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The Rockefeller University Press, 0021-9525/2001/06/1479/19 $5.00
The Journal of Cell Biology, Volume 153, Number 7, June 25, 2001 1479–1497
http://www.jcb.org/cgi/content/full/153/7/1479
1479
The
Dictyostelium
CARMIL Protein Links Capping Protein and the Arp2/3
Complex to Type I Myosins through Their SH3 Domains
Goeh Jung, Kirsten Remmert, Xufeng Wu, Joanne M. Volosky, and John A. Hammer III
Laboratory of Cell Biology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892
Abstract.
Fusion proteins containing the Src homology
(SH)3 domains of
Dictyostelium
myosin IB (myoB) and
IC (myoC) bind a 116-kD protein (p116), plus nine
other proteins identified as the seven member Arp2/3
complex, and the
and
subunits of capping protein.
Immunoprecipitation reactions indicate that myoB and
myoC form a complex with p116, Arp2/3, and capping
protein in vivo, that the myosins bind to p116 through
their SH3 domains, and that capping protein and the
Arp2/3 complex in turn bind to p116. Cloning of p116
reveals a protein dominated by leucine-rich repeats and
proline-rich sequences, and indicates that it is a homo-
logue of Acan 125. Studies using p116 fusion proteins
confirm the location of the myosin I SH3 domain bind-
ing site, implicate NH
2
-terminal sequences in binding
capping protein, and show that a region containing a
short sequence found in several G-actin binding pro-
teins, as well as an acidic stretch, can activate Arp2/3-
dependent actin nucleation. p116 localizes along with the
Arp2/3 complex, myoB, and myoC in dynamic actin-rich
cellular extensions, including the leading edge of cells
undergoing chemotactic migration, and dorsal, cup-like,
macropinocytic extensions. Cells lacking p116 exhibit a
striking defect in the formation of these macropinocytic
structures, a concomitant reduction in the rate of fluid
phase pinocytosis, a significant decrease in the efficiency
of chemotactic aggregation, and a decrease in cellular
F-actin content. These results identify a complex that links
key players in the nucleation and termination of actin
filament assembly with a ubiquitous barbed end–directed
motor, indicate that the protein responsible for the
formation of this complex is physiologically impor-
tant, and suggest that previously reported myosin I mu-
tant phenotypes in
Dictyostelium
may be due, at least in
part, to defects in the assembly state of actin. We pro-
pose that p116 and Acan 125, along with homologues
identified in
Caenorhabditis elegans
,
Drosophila
, mouse,
and man, be named CARMIL proteins, for capping pro-
tein, Arp2/3, and myosin I linker.
Key words: myosin I • Arp2/3 complex • capping pro-
tein • leucine-rich repeats •
Dictyostelium
Introduction
Type I myosins are ubiquitous, nonfilamentous, actin-
based mechanoenzymes that play important roles in sev-
eral actin-dependent processes, including endocytosis and
cell locomotion (Coluccio, 1997; Mermall et al., 1998; Wu
et al., 2000). The members of one myosin I subfamily,
which include examples from
Acanthamoeba
,
Dictyostel-
ium
, yeast,
Aspergillus
, rat, and human (Sellers, 1999),
contain an Src homology (SH)3
1
domain within their tail
domain. These
55-residue protein modules are present
in a diverse array of cytoskeletal and signaling proteins
and mediate protein–protein interactions of moderate af-
finity and high specificity with proline-rich target se-
quences containing the core element PXXP (Kuriyan and
Cowburn, 1997). In the case of
Dictyostelium
myosin IB
(myoB), yeast Myo3p, and yeast Myo5p, the presence of
the SH3 domain has been shown to be critical for the func-
tion of the myosin in vivo (Anderson et al., 1998; Novak
and Titus, 1998; Evangelista et al., 2000; Lechler et al.,
2000). Therefore, for these myosins, full expression of
function appears to depend on the SH3 domain–depen-
dent interaction with a target protein.
To date, three myosin I SH3 domain target proteins
have been identified: Acan 125, verprolin (Vrp1p), and
Las17p/Bee1p. The
Acanthamoeba
protein Acan 125, the
first to be identified (Xu et al., 1995), binds to the SH3 do-
mains of
Acanthamoeba
myosins IA and IC through
PXXP motifs located near its COOH terminus, and coim-
munoprecipitates with myosin IC (Xu et al., 1997; Lee et
al., 1999; Zot et al., 2000). The cellular function of Acan
125, a member of the leucine-rich repeat family of proteins
Address correspondence to John A. Hammer III, Laboratory of Cell Biol-
ogy, Building 3, Room B1-22, National Institutes of Health, Bethesda,
MD 20892. Tel.: (301) 496-8960. Fax: (301) 402-1519. E-mail: hammerj
@nhlbi.nih.gov
1
Abbreviations used in this paper:
LRR, leucine-rich repeat; myo, myo-
sin I; RT, reverse transcription; SB, starvation buffer; SH, Src homology.
The Journal of Cell Biology, Volume 153, 2001 1480
(Kobe and Deisenhofer, 1995), is unknown. The yeast pro-
teins verprolin and Las17/Bee1p bind to the SH3 domains
of both Myo3p and Myo5p (Anderson et al., 1998; Evan-
gelista et al., 2000; Lechler et al., 2000). Verprolin is pro-
line-rich, binds actin monomers, and is required for en-
docytosis and the establishment of a polarized cortical
actin cytoskeleton (Vaduva et al., 1997; Naqvi et al., 1998).
Las17p/Bee1p (Li, 1997) is the yeast homologue of WASp,
the product of the Wiskott-Aldrich syndrome gene in hu-
mans (Derry et al., 1994), and is essential for assembly of
the cortical actin cytoskeleton and for endocytosis (Li,
1997; Naqvi et al., 1998). Both Las17p/Bee1p and WASp
bind to the Arp2/3 complex, a highly conserved seven
member complex containing the actin-related proteins
Arp2 and Arp3 (Machesky et al., 1994), and accelerate
Arp2/3-dependent nucleation of actin filament assembly
(Machesky and Insall, 1998; Machesky et al., 1999; Ro-
hatgi et al., 1999; Winter et al., 1999; Yarar et al., 1999).
Furthermore, verprolin interacts physically and genetically
with Las17p/Bee1p (Naqvi et al., 1998). Similarly, the ver-
tebrate homologue of verprolin, WIP, interacts physically
and functionally with WASp (Ramesh et al., 1997; Vaduva
et al., 1999; Moreau et al., 2000).
The purpose of the current study was to purify and char-
acterize proteins that bind to the SH3 domains of
Dictyo-
stelium
myoB (Jung et al., 1989) and myoC (Peterson et al.,
1995). Our biochemical efforts resulted in the identification
of p116, the
Dictyostelium
homologue of Acan 125. Like
Acan 125, p116 binds to myoB and myoC through their
SH3 domains. Surprisingly, p116 was also found to bind the
seven member Arp2/3 complex, the central player in the de
novo nucleation of actin filament assembly and in the for-
mation of branched filament networks, and capping pro-
tein, the central player in the termination of actin filament
assembly (Eddy et al., 1997; Carlier et al., 1999; Higgs and
Pollard, 1999; Machesky and Insall, 1999; Welch, 1999; Blan-
choin et al., 2000; Cooper and Schafer, 2000; Pantaloni et
al., 2000). These interactions drive the formation of a cellu-
lar complex in which myoB and myoC are linked via their
association with p116 to two key regulators of actin poly-
merization and three-dimensional organization. We show
that p116, Arp2/3, myoB, and myoC are all concentrated in
regions of active actin assembly, and that the normal func-
tioning of these actin-rich domains depends on the pres-
ence of p116. Together, these results suggest that myoB
and myoC support actin-dependent cellular processes not
only through their roles as actin cross linkers and actin-
based mechanoenzymes, but also through effects they may
have on the assembly state and organization of actin. Such
effects could arise from myosin I–dependent changes in the
location, movement, and/or activity of the complex formed
by the interaction of Arp2/3, capping protein, and myosin I
with p116, which we have named the CARMIL protein, for
capping protein, Arp2/3, and myosin I linker.
Materials and Methods
Identification of SH3 Domain Binding Proteins
The SH3 domains of
Dictyostelium
myoB (residues 1,057–1,111) and
myoC (residues 1,126–1,181) were amplified by PCR using Pfu polymer-
ase (600135; Stratagene) and the following oligonucleotides: 5
-
CTAGGATCCACTGCAAAAGCACTCTACGATTATGAT-3
and
5
-TGCGAATTCTTAATTATATTGTAAATAATTTGTTGG-3
for
myoB; 5
-CTAGGATCCCAATATATCGCTCTTTACGAGTACGAC-
3
and 5
-TGCGAATTCTTAAATTTGTTGAACATAATTTGAAGG-
3
for myoC. For the proline to leucine point mutation, the 3
oligonucle-
otides for myoB and myoC were 5
-TGCGAATTCTTAATTATATTG-
TAAATAATTTGTTAGAGCCCAACCTTTTTG-3
and 5
-TGCGAA-
TTCTTAAATTTGTTGAACATAATTTGAAAGTAACATACCAA-
TTTG-3
, respectively. The SH3 domain of
Acanthamoeba
myoC (resi-
dues 980–1,033) was amplified using the following oligonucleotides: 5
-
CTAGGATCCCAGGCGCGTGCGCTGTATGACTTTGCG-3
and 5
-
TGCGAATTCTTAGATGAGTTCGACGTAGGACGCGGG-3
. All
three products were digested with BamH1 and EcoRI, ligated into
pGEX-KT, and transformed into
Escherichia coli
strain HB101. Over-
night cultures were diluted 1:10 into 450 ml of fresh LB media containing
100
g/ml ampicillin, grown at 37
C to OD 1.0 at 600 nm, and induced
with IPTG (0.5 mM final). After a 3 h incubation, the cells were harvested,
resuspended in 20 ml of PBS containing protease inhibitors (0.1 mM
PMSF, 5
g/ml AEBSF, 50
g/ml leupeptin, 10
g/ml aprotinin, and 10
g/ml pepstatin A) and 0.1 mM DTT, and broken in a French press. Tri-
ton X-100 was added to a final concentration of 1%, at which point the ly-
sate was mixed for 20 min at 4
C and centrifuged at 15,000
g
for 15 min.
