturated.
The basic domain has a broader specificity to phosphoinositides
Previous reports have suggested that the B domain is important for binding PI(4,5)P2,
which is thought to be involved with relieving the autoinhibited conformational state of WASP
(Higgs and Pollard, 2000; Prehoda et al., 2000; Rohatgi et al., 2000). However, the range of
phosphoinositide species capable of binding this domain has not been determined thoroughly.
To gain a better understanding of the profile of phosphoinositides capable of binding the B
domain of WASP, we used a protein–lipid overlay assay previously used to determine specific
phosphoinositide binding by the pleckstrin homology (PH) domain (Deak et al., 1999). This
takes advantage of commercially available PIP-Strips (Echelon, UT), which consist of
phospholipids immobilized onto nitrocellulose. The PIP strips were incubated with purified
GST-B-GBD or GST-WH1-B-GBD fusion proteins and the binding was determined by
conventional Western blotting with an anti-GST antibody. As a positive control, we used GST-
fusion of PH domain of human PLCδ1 which has been known to have a strong specificity to
PI(4,5)P2 and it showed strong preference to PI(4,5)P2. Both WH1-B-GBD and GST-B-GBD
fusion proteins showed significant binding to three phosphatidylinositol lipids, PI(3,4)P2,
PI(3,5)P2 and PI(3,4,5)P3, but moderate binding to PI(4,5)P2 (Fig. 6A). The absolute binding
intensity of GST-B-GBD to PI(3,4,5)P3 is similar to that of PLCδPH to PI(4,5)P2. However,
GST-B-GBD had higher background binding to membrane than PLCδPH, presumably due to the
difference of affinity. To show specific binding intensity, we subtracted the background
intensity from the absolute intensity, which results in relatively low binding intensity of GST-B-
GBD. GST and GST-WH1 did not show specific binding to any of phospholipids on the strip
(data not shown). The binding of GST-B-GBD protein to PI(3,4,5)P3 was also confirmed by a
protein pull-down experiment using PIP beads (Echelon, UT). GST-B-GBD showed binding to
PI(3,4,5)P3-beads with slightly higher affinity than to PI(4,5)P2-beads whereas control beads
show minimal binding (Fig. 6B). To verify specific binding of phosphoinositides to the B-GBD
domain of WASP in a more physiological condition, we performed a liposome co-sedimentation
assay with purified GST-YFP-BG protein and liposomes composed of 95% of
phosphatidylcholine (PC) and 5% of either PI, PI(4)P, PI(4,5)P2, or PI(3,4,5)P3. Binding of the
B-GBD domain to a specific phosphoinositide would result in the sedimentation of a protein-
liposome complex that can be recovered at a higher concentration of Optiprep (Greiner Biotech
Co.) fractions. GST-YFP-BGBD proteins and liposomes were mixed and subjected to
centrifugation on a Optiprep gradient (5-30% Optiprep). Fractions were collected and resolved
on SDS-PAGE gel and GST-YFP-B-GBD protein was probed by a western blot. As presented in
Figure 6C, GST-YFP-B-GBD protein mixed with liposome composed entirely of PC or
liposomes containing 5% PI and PI(4)P was found in lower Optiprep concentration fractions,
indicating little binding of B-GBD to PC, PI and PI(4)P. With liposomes containing 5%
PI(3,4,5)P3 and PI(4,5)P2, GST-YFP-B-GBD was found at the higher (~30% Optiprep)
concentration of gradient, indicating that B-GBD domain bound with higher affinity to
PI(3,4,5)P3 and PI(4,5)P2. Ovalbumin was added in the binding mixture to block non-specific
binding and it also serves as an internal control for lower concentration of Optiprep fraction. We
quantified specific binding efficiency by summing of protein band intensity in the higher
fractions than fractions where ovalbumin was present (Fig. 6C). The binding efficiency of B-
GBD domain was almost equal to PI(3,4,5)P3 and PI(4,5)P2 and was significantly higher than to
PI, PI(4)P, and PC. We examined the binding specificity of GST-WASP to phosphoinositides
and also found preference to PI(3,4,5)P3 or PI(4,5)P2 but not as significant as the B domain.
This is presumably due to the autoinhibitory conformation of WASP requiring Cdc42 or Rac to
bind to the GBD domain for full activation. Together these experiments suggest that the B
domain has a more diverse phospholipid binding profile than implied by previous reports and
that the interaction of the B domain with PI(3,4,5)P3 is important for the localization and
activation of WASP to the leading edge of chemotaxing cells. To confirm if PI3K is required for
the localization of WASP and F-actin polymerization at the leading edge, we used the PI3K
inhibitor LY294002 and examine the localization of GFP-coronin and YFP-WASP. Prior to
LY294002 addition, GFP-coronin, and therefore F-actin, is highly localized to the leading edge
of chemotaxing cells (Figure 6D). Addition of LY294002 to polarized cells results in a loss of
the polarized actin cytoskeleton as determined by a rapid loss of the localization of GFP-coronin.
Similarly, addition of LY294002 to polarized cells cause rapid loss of the polarized distribution
of GFP-WASP and uniform distribution of GFP-WASP in the cytoplasm, suggesting that PI3K
activity is essential for maintaining polarized F-actin assembly and WASP localization. It should
be noted that the redistribution of WASP upon LY294002 treatment is slower than that of
coronin, which might be due to the association of WASP with vesicles that might be tethered to
microtubule or diffusing significantly slower. These results strongly suggest that the interaction
of the B domain with PI(3,4,5)P3 is important for the localization and activation of WASP to the
leading edge of chemotaxing cells. Interactions of B domain with PI(4,5)P2 and PI(3,4,5)P3 in
lipid binding assays prompted us to examine the co-localization of YFP-B-GBD with CFP-
PLCδ1PH, a probe for PI(4,5)P2 in chemotaxing cells by performing time-lapse microscopy
(Fig. 7A). Surprisingly, CFP-PLCδ1PH primarily showed punctate vesicle labeling similar to
localization of YFP-G-GBD and CFP-PLCδ1PH and YFP-G-GBD appeared to co-localize on
many vesicles. The distribution of these PIP2-enriched vesicles somewhat polarized as vesicles
mainly showed biased distribution at the leading edge and uropod area, but few vesicles labeled
with CFP-PLCδ1PH can be found at the proximity of the leading edge membrane. However,
YFP-B-GBD signal was found at the leading edge and not co-localized with CFP-PLCδ1PH. To
determine if YFP-B-GBD localizes to the PI(3,4,5)P3-enriched membrane, we examined the
colocalization of YFP-B-GBD and CFP-PhdA which selectively binds to PI(3,4,5)P3 (Funamoto
et al., 2001) (Fig. 7B). CFP-PhdA temporarily localizes to the leading edge of chemotaxing cells,
but hardly showed association with intracellular vesicles. Most of YFP-B-GBD signal were
associated with vesicles, but some of YFP-G-GBD signal clearly co-localizes with CFP-PhdA at
the leading edge, indicating that the B domain is recruited to the membrane where local
PI(3,4,5)P3 concentration is elevated by the activation of PI3 kinase.
The B domain of WASP is highly enriched in positively charged Lys residues. To
determine which residues might be important for phospholipid binding, we mutated these Lys to
Ala residues and the specificity of each mutant was tested with the lipid overlay assay (Fig. 7C).
Surprisingly, only two mutations significantly hampered the binding of GST-B-GBD to
phosphoinositides. Mutation on Lys156 to Ala appears to be important for binding all three
phosphoinositides tested (PI(3,4)P2, PI(4,5)P2, PI(3,4,5)P3) while mutation of lysine 157 reduces
binding to those lipids with a phosphate at the 3’ position (PI(3,4)P2 and PI(3,4,5)P3),
suggesting Lys157 is important for an interaction with 3’-position phosphate . The selective
reduction of this mutant to phosphoinositide with the 3’ position phosphate further validates the
specificity of binding of the B domain to PI(3,4,5)P3.
Since mutations on Lys157A specifically reduced the binding of B domain to PI(3,4,5)P3,
we compared the intracellular localization of the YFP-B-GBD with the YFP-B157A-GBD mutant
to determine whether Lys157 residue is important for the localization of YFP-B-GBD to the
leading edge. Highly polarized, chemotaxing cells exhibit YFP-B-GBD localization at the
leading edge. Mutation of Lys157 to Ala appears to decrease YFP-B-GBD localization at the
leading edge of polarized cells, but did not significantly affect the association of YFP-B-GBD
with intracellular vesicles (Fig. 7D). YFP-B156,157A-GBD showed essentially the same
distribution as YFP-B157A-GBD. These results strongly suggest that the interaction of the B
domain with PI(3,4,5)P3 is important for the localization and activation of WASP to the leading
edge of chemotaxing cells. We further elucidate the functional significance of these mutations in
the B domain by determining if WASP carrying these mutations is able to rescue the phenotype
of WASPTK. K156AWASP expressed in WASPTK minimally rescued chemotaxis defects (Fig. 7E)
but K156,157AWASP failed to rescue the phenotype of WASPTK. YFP-K156AWASP did not show
any preferential localization in the cell nor showed punctate vesicular distribution.
WASP subdomain deletion mutants
We created deletion mutations that are expected to abrogate the function of specific
domains of WASP. Cells overexpressing the WASP VCA domain, which stimulates F-actin
nucleation activity in vitro in association with the Arp2/3 complex(Machesky and Insall, 1998),
are not well-polarized compared to wild-type cells and do not exhibit a prominent F-actin-rich
cortical region with phalloidin staining of starved cells (Fig. 8A). This presumably results from
the non-localized and unregulated nucleation of F-actin. This result is consistent with a previous
study demonstrating that overexpression of carboxy-terminal fragments of SCAR1 or WASP in
cells caused a disruption in the localization of the Arp2/3 complex and, concomitantly, induced a
complete loss of lamellipodia and actin spots (Machesky and Insall, 1998). The poly-proline
domain of mammalian WASP binds a number of SH3 domain-containing proteins(Badour et al.,
2003). Mutations within this domain of human WASP result in a severe WAS phenotype(Ochs,
1998), indicating that the poly-proline region plays an important role in the regulation of WASP
function. Dictyostelium cells expressing a WASP construct lacking the whole polyproline region
and the V domain (WASP∆Pro-V) exhibit no polarized F-actin organization and up-regulated
cortical F-actin filaments along the periphery of cells under the gradient (Fig. 8A). These cells,
not unexpectedly, have severe defects in the ability to polarize and chemotax in a response to a
chemoattractant gradient (Fig. 8B). Cells overexpressing WASP∆Pro-V do not polarize nor
extend pseudopodia effectively, probably because they lack dynamic regulation of the F-actin
cytoskeleton. In contrast, cells expressing WASP lacking the last two polyproline repeats and
the V domain (WASP∆V) have polarized F-actin organization and extend pseudopodia toward
the cAMP gradient and show some polarization, although not to the extent as wild-type cells
(Fig. 8A and B). Defects of chemotactic movement of these mutants are also manifested in the
development of these mutants (Fig. 8C). Cells expressing WASP∆Pro-V did not aggregate, even
after 24 hours, consistent with an inability to chemotax. However, cells expressing WASP∆V
aggregate through limited chemotaxis and cell-cell adhesion with neighboring cells to form many
small mounds, which eventually develop into small fruiting bodies. These results suggest that
the poly-proline region of WASP and its presumed interaction with SH3 domain-containing
proteins might play an important role in controlling WASP function and localization and thus,
polarized F-actin organization.