The supernatant was then mixed with 1 ml of glutathione-Sepharose 4B
(17-0756-01; Amersham Pharmacia Biotech) for 3 h at 4
C, at which point
the resin was washed five times with PBS (15 ml and 5 min per wash, with
rotation at 4
C). To generate the high-speed supernatant of lysed
Dictyo-
stelium
, 10 g of wet cell pellet was broken by sonication in 50 ml of
Dictyo-
stelium
lysis buffer (0.5
TBS containing 5 mM sodium pyrophosphate, 1
mM DTT, and protease inhibitors) and centrifuged at 90,000
g
for 1 h at
4
C. Approximately 40 ml of supernatant was incubated on a rotating
mixer with
1 ml of washed GST-SH3 domain resin for 3 h at 4
C. The
resin was then washed five times in 1
TBS (10 ml and 5 min per wash
with rotation 4
C). Bound proteins were eluted by incubation with 2 ml of
5
TBS for 30 min at 20
C. After centrifugation for 2 min at 15,000
g
, the
supernatant was mixed with 0.5 vol of SDS-PAGE sample buffer, resolved
on 4–20% gradient gels, visualized by silver staining or Coomassie blue
staining, and subjected to Western blot and microsequence analyses. For
the latter, Coomassie blue–stained bands were excised from the gel, di-
gested in situ with trypsin, and HPLC-purified tryptic peptides subjected
to Edman degradation (Harvard Microsequencing Facility). For the far
Western blot shown in Fig. 3 A,
1 mg of myoB or myoC SH3 domain fu-
sion protein was incubated with 0.1 mg of Sulfo-NHS-LC biotin in 0.5 ml
of PBS for 2 h at 4
C (21420; Pierce Chemical Co.). Unincorporated biotin
was removed by chromatography on Sephadex G-25. The stoichiometry of
biotin labeling, as determined using HABA reagent (28010; Pierce Chem-
ical Co.), was
1.5 moles biotin per mole of fusion protein. Blots to be
probed were blocked in TBST containing 0.2% gelatin, and incubated for
3 h at 4
C in TBST containing 0.2% gelatin and 10
g/ml of biotin-
ylated SH3 domain fusion protein. After four 5 min washes in TBST at
4
C, blots were incubated with streptavidin-conjugated alkaline phos-
phatase, washed, and developed using ECL reagents (RPN 2108; Amer-
sham Pharmacia Biotech).
Isolation of the p116 Gene
The gene for p116 was identified using four pooled degenerate oligonucle-
otides (5
-GARGARAGYCAAGCICAAAAYGARGC-3
; 5
-GAR-
GARTCICAAGCICAAAAYGARGC-3
; 5
-CAAGCICAAAAYGA-
RGCIAGYGGWGCIAC-3
; and 5
-CAAGCICAAAAYGARGCIT-
CIGGWGCIAC-3
) corresponding to residues EESQAQNEA and
QAQNEASGAT present in the longer peptide sequence obtained from
p116 (Fig. 2). The PAGE-purified oligonucleotides were end labeled using
T4 polynucleotide kinase (18004-010; GIBCO BRL) and
[P
32
]ATP
(35020; ICN Biomedicals), and purified by gel filtration on Sephadex G25
superfine. To create the genomic sub library enriched for EcoR1 frag-
ments of
5 kb, EcoR1-digested
Dictyostelium
genomic DNA was re-
solved in a 0.9% agarose gel, and the DNA in the size range of 4.6–5.4 kb
was collected by electroelution. These fragments were ligated into EcoR1-
digested
Zap II (236211; Stratagene), the ligation packaged using Giga-
pack III gold packaging extracts (200202; Stratagene), and the library
plated using strain XL1-Blue MRF. Plaque lift-offs were prehybridized at
30
C in a solution containing 6
SSPE, 5
Denhardt’s, 0.5% SDS, and 0.2
mg/ml salmon sperm DNA, and hybridized at 30
C in a solution contain-
ing 30% formamide, 5
SSPE, 2
Denhardt’s, 0.1% SDS, 0.2 mg/ml
salmon sperm DNA, and the pooled, end-labeled oligonucleotides. Filters
were washed in 6
SSPE containing 0.1% SDS at 50
C. Bluescript plas-
mids were excised using helper phage RE704. The 4.8-kb EcoR1 insert
Jung et al.
Complex Containing Capping Protein, Arp2/3, and Myosin I
1481
was sequenced on both strands using a combination of restriction frag-
ments and primer extension reactions. The precise boundaries of the four
introns were obtained by sequencing reverse transcription (RT)-PCR
products that spanned their splice donor and acceptor sites. Northern blots
of total RNA from vegetative
Dictyostelium
and Southern blots of EcoRI-
digested
Dictyostelium
genomic DNA were probed with the labeled oligo-
nucleotides exactly as described above. General DNA sequence analyses
were performed using the program GCG (University of Wisconsin Genet-
ics Computer Group sequence analysis software). Data base searches were
performed using the programs BLAST and FASTA. Dot matrix compari-
sons were performed using the DOT PLOT menu of GCG.
Creation of MyoB
/MyoC
Double Mutants,
P116
Mutants, and Cells Expressing
MyoB without an SH3 Domain
To create myoB
/myoC
double mutants, plasmid pBsr2 (gift of Dr. Ka-
zuo Sutoh, University of Tokyo, Tokyo, Japan), which confers resistance
to blasticidin S, was linearized with HindIII, converted to blunt ends by T4
polymerase fill-in, and treated with phosphatase, creating plasmid A. A 3
portion of the genomic sequence of myoB (nucleotides 2,330–3,086) was
amplified by PCR using Pfu polymerase and the following oligonu-
cleotides: 5
-CTAGCTGGTCAATGGCAATTGGG-3
and 5
-CAT-
AAGCTTGGTAAACCAGAAGCGATTG-3
. The purified product,
which contains a nested HindIII site at the 3
end, was cloned in the 5
to
3
orientation into plasmid A, creating plasmid B. Plasmid B was linear-
ized with XbaI, converted to blunt ends by T4 polymerase fill-in, and
treated with phosphatase, creating plasmid C. A 5
portion of the genomic
sequence of myoB (nucleotides 532–1,247) was amplified by PCR using
the following oligonucleotides: 5
-GCATCTAGAGCATATAGAGG-
TAAACATGC-3
and 5
-GTAGCATACTTGCAGCCAATTC-3
. The
purified product, which contains a nested XbaI site at the 5
end, was
cloned into plasmid C in the 5
to 3
orientation, creating the final disrup-
tion vector (plasmid D). Plasmid D was digested with XbaI and HindIII,
releasing a linear disruption fragment in which the 5
and 3
myoB coding
fragments bracket the Bsr resistance cassette. After purification by pro-
teinase K treatment and phenol/chloroform extraction, the mixture of vec-
tor backbone and disruption fragment was introduced by electroporation
into a representative myoC
line created previously in axenic strain JH10
(Jung et al., 1996). Transformed cells were selected in 10
g/ml blasticidin
(150477; ICN Biomedicals), and independent clones purified by serial di-
lution. MyoB
knockout lines were identified by Western blot analysis
and confirmed by Southern blot analysis.
The exact same strategy was used to create p116
cell lines. The 5
and
3
genomic fragments with nested XbaI and HindIII sites, respectively,
were generated by PCR using the following oligonucleotides: 5
-
TGCTCTAGAGAAACCAAGTCAAAACAAAGTAAAG-3
and 5
-
AGCACCTAATAAACATCTAATAGTATC-3
for the 5
fragment
(nucleotides 1,316–1,766); 5
-ACCCTCAAGAATATGCCAACACCT-
3
and 5
-GCATAAGCTTAGTTGATCTTGGAGCAACAACAGG-3
for the 3 fragment (nucleotides 3,321–4,427). The digested disruption vec-
tor was introduced by electroporation into axenic strain Ax3. Whole cell
extracts from independent, purified, blasticidin-resistant transformants
were screened by Western blot analysis using p116 antibody -p116-1 to
identify knockout lines. Homologous recombination was confirmed by
Southern blot analyses using probes to the blasticidin cassette, the 5 ge-
nomic fragment used in the disruption construct, and the portion of the
p116 gene lost from the genome as a consequence of the double-cross-
over, gene replacement event.
The creation of cells lines expressing myoB without its SH3 domain will
be presented in detail elsewhere. In brief, the myoB null cell line null 6
(Jung and Hammer, 1990) was transformed with the integrating plasmid
pDEX containing the coding sequence for all but the COOH-terminal 54
amino acids of the myoB heavy chain. Several stable transformants were
selected that expressed the truncated heavy chain at 1.5 times normal
vegetative cell levels, as determined by quantitative Western blotting,
which included a correction for the loss of epitopes due to the absence of
the SH3 domain.
Generation of Antibodies to Dictyostelium p116, Arp3,
and p19 (p21-Arc), and to Acanthamoeba Acan 125
Antibody -p116-1 was generated using the Dictyostelium 116-kD protein
present in myoB SH3 domain fusion protein column eluates. In brief,
SDS-PAGE slices containing 70 g of p116 were frozen, crushed, mixed
with Freunds complete adjuvant, and injected subcutaneously into 15 sites
on one rabbit. This animal was boosted using incomplete adjuvant at 4 wk,
and bled at 8 wk. Antibody -p116-2 was raised against a GST fusion pro-
tein containing p116 residues 847 to 1,050, which was generated by PCR
using Pfu polymerase and the following oligonucleotides: 5-TCAG-
GATCCGAAGAATCTCAAGCTCAAAATGAAGC-3 and 5-
CAGGAATTCTTAATTTTCGGTCGGTGGTCTTGGTC-3. This frag-
ment was digested with BamHI and EcoRI and cloned into pGEX 2TK,
and the fusion protein, referred to below as “GST-CT PRO,” was purified
by chromatography on glutathione-Sepharose 4B. Rabbits were immu-
nized as described above, except that 400 g of protein was used per injec-
tion. To generate antibody -APp116-2, -p116-2 serum was incubated
with a p116 null cell extract bound to nitrocellulose exactly as described
previously (Jung et al., 1993). Antibody to Dictyostelium Arp3 was raised
against a GST fusion protein containing Arp3 residues 169–312 (Murgia et
al., 1995) generated by RT-PCR using Pfu polymerase, total Dictyostel-
ium RNA, a 3’ RACE kit (18373-019; GIBCO BRL), and the following
oligonucleotides: 5-ATCGGATCCGATAGTGGTGATGGTGTAAC-
TCAT-3, and 5-GATGAATTCTTAACGACAATCGATTGGACA-
GGATTG-3. The PCR product was cloned into pGEX 4T-1 and rabbits
immunized with the purified fusion protein as described for -p116-2. An-
tibody to Dictyostelium p19 (p21-Arc) was raised against a GST fusion
protein containing the entire coding sequence of p19, obtained by RT-
PCR using oligonucleotides 5-TCAGGATCCATGGTTTATCACTCA-
CAATTCAAC-3 and 5-CAGGAATTCTTATAAGGCTTTGTTTAA-
GAATTTTCTC-3. Antibody to Acanthamoeba Acan 125 was raised
against a GST fusion protein containing Acan 125 residues 221–452, ob-
tained by RT-PCR as described above, except that total Acanthamoeba
RNA was used, and the oligonucleotides were 5-TCAGGATCCCTC-
CACTTCAACAACTACTTCATCGGC-3 and 5-CTCGAATTCTTA-
GCCGATGATGGCCTTGATGACATC-3. The insoluble fusion pro-
tein was purified from a detergent resistant pellet of lysed bacteria by
preparative SDS PAGE and electro elution (model 422; Bio-Rad Labo-
ratories).