The actin cytoskeleton controls the overall structure of cells and is highly polarized in
chemotaxing cells, with F-actin localized predominantly in the anterior leading edge and to a
lesser degree in the cell’s posterior (Borisy and Svitkina, 2000; Firtel and Chung, 2000; Gerisch
et al., 1995; Parent et al., 1998). In the present study, we demonstrated the specific and essential
role of WASP in organizing polarized F-actin assembly in chemotaxing Dictyostelium cells.
WASPTK cells, which express very low levels of WASP, show reduced F-actin levels in
unstimulated cells and significant defects in polarized F-actin assembly in migrating cells in
response to chemoattractant stimulation. These cells are unable to establish axial polarity in the
chemoattractant gradient and their motility is also severely impaired. The lack of an F-actin-
enriched lamellipodia and uropod in WASPTK cells in a cAMP gradient indicates that WASP is
essential for polarized assembly of F-actin. Our studies are consistent with the findings that
macrophages from WAS patients are less persistent in their locomotion and engage in more
frequent turning behavior compared with normal counterparts (Thrasher et al., 2000). Lack of
cell polarization and maintaining directional cues in WAS macrophage may underlie the failure
of these cells to migrate normally. Induction of monocyte chemotaxis by chemokines is
preceded by cell polarization, marked by several morphologic changes, including the formation
of cell protrusions and pseudopodia. Staining of F-actin in monocytes from WAS patients
stimulated with chemoattractants shows a severe defect in cell polarization after stimulation with
either FMLP or MCP-1 compared with the response of monocytes from normal donors (Badolato
et al., 1998). Bee1, a yeast homologue of WASP, has been implicated in the regulation of
cellular polarity as it is involved in the formation of actin patches. The lack of cortical patches in
bee1 cells suggests that disruption of cortical patches slows down polarized cell surface growth
and cytokinesis (Li, 1997). IL-4 and anti-CD40 induction of B cell polarization from WASP
deficient mice was impaired in comparison to WASP+ cells(Westerberg et al., 2001).
Interestingly, N-WASP was clearly detected in both WASP+ and WASP- B cells, in similar
concentrations, indicating that they regulate different processes despite the high degree of
Our data also suggest that WASP and SCAR might be controlled by different signaling
pathways, leading F-actin assembly for different cellular processes. WASP might be more
important in the regulation of cell polarity than SCAR since WASPTK cells showed significant
lack of cell polarity and aggregation while scar null cells become polarized, roughly orienting
towards the aggregation center and forming visible aggregation streams (Bear et al., 1998).
Dictyostelium WASP is more closely related to the mammalian WASP that is only expressed in
hematopoeitic cells. It is conceivable that WASP function in highly motile cells such as
Dictyostelium, neutrophils, and macrophages is more essential than SCAR in order to provide
highly dynamic regulation of actin cytoskeleton. Thus, lack of WASP function might cause
more severe phenotypes in highly motile cells. Recent studies in Drosophila have shown that
WASP is largely dispensable for Arp2/3-dependent control of dynamically regulated actin
structures including cortical F-actin, whereas SCAR does not contribute to cell fate decisions in
which WASP and Arp2/3 play an essential role(Zallen et al., 2002). Further, it has been
demonstrated that the VCA domain of SCAR shows different potency in stimulating Arp2/3 than
that of WASP or N-WASP, at least in in vitro systems. In a bead motility assay, the minimal
region of WASP sufficient to direct movement of beads was the C-terminal VCA fragment,
whereas the corresponding region of SCAR1 was insufficient (Yarar et al., 2002). VCA domains
of WASP, N-WASP, and SCAR1 were found to bind actin and Arp2/3 with nearly identical
affinities but to induce unique kinetics of actin assembly (Zalevsky et al., 2001). N-WASP and
WASP induce rapid actin polymerization, while SCAR1 either fails to induce detectable
polymerization or to induce the slowest rate of nucleation. Based on these results, it is
conceivable to expect WASP or N-WASP to be the primary regulators of dynamic F-actin
assembly. It is clear that differential activation of Arp2/3 complex would be required for
regulation of cellular processes as it has been suggested that diffrent rates of filament formation
may help determine the architecture of actin networks produced by different nucleation-
promoting factors(Zalevsky et al., 2001).
Our data are the first demonstration that GFP-WASP preferentially localizes at the
leading edge and uropod of chemotaxing cells and B domain is required for the localization of
WASP via binding to phosphoinositides. We show that WASP lacking the WH1 and B domains
is cytosolic as is a GFP-WH1 domain fusion. We further show that a GFP-B-GBD fusion shows
the same leading edge and to a lesser degree uropod localization, as does full-length WASP.
Combined, these data are consistent with an essential role for the B domain in the localization.
The subcellular localization of WASP is consistent with the organization of the actin
cytoskeleton shown by phalloidin staining and localization of CFP-coronin in migrating cells. It
was rather unexpected to observe that either GFP-WASP or YFP-B-GBD are associated with
vesicles that can be labeled with CFP-PLCδ1PH, indicating that vesicles are enriched with
PI(4,5)P2. The recruitment of N-WASP to the PI(4,5)P2-enriched vesicles has been previously
demonstrated and mutational analysis of N-WASP has demonstrated that WH1, B, and
polyproline domains are capable of interaction with the PI(4,5)P2 vesicles(Benesch et al., 2002).
The vesicular targeting of the polyproline domain was suggested to be mediated through the
interactions with SH3 domain-containing proteins. The recruitment of GFP-WASP to
intracellular vesicles in Dictyostelium might also require SH3 domain-containing proteins as N-
WASP requires Nck and WIP. However, B domain must play a major role for vesicle
recruitment since we observed the association of YFP-B-GBD with vesicles. It is not clear how
chemotaxing cells control biased distribution of these vesicles.
Previous studies demonstrated that the B domain of WASP interacts with PI(4,5)P2
(Higgs and Pollard, 2000; Rohatgi et al., 2000), resulting in the activation of N-WASP by
changing its conformation from auto-inhibited to active (Prehoda et al., 2000). The auto-
inhibited conformation is acquired and maintained by the binding of the autoinhibitory region
near the GBD domain to C region of the VCA domain. However, subcellular distribution of
PI(4,5)P2 has not been extensively examined in chemotaxing cells, but it has been suggested that
PI(4,5)P2 acts permissively, restricting new F-actin polymerization to the region of the plasma
membrane (Insall and Weiner, 2001). In the present study, we demonstrated that the B domain
has an equal or higher affinity to PI(3,4,5)P3 than PI(4,5)P2 in lipid binding assays and that a
mutation on Lys157 residue in the B domain specifically reduces the binding to PI(3,4,5)P3 and
the localization of the B domain to the leading edge membrane without hampering the
association of YFP-B-GBD with vesicles enriched with PI(4,5)P2. Our results suggest a very
interesting possibility that B domain direct the localization of WASP to vesicles via interaction
with PI(4,5)P2, but the interaction of the B domain with PI(3,4,5)P3 is required for the
localization of WASP to the leading edge where vigorous F-actin polymerization occurs. In a
previous study, it has been shown that inhibiting PI3K in polarized, chemotaxing cells through
the addition of LY294002 results in a loss of the polarized actin cytoskeleton (Chung et al.,
2001). Reduced polarized F-actin organization in cells lacking two of the five Class I PI3K
isoforms in Dictyostelium pi3k1/2 null cells or cells treated with LY294002 (Chung et al., 2001;
Funamoto et al., 2001) (K. Takeda and RAF, unpubl. data) is consistent with PI3 kinase playing
an important role in the regulation of the actin cytoskeleton in chemotaxing cells, possibly
through the control of WASP. PI(3,4)P2 and PI(3,4,5)P3 produced by PI3 kinase activity
function as docking sites for diverse PH domain-containing proteins(Funamoto et al., 2001;
Meili et al., 1999b; Parent et al., 1998; Servant et al., 2000). We suggest that the localization of
WASP to membrane of the leading edge where active F-actin polymerization occurs requires the
formation of PI(3,4,5)P3-enriched domains, which would provide docking sites for the WASP B
domain. In a recent study, binding of WAVE2 to PI(3,4,5)P3 through its basic domain was
demonstrated to be important for the localization of WAVE2 to the lamellipodia of cells
stimulated with PDGF (Oikawa et al., 2004), also suggesting a comparable linkage between PI3
kinase and regulation of WAVE2 localization. Local activation of WASP at the leading edge
can be achieved by interaction with PI(3,4,5)P3 and/or locally activated Rac molecules. While
previous study (Funamoto et al., 2002) suggested that PI3 Kinase is only enriched at the leading
edge, more recent studies using more sensitive imagining suggest that PI3 Kinase also shows an
enrichment in the uropod, although the level of PI3 Kinase in the uropod is significantly less than
at the leading edge (S. Lee and RAF, unpubl. obser.). Thus, PI3 Kinase may also control WASP
localization to the uropod as well as to the leading edge. Both in Dictyostelium and neutrophil,
signaling asymmetry including the activation of PI3 kinase can be achieved in the absence of F-
actin polymerization(Parent et al., 1998; Wang et al., 2002). We demonstrated that the
translocation of Akt/PH-GFP to the membrane of WASPTK cells upon cAMP stimulation and the
kinetics of translocation was not altered, indicating that signaling pathways for polarity are
intact. Reciprocal interplay between PI(3,4,5)P3 and actin polymerization appears to be required
for maintaining the asymmetry of intracellular signals responsible for cell polarity and directed
motility as amplification of the internal PtdIns(3,4,5)P3 gradient is markedly impaired by agents
inhibiting actin polymerization(Wang et al., 2002). Thus, polarity defects of WASPTK cells
might be caused by the defects in persistence in cell polarity due to the lack of the sustained
stimulation of F-actin polymerization. This hypothesis is consistent with our observation that the
interaction of the B domain with PI(3,4,5)P3 is required for the targeting of WASP to the leading
The presence of a poly-proline region in all WASP family proteins (WASP, N-WASP,
and SCAR/WAVE) and identification of many SH3 domain-containing proteins as binding
partners for the poly-proline region in mammalian cells suggests that the interaction between the
poly-proline region and SH3 domain-containing protein(s) might play an important role in the
regulation of WASP function and localization. Furthermore, previous studies show that
mutations within the SH3 binding domain of WASP result in a severe WAS phenotype (Ochs,
1998). We demonstrated that cells overexpressing WASP∆Pro-V are not polarized and cannot
extend pseudopodia effectively due to aberrant regulation of F-actin organization, and cells
overexpressing DdWASP∆V exhibited similar but less severe defects in polarity and chemotaxis.
It was unexpected to observe a strong phenotype resulting from these mutants since they cannot
stimulate F-actin nucleation due to the lack of V domain. Mutational analysis demonstrated that
both WH1- and polyproline-dependent interactions of N-WASP with WIP and SH3 domain-
containing proteins contribute to recruitment of N-WASP to the PI(4,5)P2 vesicle surface
(Benesch et al., 2002). In a yeast two-hybrid assay, WASP∆V can interact with SH3 domains of
human Nck that is known to interact with the poly-proline region of WASP (CYC and RAF,
unpubl. obser.), but WASP∆Pro-V cannot. The phenotypic differences between these two
mutants might result from the polyproline repeats interacting with SH3 domain-containing
proteins that are possibly acting as an adaptor for other components required for general
targeting of WASP to the leading edge area. More precise targeting of WASP to the membrane
of the leading edge might be acquired by the interaction of the B domain with PI(3,4,5)P3. It has
been shown that the proline-rich regions of WASP and SCAR1 and the WH1 domain of WASP
independently enhanced motility rates of beads in the beads motility assays (Yarar et al., 2002).