Immunofluorescence Light Microscopy
Vegetative cells adhered to coverslips were fixed for 5 min in 99% metha-
nol/1% formaldehyde at 15C and stained exactly as described previ-
ously (Jung et al., 1993). Crude rabbit sera against Dictyostelium Arp3 and
p19 were diluted 1:300, whereas rabbit sera against Dictyostelium myoC
and p116, which were first purified by incubation with their corresponding
null cell extract (see above and Jung et al., 1996), were diluted 1:30 (repre-
senting an 1:500 dilution of the crude sera). The FITC-labeled goat anti–
rabbit and LRSC-labeled goat anti–mouse secondary antibodies were
from Jackson ImmunoResearch Laboratories (111-095-144 and 115-085-
146, respectively) and were used at a dilution of 1:100. Neither showed
any reactivity in the absence of primary antibody, or any cross species re-
activity. The antiactin monoclonal antibody C4 (1378996; Boehringer) was
diluted 1:300. The anticoronin monoclonal antibody (gift of Eugenio De
Hostos, University of California at San Francisco, San Francisco, CA) was
diluted 1:50. For staining of starved cells undergoing chemotactic aggrega-
tion, suspension-grown cells at late log were suspended at a density of 3
10 cells/ml in starvation buffer (SB) (20 mM potassium phosphate, pH 6.7,
2 mM MgCl
2
, and 0.2 mM CaCl
2
), and 50 l was spotted in the center of a
coverslip. Coverslips were incubated in a humidified chamber at 20C un-
til cells were actively streaming, at which point the cells were briefly over-
laid with an agar sheet, fixed, and stained as described previously (Fukui
et al., 1989; Jung et al., 1993). Confocal images were obtained on a ZEISS
410 LSM using a 63 Plan-Apo objective and 0.2–0.7-m steps. Projected
images and sections rendered in three dimensions were created using
ZEISS and Metamorph (Universal Imaging) software.
Immunoprecipitations
Vegetative Dictyostelium cells were incubated for 10 min at 4C in lysis
buffer (100 mM NaCl, 20 mM Tris, pH 7.5, 2 mM ATP, pH 7.0, 1% (vol/
vol) NP-40, and protease inhibitors) and centrifuged at 100,000 g for 15
min at 4C. Approximately 20 ml of lysis buffer was used per gram of cell
pellet. Similar results were obtained when cells were lysed by sonication in
buffer lacking NP-40 and containing isotonic sucrose (0.25 M). Approxi-
mately 1 ml of high speed supernatant was mixed for 3 h at 4C with 40 l
of protein A agarose (15918-014; GIBCO BRL) that had previously been
incubated with 40 l of primary antibody against Dictyostelium myoB
The Journal of Cell Biology, Volume 153, 2001 1482
(Jung and Hammer, 1990), myoC (Jung et al., 1996), or p116 (-p116-1) in 0.5
ml of 1X TBS for 3 h at 4C. Beads were washed five times with 1 TBS (2
ml and 5 min per wash, with rotation at 4C), boiled for 5 min in SDS-
PAGE sample buffer, and the supernatants used for Western blot analy-
ses. Blots were probed with antibodies to Dictyostelium myoB, myoC,
p116 (-p116-1), and capping protein (gift of Dr. John Cooper, Wash-
ington University, St. Louis, MO), and to yeast Arp3 (gift of Dr. Kathy
Gould, Vanderbilt University, Nashville, TN), at dilutions of 1:20,000, 1:
30,000, 1:2,000, 1:5,000, and 1:3,000, respectively.
Identification in p116 of the Binding Sites
for Myosin I and Capping Protein
To identify the myosin I binding site, two adjacent PXXP motifs present
between residues 956 and 973 in p116 were deleted from the GST fusion
protein GST-CT PRO (see above) by overlap extension PCR using Pfu
polymerase and the following oligonucleotides: 5-TCAGGATCCGAA-
GAATCTCAAGCTCAAAATGAAGC-3 and 5-GGTTTTGATTTA-
AGTGGTGTAACATCTGGTGGCGCACCTCCACCCATATTTC-3
for the 5 fragment, and 5-GAAATATGGGTGGAGGTGCGCCAC-
CAGATGTTACACCACTTAAATCAAAACC-3 and 5-CAGGAAT-
TCTTAATTTTCGGTCGGTGGTCTTGGTC-3 for the 3 fragment.
The complete insert obtained using the outside primers and a mixture of
the 5 and 3 fragments was digested with BamH1 and EcoR1 and cloned
into pGEX 2TK. To test for the ability to interact with myosin I in whole
cell extracts, glutathione-Sepharose resins loaded with the deletion-con-
taining fusion protein (referred to below as “GST-CT PRO PXXP
”), the
undeleted version, or GST alone (0.8 ml resin) were incubated at 4C for
4 h with 8 ml of a high speed supernatant of lysed Dictyostelium. The prepa-
ration of this supernatant, the washing of the resin, and the elution of
bound proteins were performed as described above for the identification
of SH3 domain binding proteins. Eluates were then probed for the pres-
ence of myoB and myoC by Western blotting. To search for the binding
site for capping protein, a fusion protein spanning residues 1–179 of p116
(referred to below as “GST-NT”) was generated using pGEX 2TK, Pfu
polymerase, and the following oligonucleotides: 5-TCAGGATCCAT-
GTCAGAAGAAATATCACCAAATG-3 and 5-GTCGAATTCTTA-
AGAGGAGATAATATTTGTCATATCCC-3. Glutathione-Sepharose
resins loaded with GST-NT or GST alone were incubated with a high-
speed supernatant of lysed Dictyostelium and processed as described
above. Eluates were probed for the presence of the and subunits of
capping protein.
Purification of Actin and the Arp2/3 Complex,
and Actin Nucleation Assays
Actin was purified from Acanthamoeba castellanii according to Gordon et
al. (1976), followed by gel filtration on HiPrep Sephacryl S200 (17-1195-
01; Amersham Pharmacia Biotech). Monomeric actin was labeled with
N-(1-pyrene)-iodoacetamide (P29; Molecular Probes) and stored lyophil-
ized at 80C. Before use it was resuspended and dialyzed in G buffer (0.1
mM CaCl
2
, 0.5 mM ATP, pH 7.0, 0.75 mM -mercaptoethanol, and 3 mM
imidazole, pH 7.5). The Arp2/3 complex was purified from Acanthamoeba as
described by Kelleher et al. (1998) and stored at 70C in complex stor-
age buffer supplemented with 200 mM sucrose. The portion of p116 (resi-
dues 644–863) that contains a short sequence conserved among G-actin
binding proteins (including verprolin), plus an acidic region was amplified
by PCR using Pfu polymerase and the following oligonucleotides: 5-
TCAGGATCCATTTGGAAGGAAATCGATTCATGTATC-3 and
5-ACTGAATTCTTAATCTGGAATTGGGGTGGCACCACT-3. The
product was digested with BamH1 and EcoR1, cloned into pGEX 2TK
(27-4587-01; Amersham Pharmacia Biotech), and the fusion protein, re-
ferred to below as “GST VA,” expressed and purified as described above.
Actin nucleation assays were performed essentially as described by Higgs
et al. (1999). In brief, G-actin was mixed with pyrene-labeled actin at a ra-
tio of 20:1, converted to Mg
-actin, and polymerized at a final concentra-
tion of 4 M by the addition of 10 polymerization buffer (500 mM KCL,
10 mM MgCl
2
, 10 mM EGTA, and 100 mM imidazole, pH 7.0). Additional
proteins (e.g., Arp2/3, GST VA; see the legend to Fig. 5) were dialyzed
into G buffer supplemented with 1 mM MgCl
2
and added to the actin solu-
tion just before the initiation of polymerization. Fluorescence intensities
were measured every second for 10 min using a PTI QuantaMaster fluo-
rometer (Photon Technology International) and wavelengths of 365 and
407 nm for excitation and emission, respectively.
Estimation of Cellular F-Actin Content
The amount of F-actin in cells was estimated by a modification of previous
methods (Podolski and Steck, 1990; Watts and Howard, 1994). In brief, 2.5
10
7
log phase cells were seeded in a tissue culture dish (3003; Falcon) in
SB and incubated until streams were evident (6 h for wild-type strain
Ax3; 10 h for p116 null cells). Cells were lysed by the addition of 2 ml of
ice cold buffer A (50 mM KCL, 10 mM imidazole. pH 7.5, 3.3 mM Tris-
acetate, pH 7.5, 2.2 mM magnesium acetate, 1 mM EGTA, 0.5 mM ATP, pH
7.0, 1% Triton X-100, and protease inhibitors), followed by incubation on
ice for 5 min. Triton-insoluble cytoskeletons were collected by centrifuga-
tion at 1,000 g for 2 min at 4C. To estimate the amount of actin in this pel-
let, which should be predominantly in filamentous form, samples were re-
solved by SDS-PAGE and the amount of Coomassie blue–stained
material present at the position of actin quantitated by densitometry
(ChemImager 4400; Alpha Innotech). A second estimate of cellular
F-actin content was obtained by lysing cells on ice in buffer A containing 20
M FITC-labeled phalloidin (P5282; Sigma-Aldrich), washing the result-
ing Triton-insoluble cytoskeletons twice with ice-cold buffer A, dissolving
the pellets in 50 mM sodium phosphate buffer, pH 9.2, and measuring the
fluorescence intensities using wavelengths of 485 and 520 nm for excita-
tion and emission, respectively. Essentially identical results were obtained
using these two methods. The values presented in the text are those ob-
tained using fluorescence measurements, and are given for p116 null cells
as the percent of the value obtained for the parental strain Ax3.
Cell Biological Assays and General Methods
To perform streaming assays, midlog phase cells (4 10
6
/ml) were pel-
leted at 500 g for 5 min, washed in SB, resuspended in SB at 1.2 10
6
/ml,
and seeded in 60 mm tissue culture dishes (3002; Falcon) (2.5 ml/dish). Es-
sentially all of the cells (both control and p116
) adhered to the dish. Im-
ages were obtained using a Nikon ZMU stereo microscope coupled to a
CCD camera (model C2400; Hamamatsu) and an optical disc recorder
(TQ3031F; Panasonic). The rate of fluid phase pinocytosis was deter-
mined on cells in suspension using FITC-labeled dextran and centrifuga-
tion through PEG 8000 cushions exactly as described previously (Jung et
al., 1996). Other general methods were performed as described previously
(Jung et al., 1996).