The contributions of these regions to motility could not be accounted for by their direct effects
on actin nucleation with the Arp2/3 complex, suggesting that they stimulate motility by
recruiting additional factors. WASP∆Pro-V and WASP∆V may cause dominant negative effect
due to inability to recruit additional factors or by sequestering signaling proteins in non-
functional complexes. A recent study demonstrated that two of the SH3 domain-containing
ligands, Sla1 and Bbc1, cooperate to inhibit Las17, a yeast homologue of WASP, activity in vitro
and are required for a shared function in actin organization in vivo (Rodal et al., 2003).
Moreover, a recent paper demonstrated that N-WASP predominantly present as an inactive
complex with WIP whose poyproline domain presumably binds to WH1 domain of WASP (Ho
et al., 2004). Dictyostelium has a homologue of WIP (JH and CYC, unpubl. obser.) and
expression of WASP∆Pro-V might disrupt this multiprotein complex resulting in aberrant
regulation of WASP activity.
In the future, it will be important to examine how PI(3,4,5)P3 induce translocation of
WASP from vesicles to the leading edge membrane and how the binding of SH3 proteins to the
polyproline region of WASP affects the localization of WASP to vesicles and the leading edge.
MATERIALS AND METHODS
Cell Culture and Development
Dictyostelium cells were cultured axenically in HL5 medium supplemented with 60 units of
penicillin and 60 µg of streptomycin per ml. For examining developmental phenotypes, cells
were washed twice with 12mM Na/K Phosphate buffer and plated on non-nutrient agar plates.
Dictyostelium WASP was originally identified in a two-hybrid screen with constitutively
active human Cdc42 as a bait. The screening was essentially done as previously described
(Chung et al., 1998). A cDNA containing 1200 bp ORF for WASP was isolated. The plasmid
for homologous recombination was constructed by insertion of Bsr gene cassette into the WASP
open reading frame (ORF) at the BamHI site or THY1 gene at the EcoRI site of WASP genomic
DNA. The Bsr targeting construct was linearized with SacI and XhoI, electroporated into Ax2
wild type cells, and transformants were selected and screened for disruption of WASP by
Southern blot analysis (WASPhypo strain). The THY targeting construct was linearized with
BglII/XhoI, electroporated into the thymidine auxotrophic strain, JH10, and transformants were
selected in the absence of thymidine in the medium and screened for disruption of WASP.
Southern blot analysis showed this strain of cells (WASPTHY) still has a copy of WASP gene not
disrupted and this strain was used to create a knock-in strain.
We used a knock-in approach to acquire a strain that has very low level of WASP
expression. The targeting construct was made by cloning 500 base pair of WASP genomic DNA
encompassing the start codon in SpeI-HindIII site of pBSSK(+). A Bsr gene cassette was cloned
into BamHI site and WASP cDNA fused with YFP at the N-terminus under the control of a
tetracycline-responsive element (TRE) was cloned into EcoRI-XhoI. TRE is the binding site for
a chimeric tetracycline-controlled transcriptional activator protein (tTA). In the absence of
tetracycline, tTA binds to its target sequence and strongly induces gene expression. Tetracycline
prevents tTA from binding to the tetracycline-responsive element, rendering the promoter
virtually silent, enabling us to get a strain expressing very low level of WASP. The targeting
vector was cut with SpeI-XhoI and transformed into WASPTHYcells and positive clones were
selected in the presence of blasiticidine and identified by Southern blot. By homologous
recombination, the knock-in construct replaced the WASP gene with a WASP allele under TRE
regulation. By differing concentration of tetracycline in the medium, we were able to elucidate
defects in actin polymerization and chemotactic motility in different level of WASP expression.
Cells were transformed with a vector encoding RNA complementary to WASP mRNA under the
control of the discoidin promoter that can be negatively regulated by folic acid and turned on by
starvation. The GFP-WASP vector was constructed by cloning the sequence encoding eGFP in-
frame with the entire coding sequence of WASP that was amplified by PCR. The construct
encoded fusion proteins with GFP at the N-terminus, separated by the flexible linker GSGSG
from the entire coding sequence of WASP. The GFP fusion proteins were expressed under
control of the actin-15 promoter using the pEXP4(+) vector.
In vivo actin polymerization assay
F-actin was quantified from TRITC-phalloidin staining of Dictyostelium cells as
described in the previous study (Zigmond et al., 1997). Cells were pulsed with 30 nM cAMP at
6 min intervals for 5 hr. Cells were diluted to 1 X 107 cells/ml and shaken at 200 rpm with 2
mM caffeine for 20 min to synchronize the signaling of the cells. Cells were spun and
resuspended with phosphate buffer (10 mM PO4 buffer, pH 6.1, and 2 mM MgSO4) at 5 X 107
cells/ml and stimulated with 100 µM cAMP. 500 µl of cells were taken at 5, 10, 20, 30, 50,
and 80 second time points and mixed with actin buffer (20 mM KH2PO4, 10 mM PIPES, pH
6.8, 5 mM EGTA, 2 mM MgCl2) containing 6% formaldehyde, 0.15% Triton X-100, 1µM
TRITC phalloidin. Cells were fixed and stained for 1 hr and spun down at 14,000 rpm for 5
min in the microfuge. Pelleted cells were extracted with 1 ml of 100% methanol and
fluorescence was measured (540ex/575em). To determine nonsaturable binding, 100 µM
unlabeled phalloidin was included.
For phalloidin staining, cells were starved in 12 mM sodium phosphate buffer (pH 6.2)
for more than 5 h and fixed with 3.7% formaldehyde for 5 min. Cells were permeabilized with
0.5% Triton X-100, washed, and incubated with FITC or TRITC-labeled phalloidin (Sigma) in
PBS containing 0.5% BSA and 0.05% Tween-20 for 1 h. Cells were washed in PBS containing
0.5% Tween-20. Images were captured with Roper Coolsnap camera and Metamorph software.
For labeling barbed-ends, aggregation competent cells were permeabilized with
100mM PIPES pH 6.9, 1% Triton X-100, 4% PEG, 1mM EGTA, 1mM MgCl2, 3µM
phalloidin for 3 min and 0.4µM Rhodamine-labeled actin in 1µM ATP solution was added.
After 5 min staining, cells were washed 3 times with PIPES buffer and fixed with 3.7%
Cells competent to chemotax toward cAMP (aggregation-competent cells) were
obtained by pulsing cells in suspension for 5 h with 30 nM cAMP, conditions that maximally
induce the expression of aggregation-stage genes required for aggregation, including the cAMP
receptor cAR1 and the coupled G protein α subunit Gα2. The chemotaxis assays were done as
previously described (Chung and Firtel, 1999; Meili et al., 1999a). Cells were pulsed with 30
nM cAMP at 6 min intervals for 5 hr and plated on glass-bottomed microwell dishes (MarTek,
Inc., Ashland, MA). A micropipette filled with 100 µM cAMP was positioned to stimulate cells
by using a micromanipulator (Eppendorf, Germany) and the response and movement of cells
were recorded by using Metamorph software (1 image per every 6 sec). Cell movement was
examined by tracing the movement of a single cell in a stack of images.
Lipid binding assay
PIP strips (Echelon Inc., Utah) were blocked with 3% (w/v) fatty-acid-free bovine
serum albumin in TBST (10 mM Tris-HCl, pH 8.0, 150 mM NaCl and 0.1% (v/v) Tween-20)
for 1 h. Blocked membranes were incubated for 2 h at 4 °C with 100 ng/ml of GST fusion
protein, then washed five times for 5 min each with TBST. After washing, membranes were
incubated with anti-GST monoclonal antibody for 1 h (Santa Cruz Biotechnology, Santa Cruz,
California, USA), followed by additional washing and incubation with an anti-mouse
horseradish-peroxidase conjugate (Amersham). Following final washing, chemiluminescence
was then used to detect binding of GST fusions to phospholipids. Densitometry was conducted
using NIH Image.
PIP beads (Echelon, Utah) were washed twice with 5 volumes of wash/binding buffer
(20 mM Tris, pH 7.4, 150 mM NaCl, 0.05% Tween 20, 1% Ovalbumin) for 1 hour. Control
beads without crosslinked lipid were prepared in an identical manner. 5 µg of GST-B-GBD
protein was then added to the tubes, and the protein was allowed to bind for 2 h at 4 °C with
gentle agitation. The tubes were then centrifuged briefly, the beads were washed two times with
0.5 ml of wash/binding buffer, and the bound proteins were eluted by boiling the beads in 20 µl
of Laemmli sample buffer containing 5% 2-mercaptoethanol. The proteins were then separated
on a 10% SDS-polyacrylamide gel electrophoresis gel. GST-B-GBD protein was detected by
Liposomes were prepared using phosphatidylcholine (PC) (sigma) or synthetic PC
phosphatidylcholine (Avanti Polar Lipids) and 5 % of the phospholipid of interest (150 µg of
total phospholipid). Phospholipids were resuspended in chloroform, mixed, and dried down in
glass tubes under nitrogen. The lipid film was resuspended in resuspension buffer (50mM
HEPES, KOH (pH 7.6), 100 mM KCl, 1 mM EGTA, and 1 mM DTT) at a final concentration
of 1 µg/ul, vortexed vigorously for 5 minutes, and then placed in a bath sonicator for 5 minutes
at 4 degrees C. To perform binding assay, 25 µg of GST-YFP-BG or GST proteins, 50 µg of
the appropriate liposomes, 50 µl of 2% ovalbumin (Sigma), and 10 µl of protease inhibitor
cocktail (Sigma) were mixed in binding buffer (0.25M sucrose, 1mM EDTA, and 20mM
Tricine, pH 7.8). Immediately after incubation, GST fusion protein and liposome complex were
overlaid on an Optiprep (Greiner Biotech.) step gradient (5ml of 40 % : 2ml of 30 % : 1.6ml of
15 % : 1.6ml of 5 % of Optiprep in binding buffer) and centrifuged in a Beckman SW40Ti rotor
for 18h at 35,000 rpm at 4oC. 500 µl fractions were collected from the top of tubes and analyzed
by SDS-PAGE followed by either immunoblot with anti-GST polyclonal antibody (Santa Cruz
Biotechnology) or Coomassie-stained analysis. Protein bands were quantified using NIH image
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Figure 1. Deduced sequence of Dictyostelium WASP (A) Sequence comparison of
Dictyostelium WASP with WASPs from other species. Deduced amino acid sequence of WASP
from the 1200 base pairs DNA clone is aligned with human (Hs), mouse (Mm), C. Elegans (CE),
Drosophila (Dm) WASP. (B) Schematic diagram of WASP domain structure. WH1: WASP
homology I domain; B: Basic domain; GBD: Cdc42/Rac binding domain; V: Verprolin
homology domain; C: central domain; A: acidic domain. (C) Northern blot showing the
developmental time course of WASP expression. Total RNA of 16 µg/sample was resolved on a
1.0% denaturing agarose gel, blotted, and probed as described previously (Datta and Firtel,
1987). The 0-h time point is for vegetative cells. (D) Northern blot showing reduction of WASP
transcript in WASPhypo cells and the lack of WASP transcript in WASPTK cells.
Figure 2. F-actin organization of cells expressing low level of WASP. (A) F-actin organization
revealed by phallioidin staining in WASPhypo and WASPTK cells under cAMP gradient.