Results
The SH3 Domains of MyoB and MyoC Interact In Vitro
with a Complex Mixture of Polypeptides Comprised
of the Seven-Member Arp2/3 Complex, Capping
Protein, and a Protein of 116 kD
To look for soluble proteins that interact with the SH3 do-
mains of myoB and myoC, these domains were expressed
as GST fusion proteins, incubated with high speed super-
natants of lysed Dictyostelium, washed extensively at phys-
iologic ionic strength, and eluted with high salt. Eluates
were then resolved by SDS-PAGE and visualized by silver
staining (Fig. 1). Relative to controls (beads alone, lane 2;
beads coupled to GST only, lane 3), the myoB SH3-domain
fusion protein bound nine polypeptides with molecu-
lar masses of 116, 47, 44, 41, 35, 33, 21, 19, and 16 kD (lane
4, see arrowheads). Introduction into the myoB SH3 do-
main of a proline to leucine (PL) point mutation, which
is function blocking when present in the SH3 domain of
the Caenorhabditis elegans adaptor protein SEM 5 (Stern
et al., 1993), completely abrogated the interaction with
these nine polypeptides (lane 5), suggesting that the inter-
actions are specific. Similar results were obtained with the
myoC SH3-domain fusion protein (lane 6) and its corre-
sponding point mutant (lane 7).
All nine polypeptides described above were subjected to
protein microsequencing. Unambiguous sequences were
obtained for p116, p47, p41, p35, p21, p19, and p16 (Fig. 2).
Jung et al. Complex Containing Capping Protein, Arp2/3, and Myosin I 1483
Blast searches failed to detect any homologues to the se-
quences of two peptides obtained from p116, but identi-
fied the 35-kD band as the subunit of capping protein,
and revealed that the remaining five sequenced proteins
correspond to the Dictyostelium homologues of the Arp2/3
complex components Arp3 (47-kD band), p41-Arc (41-
kD band), p21-Arc (19-kD band), p20-Arc (21-kD band)
and p16-Arc (16-kD band) (Fig. 2 and Fig. 1, lane 8).
Given that a tight association exists between all seven
members of the Arp2/3 complex (Higgs and Pollard,
1999), and that the subunit of capping protein should be
present along with the subunit, we conclude that the re-
maining two unaccounted for bands at 44 and 33 kD corre-
spond to Arp2 and a mixture of the 34-kD Arp2/3 com-
plex component p34-Arc and the 33-kD subunit of
capping protein (Fig. 1, lane 8). This latter conclusion is
supported by the fact that an antibody specific for the
subunit of capping protein (Hug et al., 1995) recognized
the 33-kD band (data not shown). Therefore these data
indicate that the nine bands correspond to ten different
polypeptides: the and subunits of capping protein, the
seven members of the Arp2/3 complex, and an unknown
protein of 116 kD (Fig. 1, lane 8).
Given that capping protein and all seven Arp2/3 com-
plex components lack a consensus SH3 domain binding
motif, we hypothesized that p116 alone interacts directly
with the myosin I SH3 domains. To test this idea, the ma-
terial in Fig. 1, lane 8 was transferred to nitrocellulose and
probed with biotinylated myoB SH3-domain fusion pro-
tein. Development of the blot with strepavidin/alkaline
phosphatase revealed that the SH3 domain did indeed in-
teract to a significant extent with just p116 (Fig. 3 A, lane
1; lane 2, which contained the material bound to the myoB
SH3 PL column, serves as a control). This result, and
identical results obtained using the myoC SH3 domain as a
probe (data not shown), indicate that the proline-rich
binding site for the two myosin I SH3 domains resides
within p116, and suggest that p116 in turn binds capping
protein and the Arp2/3 complex. Consistent with this idea,
the abundance of p116 in many individual preparations al-
ways dictated the abundance of capping protein and the
Arp2/3 complex. When the yield of p116 was high, the
yield of capping protein and Arp2/3 could be high, some-
times approaching apparent stoichiometry with p116, but
when the yield of p116 was low, the yield of capping pro-
tein and Arp2/3 was always low.
Although not evident from the gel in Fig. 1, these com-
plexes do not contain profilin (data not shown, but see leg-
end to Fig. 1). Furthermore, we never observed bands
other than the nine shown in Fig. 1 appearing consistently
or in amounts remotely approaching stoichiometry with
p116. Finally, Western blots indicated that some or all of
the band at 45 kD in control and SH3-domain column elu-
ates (see asterisk in Fig. 1, lane 2) is conventional actin
(data not shown). This small amount of actin could not be
responsible for the presence of capping protein and Arp2/3
in the SH3 domain column eluates because all four con-
trols (Fig. 1, lanes 2, 3, 5, and 7) contain the same amount
of immunoreactive and silver-stained material at 45 kD,
and yet lack all of the complex components. To summa-
rize, these biochemical studies indicate that Arp2/3, cap-
ping protein, an unknown protein of 116 kD, and, by infer-
Figure 1. Isolation of SH3
domain binding proteins.
Shown are silver-stained
SDS-PAGE gels (4–20%) of
molecular weight standards
(lane 1), proteins eluted from
glutathione beads (lane 2)
and beads loaded with un-
fused GST (lane 3), and pro-
teins eluted from beads
loaded with the following
GST fusion proteins: GST-
myoB SH3 domain (lane 4),
GST-myoB SH3 domain con-
taining the PL point muta-
tion (lane 5), GST-myoC
SH3 domain (lane 6), GST-
myoC SH3 domain contain-
ing the PL mutation (lane
7), and GST-myoB SH3 do-
main (lane 8). The asterisk
next to lane 2 marks the posi-
tion of the silver-stained
band that reacted with an an-
tiactin monoclonal antibody.
The arrowheads adjacent to
lanes 4 and 6 point to the
nine apparent polypeptides that bound to the myoB and myoC SH3 domain fusion proteins. The identities of the 10 proteins that corre-
spond to these nine bands are shown to the right of lane 8. Using a 15% SDS-PAGE gel and authentic Dictyostelium profilin as a stan-
dard, we found that the isolated complex is devoid of profilin. Westerns performed using an antibody to Dictyostelium SCAR (Bear et
al., 1998; gift of Dr. Karl Saxe, Emory University, Atlanta, GA) show that this Arp2/3 binding protein is also not present.
The Journal of Cell Biology, Volume 153, 2001 1484
chain (lanes 1 and 3; the band at 116 kD in lane 1 is a
breakdown product of myoB), but also p116 (lanes 2 and
4). Similarly, immunoprecipitations made using wild-type
cell extracts and the antibody to p116 (lanes 5–7) con-
tained not only p116 (lane 5), but also myoB (lane 6) and
myoC (lane 7). To demonstrate that the interaction be-
tween myosin I and p116 is SH3 domain–dependent, ex-
tracts of cells that express myoB lacking its SH3 domain,
which were made by complementing a myoB null cell line
with a truncated version of myoB missing the COOH-ter-
minal 54 amino acids (Fig. 3 B, lanes 5–7 and Materials
and Methods regarding this mutant), were used. As pre-
dicted, myoB was present in immunoprecipitates made
from these cells using an antibody to myoB (Fig. 4 A, lane
8), but p116 was not (lane 9). Together, these results indi-
cate that p116 interacts with myoB and myoC in vivo, and
that these interactions require the presence of the myosin
I SH3 domain.
To look for the association of the Arp2/3 complex with
myosin I and p116 in vivo, immunoprecipitations were
made using wild-type cell extracts and antibodies to myoB,
myoC, or p116 (Fig. 4 B, lanes 1–3, respectively). Arp3 was
present in all three immunoprecipitates. Furthermore, im-
munoprecipitates made using the antibody to p116 and ex-
tracts from cells lacking both the myoB and myoC heavy
chains, which were created by targeted disruption of both
genes (Fig. 3 B, lanes 1–4, and Materials and Methods re-
garding this mutant), contained Arp3 (Fig. 4 B, lane 4).
Conversely, antibody to myoB did not immunoprecipitate
Arp3 from extracts of cells expressing myoB without its
SH3 domain (lane 5). Together, these results indicate that
Arp2/3 is associated with myosin I and p116 in vivo, and
that its presence in this complex is mediated by an interac-
tion with p116, not myosin I.
To look for the association of capping protein with myo-
sin I and p116 in vivo, immunoprecipitations were made
using wild-type cell extracts and either preimmune serum
or antibodies to myoB, myoC, or p116 (Fig. 4 C, lanes 1–4,
respectively). The subunit of capping protein was
present in all but the immunoprecipitate made using pre-
immune serum (lanes 1–4). Furthermore, immunoprecipi-
tates made using extracts of cells lacking myoB and myoC,
and the antibody to p116, contained capping protein (lane
5). Conversely, immunoprecipitates made using cells ex-
pressing myoB without its SH3 domain and the antibody
to myoB did not contain capping protein (lane 6). To-
gether, these results indicate that capping protein is associ-
ated with myosin I and p116 in vivo, and that its presence
in this complex is mediated by an interaction with p116,
not myosin I.
Cloning of the Gene Encoding p116 Indicates that
It Is the Dictyostelium Homologue of Acan 125
We used degenerate oligonucleotides to the longer of the
two p116 peptide sequences to search for the gene encod-
ing p116. These oligonucleotides recognized a single 5-
kb EcoRI band in digests of Dictyostelium genomic DNA,
as well as a single mRNA of a size (2,700 nucleotides)
consistent with a 116-kD polypeptide (data not shown).
Therefore, these oligonucleotide probes were used to
screen a genomic DNA sub library created in ZAP using
EcoRI-digested Dictyostelium genomic DNA in the size
Figure 2. Peptide microsequences. Shown are the sequences ob-
tained from single peptides derived from p47, p41, p35, p21, p19,
and p16, and two from p116. Each is aligned with the deduced
protein sequence of a cDNA present in the Japanese Dictyostel-
ium cDNA sequencing project database (the protein’s identity
and accession number are shown). Exceptions are the p35 pep-
tide sequence, which is aligned with the sequence of capping pro-
tein (Hug et al., 1995), and the two p116 peptide sequences,
where no homologues were found.
ence, myosin I, can be isolated as a complex at physiologic
ionic strength, and suggest that p116 serves as the scaffold
for assembly of the complex.
Immunoprecipitation Reactions Support the Existence of
the Complex In Vivo, Confirm That Myosin I Interacts
with p116 Through Its SH3 Domain, and Indicate that
p116 Serves as the Scaffold for Assembly of the Complex
To look for the existence of the complex between myosin
I, Arp2/3, capping protein, and p116 in vivo, we performed
immunoprecipitation experiments using wild-type and
mutant cell extracts, antibodies to Dictyostelium myoB
(Jung and Hammer, 1990), myoC (Jung et al., 1996), and
capping protein (Hug et al., 1995), an antibody to yeast Arp3
(gift of Dr. Kathy Gould, Vanderbilt University, Nashville,
TN), and an antibody raised against p116 eluted from gels
of the purified complex (-p116-1; see Fig. 3 A, lane 6).
To look for the association of p116 with myosin I in
vivo, immunoprecipitations were made using wild-type
cell extracts and antibodies to either myoB (Fig. 4 A, lanes
1 and 2) or myoC (lanes 3 and 4). These immunoprecipi-
tates contained not only the corresponding myosin I heavy
Jung et al. Complex Containing Capping Protein, Arp2/3, and Myosin I 1485
range of 4.6–5.4 kb. The 4.8-kb EcoRI insert present in
several purified phage clones obtained was sequenced
on both strands (EMBL/GenBank/DDBJ accession no.
AF388524), revealing 3,150 nucleotides of protein cod-
ing sequence interrupted by four introns, whose precise
boundaries were confirmed by the sequencing of RT-PCR
products (data not shown). The complete ORF contains
1,050 amino acids with a calculated molecular mass of 115.8
kD, and possesses both peptides obtained from the mi-
crosequencing of p116 (data not shown), indicating that
this genomic clone does encode the 116-kD polypeptide
present in the SH3 domain column eluates. Despite the fact
that the two peptides obtained from p116 were not obvi-
ously present in Acan 125, and that antibodies to both pro-
teins do not cross react (data not shown), p116 is indeed
the Dictyostelium homologue of Acan 125, as can be seen
in a dot matrix comparison between the two (Fig. 5 A),
where a line of identity spans virtually the entire matrix.
Direct pair wise comparisons (data not shown) indicate
that the two proteins are 32% identical and 65% similar.
P116 and Acan 125 Exhibit the Same Domain Structure
and Bind Myosin I in a Similar Fashion
Fig. 5 B shows a schematic of the domain structure of p116
and Acan 125 (Xu et al., 1997). Like Acan 125, the central
456 amino acids of p116 are comprised of 16 copies of a
29-residue - structural unit known as a leucine-rich re-
peat (LRR). LRRs contain a loose consensus sequence
dominated by leucines, form amphipathic - structural
units, and mediate protein–protein interactions, either by
serving as the ligand binding site itself, or by increasing the
affinity and/or specificity of binding at a separate site
(Kobe and Deisenhofer, 1995). p116 and Acan 125 are
41% identical and 77% similar in this domain. NH
2
-termi-
nal of the LRR domain in both proteins are 190 amino
Figure 3. Far Western analysis of SH3 domain: p116 interaction,
evidence for an Acanthamoeba complex, Western blot analyses of
antibodies, mutant cell lines, identification of critical PXXP mo-
tifs, and developmental Westerns. (A) Lanes 1 and 2, blots of the
material eluted from the myoB SH3 domain (lane 1) and the
myoB PL SH3 domain (lane 2), and probed with biotinylated
myoB SH3 domain. The hash marks here and elsewhere refer to
the following molecular weight markers: 200, 116, 97, 66, 55, 37,
32, and 22 kD. Lanes 3–5, Western blots of the material eluted
from the Acanthamoeba MIC SH3 domain and probed with anti-
bodies to Acanthamoeba Acan 125 (lane 3), yeast Arp3 (lane 4),
and Dictyostelium capping protein (lane 5), using dilutions of
1:5,000, 1:3,000, and 1:3,000, respectively. Lanes 6–10, Western
blots of Dictyostelium whole cell extracts probed with antibodies
against Dictyostelium p116 (-p116-1, lane 6; -p116-2, lane 7;
-AP p116-2, lane 8), Dictyostelium Arp3 (lane 9), and Dictyo-
stelium p19 (lane 10), using dilutions of 1:2,000, 1:20,000, 1:1,000,
1:20,000, and 1:10,000, respectively. (B) Lanes 1–4, Western blots
of whole cell extracts from a myoB
/myoC
double mutant (lanes 2
and 4) and its parental strain JH10 (lanes 1 and 3), probed with
affinity-purified antibodies to myoB (lanes 1 and 2) or myoC
(lanes 3 and 4). Lanes 5–7, Western blot performed on whole cell
extracts from strain Ax3 (lane 5), a myoB null cell line expressing
myoB without its SH3 domain (myoBSH3) (lane 7), and an
equal mixture of these two extracts (lane 6), and probed for
myoB. Lanes 8–11, Western blot performed on whole cell ex-
tracts from three independent p116
cell lines (10, 20, and 30;
lanes 9–11) and their parental strain Ax3 (lane 8), and probed
with the anti-p116 antibody -AP p116-2. (C) Lanes 1–3: Western
blot of the eluates from glutathione beads loaded with unfused
GST (lane 1), a GST fusion protein containing the COOH-termi-
nal, proline-rich 203 residues of p116 (GST-CTPRO; lane 2), and
GST-CTPRO in which the two PXXP motifs between residues
956–973 were deleted (GST-CTPROPXXP; lane 3) and incu-
bated with a high-speed supernatant of lysed Dictyostelium. The
eluates were probed with a mixture of antibodies to myoB and
myoC (the arrow marks the position of their comigrating heavy
chains). Lanes 4 and 5, Western blots of the eluates from glu-
tathione beads loaded with unfused GST (lane 4) and a GST fu-
sion protein containing the NH
2
-terminal 179 residues of p116
(GST-NT; lane 5), and incubated with a high speed supernatant
of lysed Dictyostelium. The elutes were probed with a mixture of
antibodies to the and subunits of capping protein. (D) West-
ern blots of whole cell extracts from vegetative Ax3 cells (VEG), and cells starved on black filter supports for the indicated lengths of
time (1.25–8.75 h) and probed with -AP p116-2 (top) and -Arp3 (bottom). The cells had reached ripple stage, the time of peak che-
motactic aggregation, at 7 h.
The Journal of Cell Biology, Volume 153, 2001 1486
acids that, while conserved (35% identical, 65% similar),
are largely unremarkable in composition. By contrast, the
COOH-terminal 189 residues of p116 and 198 residues of
Acan 125 are remarkable in being proline-rich (24% in
p116, 26% in Acan 125; Fig. 5 B). Runs of five or six pro-
lines in a row, which represent profilin binding sites in pro-
teins like WASp and VASP (Zeile et al., 1998), are not
present in either p116 or Acan 125, however, consistent
with the fact that our complex lacks profilin.
As reported by Xu et al. (1997), the proline-rich domain
of Acan 125 contains two closely spaced PXXP motifs
(residues 980–983 and 989–992) that fit the consensus for
SH3 domain target sequences. Consistent with this, a fu-
sion protein containing the COOH-terminal 344 residues
of Acan 125 was found to bind to the isolated SH3 domain
of Acanthamoeba myosin IC, and this interaction was ab-
rogated by an 18-residue deletion spanning the two PXXP
motifs (Xu et al., 1997). We find that the COOH-terminal
proline-rich domain of p116 also contains two closely
spaced PXXP motifs (residues 959–962 and 968–971; Fig. 5
B). Moreover, a GST fusion protein containing the pro-
line-rich COOH terminus of p116 (residues 847–1050;
GST-CT PRO) pulls down myoB and myoC from high-
speed supernatants of lysed Dictyostelium (Fig. 3 C, lane
2), but not when residues 956–973, which span the two
PXXP motifs, have been deleted from the fusion protein
(Fig. 3 C, lane 3). Furthermore, the wild-type fusion pro-
tein fails to pull down myoB from extracts of cells express-
ing the truncated version of the protein lacking the SH3
domain (data not shown).
Identification of an Arp2/3 Activation Domain and
the Capping Protein Interaction Site in p116
Given that p116 and Acan 125 (see below) bind the Arp2/3
complex, we searched their sequences for evidence of the
domains that in WASp/SCAR proteins are responsible for
binding Arp2/3 (the acidic or A domain) and for accel-
erating Arp2/3-dependent actin nucleation (the WH2 do-
main, in combination with the A domain and, possibly,
an intervening cofilin-like sequence) (Higgs and Pollard,
1999; Welch, 1999). Neither p116 nor Acan 125 possesses a
highly concentrated patch of acidic residues containing a
trytophan, which together comprise the WASp/SCAR A
domain. Furthermore, neither protein contains a cofilin-
like sequence. Finally, neither protein contains a stretch of
Figure 4. Immunoprecipita-
tions. Shown throughout are
ECL-based Western blots
performed on the final im-
munoprecipitate. (A) Wild-
type (WT) cell extracts
(lanes 1–7) were immunopre-
cipitated (IP) with antibodies
to myoB (lanes 1 and 2),
myoC (lanes 3 and 4), and
p116 (lanes 5–7), and the
immunoprecipitates probed
(Western) with antibodies to
myoB (lanes 1 and 6), myoC
(lanes 3 and 7), and p116
(lanes 2, 4, and 5). Extracts
from cells expressing myoB
without its SH3 domain
(BSH3
; lanes 8 and 9) were
immunoprecipitated with an
antibody to myoB (lanes 8
and 9), and the immunopre-
cipitate probed with antibod-
ies to myoB (lane 8) and
p116 (lane 9). (B) Wild-type
cell extracts (lanes 1–3) were
immunoprecipitated with an-
tibodies to myoB (lane 1),
myoC (lane 2), and p116
(lane 3), and the immunopre-
cipitates probed with an antibody to yeast Arp3 (lanes 1–3). Extracts from myoB
/myoC
double mutants (B
/C
) (lane 4) were immu-
noprecipitated with an antibody to p116 (lane 4), and the immunoprecipitate probed with an antibody to yeast Arp3 (lane 4). Extracts
from cells expressing myoB without its SH3 domain (lane 5) were immunoprecipitated with an antibody to myoB (lane 5), and the im-
munoprecipitate probed with an antibody to yeast Arp3 (lane 5). (C) Wild-type extracts (lanes 1–4) were immunoprecipitated with pre-
immune serum (PI) (lane 1) and antibodies to myoB (lane 2), myoC (lane 3), and p116 (lane 4), and the immunoprecipitates probed
with an antibody to the subunit of capping protein (lanes 1–4). Extracts from myoB
/myoC
cells (lane 5) were immunoprecipitated
with an antibody to p116 (lane 5), and the immunoprecipitate probed with an antibody to capping protein (lane 5). Extracts from cells
expressing myoB without its SH3 domain (lane 6) were immunoprecipitated with an antibody to myoB (lane 6), and the immunoprecip-
itate probed with the antibody to capping protein (lane 6). We note that immunoprecipitates made from p116
cells using antibodies
to myoB or myoC were devoid of Arp3 and capping protein, consistent with the idea that the association of Arp2/3 and capping protein
with myosin I is via their interaction with p116 (data not shown).