Stainings of two cells with representative phenotype are shown. Arrows indicate the direction of
cAMP gradient. Right panel shows ratio of intensity of F-actin staining at the foremost part
(front) or rear end (back) of cells divided by the intensity of the center of cells. Intensity value
was acquired by linescan of images in the direction of the gradient (n=10). (B) In vivo actin
polymerization assay measuring F-actin assembled in response to the chemoattractant (cAMP)
stimulation. Note that F-actin polymerization upon cAMP stimulation is defective in WASPTK
cells. (C) F-actin organization revealed with ABP-GFP in live cells. ABP-GFP fusion protein
binds specifically and dynamically to the same F-actin structures that phalloidin recognizes in
fixed cells. (D) Distribution of free barbed end in cells. 0.4 µM Rhodamine-labeled actin was
incorporated into permeabilized cells to visualize free barbed-ends for actin polymerization in
wild type cells or WASPTK cells. Linescans of a cell from point A to B were shown in lower
panels. Incorporation of Rhodamine-labeled G-actin was quantified by measuring the
fluorescence intensity and shown in right panel (n=6). (E) Axial polarity of wild type and
WASPTK cells. GFP fusion of N-terminal half of PAKa (PAKa-N-GFP) shows specific
localization to uropod of polarized cells and was used as a reporter to examine cellular polarity.
Aggregation competent wild type cells are well polarized and showed biased localization of
PAKa-N-GFP at the uropod, but WASPTK cells show almost equal distribution of PAKa-N-GFP,
indicating lack of axial polarity. Polarity index was calculated by the ratio of PAKa-N-GFP
intensity at the leading edge to GFP intensity at the uropod and shown in the right panel (n=6).
The polarity index of wild type cells is significantly greater than that of WASPTK cells.
Figure 3. Abnormal chemotactic movement of cells expressing low level of WASP. (A) DIC
images of cells migrating toward a cAMP gradient. Wild type cells are well polarized and retract
uropod actively. WASPhypo cells have defects in protruding lamellipod and retracting rear cell
body, resulting in narrow and long uropod. WASPTK cells do not polarize and actively migrate.
WASPTK cells expressing tTA, a chimeric tetracycline-controlled transcriptional activator
protein, recovers normal chemotactic movements. Images of cell at 0, 7, and 14 min were
shown. (B) Chemotaxis of cells were analyzed by Metamorph software and traces of cells
chemotaxing toward cAMP source were shown. Asterik represents the position of micropipette.
(C) Cell body length of WASPhypo cells migrating toward cAMP gradient. Due to lack of active
retraction at the uropod, the length of cell body extends until the uropod retracts by mechanical
force generated by pseudopod extension. As an example, variations of cell body length of a wild
type cell and a WASPhypo mutant cell are shown in the left. Differences in longest and shortest
cell body lengths of 8 cells during chemotaxis were analyzed and are shown in the right. (D)
Chemotaxis speed of wild type, WASPTK, and WASPTK cells expressing tTA (n=6). The
chemotactic speed of WASPTK cells is significantly decreased, but recovered by the expression
of tTA leading WASP expression. DIC images of migrating cells were taken in 6 sec intervals
for 15 min and analyzed with Metamorph software (Universal Imaging Corp., Downingtown,
PA). Angular deviation shows deviations of the angle of the path taken by cells from frame to
frame. Chemotaxis indices were calculated as described in Futrelle et al. (1982). If the cell
moves directly towards gradient source it is 1, if directly away it is -1. If movement is indifferent
to gradient (random movement) it is 0. (E) Translocation of Akt/PH-GFP upon cAMP
stimulation. Wild type and WASPTK cells expressing PH domain of Akt/PKB (Akt/PH-GFP)
were stimulated by the addition of a saturating dose of cAMP (10 µM). Upon cAMP
stimulation, a dramatic translocation of Akt/PH-GFP from the cytosol to the plasma membrane is
observed. Numbers in the lower right corner are seconds before and after stimulation. Right
panel shows the translocation kinetics of Akt/PH-GFP obtained from time-lapse recordings. The
fluorescence intensity of membrane-localized GFP fusion protein was quantitated as E(t) using
the linescan module of Metamorph software. Et/Eo is plotted as a measure of the amount of
membrane-associated protein relative to the starting conditions.
Figure 4. Motility defects of cells expressing low level of WASP in multicellular development.
(A) Development of cells expressing low level of WASP. Cells grown in axenic medium were
washed and plated on non-nutrient agar plate. Photographs were taken at various developmental
stages thereafter. Development of WASPhypo cells was delayed in mound stage, resulting in
delayed formation of slugs and fruiting bodies. WASPTK cells showed severe defect in
chemotactic aggregation, resulting in few fruits formed. Ectopic expression of GFP-WASP
rescued defects of WASPTK cells. (B) Motility defect of WASPhypo cells. Wild type, WASPhypo,
WASPhypo cells expressing GFP were mixed in a ratio of 8:1:1. GFP-labeled WASPhypo cells
were uniformly distributed in the loose-aggregate at 8hrs after the onset of development, but
sorted out to the periphery of the tight mound (12 hr), presumably due to the motility defect. (C)
Motility defects of WASPTK cells. GFP-labeled WASPTK cells were mixed with unlabeled wild
type cells and they showed significant defects in chemotactic aggregation. Most of GFP-labeled
WASPTK cells were neither present in the mound nor polarized.
Figure 5. (A) Localization of GFP-WASP in Dictyostelium cells (WASPhypo) migrating toward a
cAMP gradient. For examining GFP-WASP, cells were pulsed for 4.5 hours at 6 min intervals.
Fluorescence images were taken from live aggregation-competent cells migrating toward a
chemoattractant gradient. GFP-WASP localizes to the leading edge and the uropod, which is
recapitulated by GFP-B-GBD. GFP-WASP variant lacking WH1-B domains do not show
prominent localization as GFP-WH1 shows uniform distribution. Arrows indicate the direction
of gradient. Bar = 5 ?m. (B) Localization of GFP-WASP in WASPTK cells. GFP-WASP still
shows biased distribution at the leading edge and uropod, but appears to be associated with
vesicular structures. Higher expression of GFP-WASP showed more diffuse distribution. (C)
Localization of YFP-WASP to the leading edge. CFP-Coronin and YFP-WASP were
coexpressed in wasphypo cells and their localizations in chemotaxing cells were examined in time-
lapse recording of alternate exposures (1 sec) for CFP and YFP. Images were acquired and
analyzed with multidimensional acquisition utility of Metamorph (Universal Imaging) software.