Jung et al. Complex Containing Capping Protein, Arp2/3, and Myosin I 1487
sequence with strong similarity throughout to the 18-res-
idue WH2 domain, which participates in the binding of
G-actin by WASp/SCAR proteins (Machesky and Insall,
1998; Miki and Takenawa, 1998; Egile et al., 1999; Higgs et
al., 1999). Nevertheless, analysis of the 200 residues of
p116 and Acan 125 located between their LRR and
COOH-terminal proline-rich domains revealed two se-
quence features that could be functionally related to the
WH2 and A domains. The first is a 26-residue sequence in
p116 (residues 697–722), and the corresponding sequence
in Acan 125 (residues 712–737), both of which can be
aligned with residues 30–57 in verprolin (Fig. 5, B and C).
This portion of verprolin has been implicated in the bind-
ing of G-actin (Vaduva et al., 1997), and overlaps the por-
tion of the protein (residues 30–46) with significant homol-
ogy to the WH2 domain of WASp/SCAR (Higgs and
Pollard, 1999). Furthermore, contained within the verpro-
lin-like sequences of p116 and Acan 125 is a six-residue
motif that verprolin shares with several other G-actin-
binding proteins, including thymosin 4 and actobindin
(Vancompernolle et al., 1991; Vaduva et al., 1997) (Fig. 5
C). Cross-linking studies and mutational analyses have
shown that this six-residue sequence forms part of an actin
monomer-binding site in these latter two proteins (Van-
compernolle et al., 1991; Van Troys et al., 1996).
The second sequence feature, which lies COOH-termi-
nal of the verprolin-like sequence, is a region of 72 resi-
dues in p116 (792–863) and 55 residues in Acan 125 (871–
925), both of which exhibit a strong net negative charge
(Fig. 5 B). Specifically, these regions possess net charges of
10 for p116 (14 D/E, 4 K/R) and 16 for Acan 125 (17
D/E, 1 K/R).
To determine whether these p116/Acan 125 sequences
function like the WH2/A domains of WASp/SCAR to ac-
celerate Arp2/3-dependent actin nucleation (Machesky
and Insall, 1998; Higgs et al., 1999; Machesky et al., 1999;
Winter et al., 1999), actin assembly assays were performed
in the presence of the Arp2/3 complex, with and without
GST VA, a GST fusion protein containing the verprolin-
Figure 5. Dot matrix comparison, schematic of the domain or-
ganization of p116 and Acan 125, alignment of the verprolin-like
sequences, and acceleration of Arp2/3-dependent actin nucle-
ation by the VA domain of p116. (A) Dot matrix comparison be-
tween Dictyostelium p116 and Acanthamoeba Acan 125 (window
size, 30; stringency, 11). The dark rectangular region in the upper
right corner is due to the alignment of their repetitive proline-
rich sequences. (B) Schematic of the tripartite domain organiza-
tion of p116 and Acan 125, showing the percent identity and the
percent similarity (identities plus conservative substitutions in
parentheses) for each domain. The positions of the 16 LRRs, the
verprolin-like sequence that in verprolin has been implicated in
binding G-actin, the acidic region, the proline-rich domain, the
two PXXP motifs known to be critical for the interaction be-
tween Acan 125 and the SH3 domain of Acanthamoeba myosin
IC, and the two PXXP motifs deleted from p116 in this study, are
indicated. (C) Alignment of a portion of p116 and the homolo-
gous sequence in Acan 125 with a region of yeast verprolin that
contributes to the binding of G-actin, and with six-residue se-
quences present in thymosin 4 and actobindin that also contrib-
ute to binding monomeric actin. (D) Actin nucleation assays
were performed using 4 M G-actin (5% pyrene actin) with or
without the Arp2/3 complex and GST VA (see Materials and
Methods). Shown are the rates of polymerization for actin alone
(trace a, no symbols), actin plus 50 nM Arp2/3 complex (trace b,
squares), actin plus 3 M GST VA (trace c, open circles), and ac-
tin plus 50 nM Arp2/3 complex and 3 M GST VA (trace d,
closed circles). Similar results were obtained using two different
preparations of proteins. Addition of 3 M unfused GST did not
accelerate Arp2/3-dependent actin nucleation (data not shown).
The Journal of Cell Biology, Volume 153, 2001 1488
like and acidic sequences of p116 (residues 644–863; Fig. 5
D). As seen by others, Arp2/3 alone had little effect on ac-
tin nucleation (trace b; trace a is actin alone). Similarly,
addition of GST VA alone had no obvious effect on the
rate of actin assembly (trace c). However, the addition of
both together resulted in a significant acceleration of actin
polymerization (trace d). Although these results indicate
that the VA domain of p116 activates Arp2/3-dependent
actin nucleation, the concentration of GST VA required to
obtain this level of activation (low M) is significantly
higher than that needed for the isolated WH2/A domain
of WASp/SCAR (Higgs and Pollard, 1999). Therefore, ei-
ther the ability of intact p116 to activate Arp2/3-depen-
dent actin nucleation is relatively modest, or analysis of
the isolated VA domain underestimates the activating po-
tential of the intact protein (see Discussion).
In an effort to identify the binding site in p116 for cap-
ping protein, the NH
2
-terminal 179 residues of p116, which
spans the region from the ATG to the start of the LRR do-
main, was expressed as a GST fusion protein (GST NT)
and incubated with a high speed supernatant of lysed Dic-
tyostelium. Western blots of the material eluted from this
resin revealed the presence of the and subunits of cap-
ping protein (Fig. 3 C, lane 5). By contrast, the material
eluted from GST alone (Fig. 3 C, lane 4), GST VA, or
GST CT PRO (data not shown) did not contain capping
protein. These results are consistent with the presence of a
capping protein interaction site within the NH
2
-terminal
179 residues of p116.
Acan 125 Also Interacts with the Arp2/3
Complex and Capping Protein
The identification of p116 as a homologue of Acan 125 im-
plies that Acan 125 should also interact with the Arp2/3
complex and capping protein, in addition to myosin I. To
test for this, we performed biochemical experiments like
those in Fig. 1 using the SH3 domain of Acanthamoeba
myosin IC and Acanthamoeba cell lysates. Western blots
(Fig. 3 A, lanes 3–5) indicated that the eluate contained
not only Acan 125 (lane 3), as expected, but also Arp 3
(lane 4) and the subunit of capping protein (lane 5).
These results are consistent with the idea that a portion of
cellular Acan 125 is present in a complex similar to the one
identified here for p116.
Homologues of p116 and Acan 125
Are Present in Metazoans
A previous search for metazoan homologues to Acan 125
(Xu et al., 1997) revealed a Caenorhabditis elegans ORF of
1,000 residues (EMBL/GenBank/DDBJ accession no.
Z71264) that showed significant similarity to Acan 125 pri-
marily over the NH
2
-terminal 640 amino acids of both
proteins. Our analysis of this C. elegans sequence using an
updated sequence file (EMBL/GenBank/DDBJ accession
no. CAA95828.1) reveals a 998-residue polypeptide that
can be aligned with p116 and Acan 125 from NH
2
to
COOH terminus (data not shown). Our searches also
identified a Drosophila melanogaster ORF of 1,369 amino
acids (EMBL/GenBank/DDBJ accession no. AE003840),
which exhibits significant sequence similarity to p116 and
Acan 125 over much of the protein (data not shown). Im-
portantly, searches performed using this D. melanogas-
ter sequence identified putative homologues in humans
(EMBL/GenBank/DDBJ accession no. CAA18156.1 and
BAA90912.1) and mouse (EMBL/GenBank/DDBJ acces-
sion no. AA407423.1 and AA118567.1). The human ORF
exhibits 24% identity and 40% similarity to residues 647–
1196 of the D. melanogaster sequence.
P116 Localizes Along with MyoB, MyoC, and the Arp2/3
Complex in Dynamic Actin-rich Cellular Extensions,
Including Macropinocytic Crowns and the Leading Edge
of Locomoting Cells
We next sought to determine the intracellular localization
of p116, and to see if it, myosin I, and the Arp2/3 complex
localize to the same cellular structures. We used previously
characterized antibodies to myoB and myoC (Jung and
Hammer, 1990; Jung et al., 1996; Fig. 3 B, lanes 1–4), newly
generated antibodies to the Dictyostelium Arp2/3 complex
components Arp3 and p19 (p21-Arc; Fig. 3 A, lanes 9 and
10, respectively), and a second new antibody to p116
(-p116-2), which was raised against the proline-rich domain
of p116 and purified by absorption against p116 null cell
extracts (-AP-p116-2; Fig. 3 A, lanes 7 and 8, respec-
tively). Given that myoB and myoC in Dictyostelium, and
the Arp2/3 complex in other cell types, have been shown to
localize to regions of active actin assembly (Uyeda and Ti-
tus, 1997; Higgs and Pollard, 1999), cells were sometimes
double-stained for actin or for the actin-binding protein
coronin, which accumulates in actin-rich structures (De
Hostos, 1999). We also examined both vegetative and
starved cells because p116 and Arp3 (Fig. 3 D), as well as
myoB and myoC (Jung et al., 1996), are expressed at both
stages, and because cells at these two stages differ signifi-
cantly in terms of shape, speed, and the site where actin as-
sembly is most pronounced (Varnum et al., 1985; Spudich,
1987). Vegetative cells are relatively round, slow moving
(1 m/min), and exhibit dramatic actin assembly prima-
rily on their dorsal surface, where cuplike membrane ex-
tensions rich in F-actin and coronin are continuously being
extended upwards (De Hostos, 1999). These structures,
known variously as cups, crowns, or amoebastomes, are
macropinocytic structures that rise up, close, and retreat
into the cell, bringing with them large fluid-filled macropi-
nosomes (Hacker et al., 1997). Starved cells undergoing
chemotactic migration towards extracellular cAMP are
highly elongated, fast moving (12 m/min), and exhibit
dramatic actin assembly primarily in the pseudopods and
lamellopods at their leading edge.
With regard to vegetative cells, Fig. 6 A shows a Z series
of a cell that had been double stained for Arp3 and actin.
Striking colocalization between the two (yellow) is seen al-
most exclusively in dorsal confocal sections, where both
are concentrated throughout a cup-shaped macropinocytic
crown. Very similar images were obtained by double stain-
ing vegetative cells for Arp3 and coronin, a bona fide marker
for these dorsal macropinocytic extensions (De Hostos,
1999), and by using the antibody to p19 instead of Arp3
(data not shown). Examination of the ventral confocal sec-
tions in Fig. 6 A, and the single ventral confocal section
shown for the two cells in Fig. 6 B (top), indicates that the
Arp2/3 complex, while present within the ventral actin–rich
lamellae of vegetative cells (marked by arrows in upper “Ac-
Jung et al. Complex Containing Capping Protein, Arp2/3, and Myosin I 1489
Figure 6. Localization of the Arp2/3
complex and p116 in vegetative cells. (A)
Z series (0.7 m sections) of a vegetative
cell double stained for actin and Arp3
(top, ventral; bottom, dorsal). The corre-
sponding overlaid (actin, red; Arp3, green;
colocalization, yellow) and DIC images
are also shown. (B) A single, ventral, 1
m-confocal section through vegetative
cells double stained for actin and Arp3
(top) or actin and p116 (bottom). The cor-
responding overlaid (red, actin; green,
Arp3 or p116; colocalization, yellow) and
DIC images are also shown. The arrows in
the two Actin panels point to the actin-
rich ventral lamella, whereas the arrow-
heads in the Arp3 and p116 panels point
to the prominent spots of staining seen for
both antibodies just inside the lamella.
Bars: (A) 4.6; (B) 7.2 m.
The Journal of Cell Biology, Volume 153, 2001 1490
tin” panel), is not highly concentrated there. This may reflect
the fact that the requirement for actin assembly in these
lamellae, which are not driving rapid cell migration, is signif-
icantly less than in dorsal crowns, which are driving constitu-
tive endocytosis. Interestingly, Arp3 is often concentrated in
bright spots that accumulate just inside the ventral lamellae
(see arrowheads in Fig. 6 B, top, “Arp3” panel). The nature
and function of these spots is currently unknown.
Previous work by Novak and Titus (1998) showed that
myoB is concentrated in macropinocytic crowns. We now
find the same result for myoC (Fig. 7 A; arrowheads point
to a myoC-positive crown). Furthermore, p116 is also
present in these macropinocytic structures, as can be seen
for two representative cells in Fig. 7 B, where the promi-
nent macropinocytic crowns evident in the DIC images
(see arrowheads) are strongly stained for p116 using anti-
body -APp116-2. Similar results were obtained using an-
tibody -p116-1, and p116 null cells showed negligible
staining with both antibodies, as expected (data not
shown). Interestingly, p116, like Arp3, is not particularly
concentrated in the ventral actin–rich lamellae of vegeta-
tive cells (Fig. 6 B, bottom). Furthermore, p116, like Arp3,
is often present in prominent spots that accumulate just in-
side these lamellae (see arrowheads in Fig. 6 B, bottom,
“p116” panel). Whether these p116-rich spots are the same
as the Arp3-rich spots described above remains to be de-
termined.
With regard to starved cells, Fig. 8 A shows a projected
image of a field of such cells that were double stained for
actin and Arp3. These cells, which were undergoing che-
motactic migration towards the upper left upon fixation,
exhibit a striking concentration of actin and Arp3 within
the pseudopods and lamellopods at their leading edge.
Similar results were obtained using the antibody to p19
(data not shown). Previous work by Fukui et al. (1989) and
Jung et al. (1996) has shown that myoB and myoC are also
concentrated in these leading edge pseudopods. Fig. 8 B
shows that p116 is concentrated there as well. Examina-
tion of the individual confocal sections that make up this
projected image indicated that actin and p116 colocalize
throughout the leading edge.
Cells Lacking p116 Exhibit Defects in Fluid Phase
Endocytosis, the Formation of Macropinocytic Crowns
and the Efficiency of Chemotactic Aggregation, and a
Reduction in Cellular F-Actin Content
We created p116
cell lines in an effort to gauge the physi-
ologic importance of the protein. Ax3 cells were trans-
formed with a linear gene disruption fragment comprised
of the selectable marker blasticidin flanked by portions of
the p116 gene (see Materials and Methods). Approxi-
mately 20% of 60 purified blasticidin-resistant transfor-
mants were found by Western blot analysis to lack p116.
Three independent clones were chosen for further study
(clones 10, 20, and 30). Fig. 3 B, lanes 8–11, show that
these clones are completely devoid of p116 protein, and
Southern blot analyses indicated that the linear disruption
fragment had indeed disrupted the p116 locus, and only
this locus (data not shown). All three clones gave essen-
tially identical results in the behavioral assays described
below.
Given that p116 localizes to macropincytic crowns,
that macropinocytosis appears to account for most of
fluid phase endocytosis in axenically grown Dictyostelium
(Hacker et al., 1997), and that axenically grown p116 null
cells exhibit a 35% reduction in growth rate relative to the
parental cell line (doubling times: 8.5 h for Ax3, 12 h
for p116
), we compared the rate of uptake of the fluid
phase marker FITC-dextran in control and p116
cells.
Figure 7. Localization of MyoC and p116 in vegetative cells.
(A) Z series (1-m sections) of a vegetative cell stained for myoC
(left, ventral; right, dorsal). Arrowheads in the corresponding
DIC images mark the prominent myoC-positive crown. (B) Two
sets of Z series (1-m sections) of vegetative cells stained for
p116 using -AP-p116-2 (top, ventral; bottom, dorsal). Arrow-
heads in the corresponding DIC images mark the dorsal p116-
positive crowns. Bars: (A) 2.2; (B) 4.4 m.
Jung et al. Complex Containing Capping Protein, Arp2/3, and Myosin I 1491
Figure 8. Localization of the Arp2/3 complex and p116 in
starved, chemotaxing cells. (A) A projected image of a field of
cells undergoing chemotactic aggregation that were double
stained for actin and Arp3 (the cells are moving towards the up-
per left). The corresponding overlaid (actin, red; Arp3, green;
colocalization, yellow) and DIC images are also shown. (B) A
projected image of a field of cells undergoing chemotactic aggre-
gation that were double stained for actin and p116 (the cells are
moving towards the left). The corresponding overlaid (actin, red;
p116, green; colocalization, yellow) and DIC images are also
shown. Bars: (A) 6.5; (B) 12 m.
Figure 9. Rate of fluid phase endocytosis, and crown size, in wild-
type and p116
cells. (A) Kinetics of accumulation of the fluid
phase pinocytic marker FITC-dextran (arbitrary units) within
three independent p116
cell lines and the parental line Ax3 (see
key). Each value is the mean of duplicate samples. (B) Histogram
of the height of crowns in Ax3 and p116
cells (see key). The val-
ues, which were taken from confocal Z series of cells stained for
actin, were binned in 0.7-m intervals and are presented as a per-
cent of total crowns counted (64 in Ax3, 58 in p116
). Crowns
were counted only if they rose almost vertically from the dorsal
surface of cells. The bottom of such crowns typically exhibited a
spherical, solid disc of actin staining. Above this the crown had the
appearance of a ring of fluorescence in each section. Similar results
were obtained using cells stained for coronin.
The Journal of Cell Biology, Volume 153, 2001 1492
The rate of accumulation of this marker within cells during
the first 60 min after its addition to the medium accu-
rately reports the rate of fluid phase endocytosis because
the time interval between endocytosis and exocytosis of
nondigestable markers in Dictyostelium, which lack a
rapid recycling component, is 60 min (Klein and Satre,
1986; Jung et al., 1996). Fig. 9 A shows that all three p116
cell lines exhibit an 45% reduction in the rate of fluid
phase endocytosis relative to the parental cell line. Mu-
tants also appear to have a smaller intracellular endocytic
compartment, as the amount of cell-associated dextran at
steady state, which is attained in 75 to 90 min, and repre-
sents the point of equivalence between marker uptake and
exocytosis, is about half the value of control cells.
To identify a structural basis for the reduced rate of
macropinocytosis, control and p116
cells in growth media
were fixed, stained for actin, optically sectioned at 0.2-m
intervals, and the sections rendered in three dimensions to
visualize the shape and size of macropinocytic crowns. Fig.
10, A and B, show control cells at 30 and 90 degrees of tilt.
These cells typically have one or two large and well-
formed macropinocytic cups on their dorsal surfaces (see
arrowheads). By contrast, p116
cells possess much less
robust crowns on their dorsal surface (Fig. 10, C and
D, see arrowheads; to view an image series that pro-
gresses gradually from 30 to 90 degrees of tilt, go to
www.nhlbi.nih.gov/labs/cellbiology/index.htm). To quan-
tify this apparent difference, we measured the height of
dorsal crowns in confocal images obtained using 0.2–0.7
m steps, binned the heights in 0.7-m intervals, and
graphed the values as a percentage of crowns counted
(Fig. 9 B). As one would predict from the fact that this ap-
proach captures crowns at all stages of formation, the val-
ues obtained exhibit a gaussian distribution. Importantly,
this distribution for p116
cells is shifted relative to con-
trol cells towards an obviously shorter height. Indeed,
p116
cells exhibit a mean crown height of 2.4 m, versus
4.3 m for Ax3 cells. Furthermore, no crowns of 3.5 m
were found in p116
cells, whereas 78% of crowns in Ax3
cells were 3.5 m in height. Therefore, although p116
cells can still form crowns, they do so less effectively than
wild-type cells, and there is a striking defect in the forma-
tion of large well-formed macropinocytic cups when p116
is absent. We suggest that this structural defect underlies
the decreased rate of endocytosis exhibited by p116
cells.
Given the striking concentration of p116 within leading
edge pseudopods, we also compared the ability of control
and p116
cells to undergo starvation-induced chemotac-
tic aggregation in streaming assays. Fig. 11 shows the typi-
cal result obtained when an initial cell density of 10
5
cells/
cm
2
was used. p116
cells began to aggregate 3 h after
control cells (compare Fig. 11, H and A), and by 13 h,
when control cells had nearly completed streaming and
the formation of large, robust aggregates (Panel D), p116
cells had formed only small, loosely organized clumps
(Fig. 11, I). Although this difference was accentuated at
lower starting cell densities, p116
cells lagged behind con-
trol cells and formed much smaller streams and aggregates
even at higher initial cell densities (e.g., 3 10
5
cells/
cm
2
; data not shown). Therefore, although p116
cells can
still undergo chemotactic aggregation, they do so much
less efficiently than wild-type cells, and form only small ag-
gregates (Fig. 11, J). At least part of this defect may be due
to a reduction in cellular F-actin content, as aggregating
(10 h starved) p116
cells contain 77 2.3% as much
F-actin as aggregating (6 h starved) control cells (n 8).
This reduction could be due to the loss of that portion of
Arp2/3-dependent actin nucleation that is stimulated by
p116, to an increase in the level of active capping protein,
or to a combination of both (see Discussion).
Figure 10. The appearance of crowns in wild-type and p116
cells.
Vegetative Ax3 and p116
cells were stained for actin, sectioned in
0.2-m intervals, and the sections rendered in three dimensions.
Shown are images at 30 and 90 degrees of tilt for Ax3 (A and B)
and p116
(C and D) cells. Arrowheads point to the dorsal
crowns. The hollow center of these crowns is especially obvious
when viewed from above (i.e., 90 degrees; see, for example, the
crown marked by the lower arrowhead in B), whereas their sides
are most evident when viewed from an angle that is somewhat
above 0 degrees (i.e., 30 degrees; see, for example, the same
crown mentioned above, but now in A). To view an image series
that progresses gradually from 30 to 90 degrees of tilt, go to
www.nhlbi.nih.gov/labs/cellbiology/index.htm. Bar, 3.4 m.