Coronin localizes to sites of dynamic actin assembly and functions as a reporter for F-actin
distribution in live cells.
Figure 6. (A) The phospholipid binding properties of the basic domain of WASP. The indicated
phospholipids were spotted onto a nitrocellulose membrane (PIP strip) that was then incubated
with GST-PH/PLCδ1 and GST-B-GBD. The membranes were washed and the GST fusion
proteins bound were detected by western blot using a GST antibody. Inlet shows a PIP strip and
bar graph is an average of four PIP strips. Note that GST-PH/PLCδ1 specifically bound to
PI(4,5)P2, but GST-B-GBD showed higher affinity to PI(3,4,5)P3 and PI(3,4)P2 that are products
of PI3 kinase. Unexpectedly higher binding to PI(4)P and PI(5)P in the graph was due to having
one blot showing unusually high binding to PI(4)P and PI(5)P. (B) PIP beads pull-down assay.
GST-B-GBD fusion protein was incubated with agarose beads cross-linked to phosphoinositides
and bound GST-fusion protein was pulled down and then probed by a western blot. GST-B-
GBD showed binding to PI(3,4,5)P3-beads with slightly higher affinity than to PI(4,5)P2-beads
whereas control beads showed minimal binding. Quantification of three experiments is shown.
(C) Liposome co-sedimentation assay with purified GST-YFP-BG or GST-WASP protein and
liposomes composed of 95% PC and 5% of either PI, PI(4)P, PI(4,5)P2, or PI(3,4,5)P3. GST-
YFP-BG protein and liposomes were mixed and subjected to centrifugation on an Optiprep
gradient (5-30% Optiprep). Collected fractions were run on SDS-PAGE gel and probed with
western blot with anti-GST antibody. Ovalbumin was added in the binding mixture to block
non-specific binding and it also serves as an internal control for lower concentration of Optiprep
fraction. Specific binding efficiency was quantified by summing of GST-YFP-B-GBD band
intensity in the higher fractions than fractions where ovalbumin was present. (D) Disruption of
polarized localization of GFP-WASP by the PI3K inhibitor LY294002. Dictyostelium
chemotaxing cells expressing the Coronin-GFP or GFP-WASP were treated with 15 µM
LY294002 or 10nM latrunculin A and the change in the subcellular localization of Coronin-GFP
and GFP-WASP was followed by time-lapse digital video microscopy. Arrows indicate the
direction of movements and numbers shown are time (seconds) after LY treatment.
Figure 7. (A) Co-localization of YFP-BG and CFP-PLCδ1PH in chemotaxing cells. CFP-
PLCδ1PH binds to PI(4,5)P2 with very high specificity. CFP-PLCδ1PH showed punctate vesicle
labeling which appears to overlap with YFP-G-GBD signal. Note that some YFP-B-GBD signal
was found at the leading edge without co-localization with CFP-PLCδ1PH. (B) Co-localization
of YFP-BG and CFP-PhdA in chemotaxing cells. CFP-PhdA selectively binds to PI(3,4,5)P3
and accumulated at the leading edge of migrating cells. Wild-type cells migrating toward cAMP
source show a distinct co-localization of PhdA–GFP and YFP-B-GBD at the leading edge. (C)
PIP strip assay of B domain mutants. Lys residues of the B domain were mutated to Ala and
binding efficiency of GST fusion protein of these mutants to phosphoinositides were assessed by
PIP strip assay. Lys156A and Lys157A appear to specifically block the binding to
phosphoinositides. (D) Localization of YFP-B-GBD and YFP-B157A-GBD in chemotaxing cells.
Phase contrast and fluorescence micrographs of cells expressing YFP-B-GBD and YFP-B157A-
GBD are shown. Note that YFP-B-GBD is localized at the leading edge, but the localization of
YFP-B157A-GBD to the leading edge is significantly reduced. Fluorescence intensities of YFP
were measured along a thin line through the central portion of the cell. Arrows indicate the
position of the leading edge. Some of YFP-B-GBD signal was from intracellular vesicles which
appears to be blurry since optical focus was at the leading edge membrane which cells tend to lift
up. YFP-B156,157A-GBD showed essentially the same distribution as YFP-B157A-GBD. (E)
Rescue of motility defects of WASPTK cells by expressing WASP B domain mutants. Wild type
WASP, K156AWASP, or K156,157AWASP were expressed in WASPTKcells and their development
were examined. Cells expressing K156AWASP showed partial rescue of development whereas
K156,157AWASP did not rescue motility defects of WASPTK cells. Compared to polarized
localization of GFP-WASP, YFP-K156AWASP did not show any biased localization.
Figure 8. F-actin organization of cells expressing WASP mutants. (A) Deletion mutations
expected to abrogate the function of specific domains of WASP were created and F-actin
organization of cells expressing these mutants were examined by TRITC-phalloidin staining.
Cells under a cAMP gradient were fixed and stained with TRITC-phalloidin. Arrows indicate
the direction of gradient. (B) Chemotaxis of cells expressing WASP∆Pro-V and GFP-WASP∆V.
DIC images of cells migrating toward a cAMP gradient at 0 and 15 min are shown. Average
speed and angular deviation of these mutants are shown. (C) Developmental phenotypes of cells
expressing WASP truncation mutants. Cells grown in axenic medium were washed and plated
on non-nutrient agar plate. Photographs were taken at various developmental stages thereafter.
We thank Joel Mulimba and Laura Leeper for excellent technical assistance. We also thank Drs.
Alissa Weaver and Ann Richmond for useful discussions and critical reading of the manuscript.
This work was supported, in part, by grants from National Institute of Health (to C.Y.C. and
R.A.F.). C.Y.C. was partially supported by a grant from The Leukemia and Lymphoma Society
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