Jung et al. Complex Containing Capping Protein, Arp2/3, and Myosin I 1493
Discussion
Identification and Organization of the Complex Containing
CARMIL, Myosin I, Arp2/3, and Capping Protein
The main goal of this study was to identify through bio-
chemical means proteins that bind to the SH3 domains of
Dictyostelium myoB and myoC. Using these domains as
affinity matrices, we isolated p116, the Dictyostelium ho-
mologue of Acan 125 (Xu et al., 1995). Our SH3 domain
column eluates also contained on a consistent basis nine
other polypeptides, which we identified as the seven mem-
bers of the Arp2/3 complex and the and subunits of
capping protein. Immunoprecipitation reactions and other
experiments provided evidence that at least some fraction
of cellular myoB and myoC is present in a complex with
p116, Arp2/3, and capping protein in vivo, and that p116
serves as a scaffold for assembly of the complex, binding
myosin I, capping protein, and Arp2/3 at independent
sites. Given its central role in complex formation, we pro-
pose the name CARMIL for p116, which stands for cap-
ping protein, Arp2/3, myosin I linker.
In terms of how the complex is organized, Fig. 12 pre-
sents a working model based on our efforts to identify the
sites in CARMIL where myosin I, capping protein, and
the Arp2/3 complex bind. First, deletion analyses indi-
cated that the myosin I SH3 domain binds to CARMIL
through one or two closely spaced PXXP motifs present
within CARMIL’s COOH-terminal, proline-rich domain.
These latter results agree with those of Xu et al. (1997)
and Zot et al. (2000), who have shown that one or both of
two PXXP motifs present in the analogous region of Acan
125 are necessary and sufficient to drive its interaction
with myosin I. Second, the ability of a fusion protein con-
taining the N-terminal 179 residues of CARMIL to bind
capping protein present in cell lysates implicates this por-
tion of CARMIL in its interaction with capping protein.
Finally, the ability of a fusion protein containing the ver-
prolin-like and acidic sequences of CARMIL to accelerate
Arp2/3-dependent actin nucleation implicates the region
between the LRR domain and COOH-terminal proline-
rich domain in binding the Arp2/3 complex.
In terms of the affinities of these interactions, Lee et al.
(1999) and Zot et al. (2000) have measured binding con-
stants in the range of 20–150 nM for the interaction be-
tween Acan 125 and the SH3 domain of myosin I. These
values, together with the cellular concentrations of myosin
I and Acan 125 (1 and 2 M, respectively), suggest
that type I myosins and the CARMIL protein may be
largely associated in vivo, barring some type of regulation
(Lee et al., 1999). Although we have not measured affini-
ties for the interaction of CARMIL with capping protein
and Arp2/3, the fact that the amount of Arp2/3 in SH3 do-
main column eluates sometimes approached apparent
stoichiometry with CARMIL, and that the amount of capping
protein almost always appeared to be approximately
stoichiometric with CARMIL, suggests that the affinities of
Arp2/3 and capping protein for CARMIL are fairly high.
Having said this, we note that the concentration of GST
VA required to activate Arp2/3-dependent actin nucle-
ation is much higher than that required for GST fusion
proteins containing the VCA domain of WASp (Higgs and
Figure 11. Streaming assays. In these assays, vegetative cells are
plated at moderate densities in the presence of a buffered salt so-
lution. After 8 h of starvation, normal cells begin to undergo
chemotactic migration towards certain cells in the population that
have begun to secrete cAMP. Cells initially migrate individually,
but soon express surface adhesion proteins, causing them to mi-
grate towards the source of extracellular cAMP as elongated ag-
gregates (“streams”) (Spudich, 1987). Shown are Ax3 cells (A–E)
and p116
cells (F–J) undergoing this starvation-induced chemo-
tactic streaming and aggregation. The numbers to the left indicate
the time in hours between when the cells were plated in SB (0 h)
and when each pair of images was taken.
The Journal of Cell Biology, Volume 153, 2001 1494
Pollard, 1999). Although this may mean that CARMIL’s
ability to activate Arp2/3 is relatively modest, it is also pos-
sible that the activity of GST VA significantly underesti-
mates the activating potential of the intact protein. For ex-
ample, the optimal conformation of the VA domain for
activation of Arp2/3 may be dependent on its presence in
the intact protein. Relevant to this, LRR domains have
been shown to increase the affinity of adjacent sites for
their ligands (Kobe and Deisenhofer, 1995). Resolution
of this question must await characterization of purified
CARMIL.
Localization of CARMIL, Phenotype of CARMIL Null
Cells, and Implications for Myosin I Mutant Phenotypes
Previous studies have shown that myoB and myoC in Dic-
tyostelium (Uyeda and Titus, 1997), the Arp2/3 complex in
other cell types (Higgs and Pollard, 1999; Machesky and
Insall, 1999), and capping protein in Acanthamoeba (Coo-
per et al., 1984) are all concentrated in dynamic, actin-rich
cellular extensions. We find that CARMIL is concentrated
along with myoB, myoC, and Arp2/3 within two such re-
gions in Dictyostelium: crown-shaped macropinocytic ex-
tensions on the dorsal surface of vegetative cells, and psue-
dopods at the leading edge of starved cells undergoing
chemotactic migration. Although this apparent colocaliza-
tion does not prove the existence of the complex, it is con-
sistent with the possibility that these proteins interact
physically at least part of the time. The localization data
also suggest that CARMIL plays a role in the structure
and function of dynamic, actin-rich extensions associated
with endocytosis and chemotaxis. Consistent with this,
cells that lack CARMIL exhibit reduced rates of fluid
phase endocytosis, a defect in the formation of the dorsal
crowns responsible for this fluid uptake, and a reduction in
the efficiency of chemotactic aggregation. Therefore, we
conclude that CARMIL is physiologically important, that
the behavioral defects exhibited by CARMIL null cells
correlate with the localization of the protein to crowns and
the leading edge, where myoB, myoC, and the Arp2/3
complex also localize, and that CARMIL plays a signifi-
cant role in the structure and function of these actin-rich
cellular extensions.
Novak and Titus (1997, 1998) have shown that the abil-
ity of Dictyostelium myoB to rescue the defect in endocy-
tosis exhibited by myoB/myoC double mutants, and the
deleterious effects of myoB overexpression on pinocytosis
and cell migration, are all eliminated by deletion of the
myosin’s SH3 domain. Similarly, the SH3 domains of the
two yeast type I myosins are required for these myosins to
exert most of their positive effects on actin cytoskeletal or-
ganization (Anderson et al., 1998; see below). Therefore,
the in vivo functions of these type I myosins appear to be
critically dependent on their ability to interact with the
targets of their SH3 domains. We now suggest, based on
our demonstration here, that the target for the SH3 do-
mains of Dictyostelium myoB and myoC also interacts
with Arp2/3 and capping protein, that the phenotypes of
Figure 12. Working model
for the complex between my-
osin I, CARMIL, Arp2/3, and
capping protein. In this work-
ing model, CARMIL serves
as the scaffold for assembly of
the complex. CARMIL, and
the most conspicuous do-
mains within it (NT, NH
2
-ter-
minal domain; LRR, leu-
cine-rich repeat domain; V,
verprolin-like sequence; A,
acidic domain; Pro-Rich, pro-
line-rich COOH-terminal do-
main), are shown approxi-
mately to scale, whereas the
remaining proteins are not.
The binding of G-actin is in-
ferred from the sequence of
CARMIL and the activation
data. The large black arrow
signifies that myosin I might
move the complex towards the barbed end of actin filaments, making this complex the “cargo”. The model also suggests that the LRR do-
main may be the site where upstream signaling molecules bind (e.g., Ras-related GTPases), based on the fact that the LRR domains in
yeast adenylate cyclase (Suzuki et al., 1990) and the mouse protein rsp-1 (Masuelli and Cutler, 1996) bind ras-related GTPases. If this were
also the case for the LRR domains of p116 and Acan 125, it would provide a mechanism through which the activities of the complex could
be regulated by signal transduction pathways known to regulate actin assembly in other contexts. The fact that we obtained complexes
from the SH3 column in which both capping protein and the Arp2/3 complex appear to be approximately stoichiometric with CARMIL
makes it very unlikely that their interaction with CARMIL is mutually exclusive. With regard to dependent binding, the fact that we
sometimes obtained complexes with much more capping protein than Arp2/3 argues that capping protein does not bind to CARMIL
through Arp2/3. Having said this, we never obtained complexes in which the amount of Arp2/3 complex exceeded that of capping protein.
Based on this observation alone, we cannot exclude the possibility that Arp2/3 binds to CARMIL through capping protein. However, this
seems unlikely based on our demonstration that different parts of CARMIL are responsible for binding capping protein and for activating
Arp2/3, and based on the fact that no one has reported that capping protein binds the pseudobarbed end of the Arp2/3 complex.
Jung et al. Complex Containing Capping Protein, Arp2/3, and Myosin I 1495
myoB
/myoC
mutants, which include reduced rates of
pinocytosis and cell migration (Ostap and Pollard, 1996a),
might be due at least in part to defects in the assembly
state and organization of actin. These defects could arise
from alterations in the localization, activity, and/or move-
ment of the complex caused by the loss of myosin I. This
role for myosin I, together with its role in generating ten-
sion within the actin cortex (Dai et al., 1999) as a result of
its ability to cross link actin filaments and collapse isotro-
pic actin mesh works (Fujisaki et al., 1985), and its role in
membrane/organelle movements (Neuhaus and Soldati,
2000), may be largely responsible for the defects exhibited
by myosin I mutants in Dictyostelium.
Biochemical Properties of the CARMIL Protein
CARMIL possesses at least three biochemical functions.
First, it binds the Arp2/3 complex and, based on analysis
of an internal fragment, has at least a modest ability to ac-
tivate Arp2/3-dependent actin nucleation. Second, it binds
capping protein. The critical question regarding this inter-
action is whether capping protein retains its ability to cap
the barbed ends of actin filaments when bound to CAR-
MIL. If this does not occur, then CARMIL’s main func-
tion as regards capping protein may be to regulate the
functional levels of the protein. Given the cellular concen-
tration of CARMIL (2 M, versus 1 M for capping
protein; Lee et al., 1999), and the likelihood that CAR-
MIL has a relatively high affinity for capping protein (see
above), CARMIL could be a very significant competitor
with actin filament barbed ends for free capping protein.
Depending on the local concentrations of reactants, and
on possible regulation of CARMIL–capping protein inter-
action, CARMIL could drive both the uncapping of fila-
ments and the accelerated capping of filaments by the
binding and release of capping protein, respectively. How-
ever, the situation will be quite different if the complex of
CARMIL and capping protein can cap, and, by extension,
if CARMIL can bind to capping protein present at the
ends of capped filaments. If these interactions occur, then
the complex could, by virtue of its ability to also recruit
and activate the Arp2/3 complex, drive the formation of a
new filament off of a capped barbed end. This could repre-
sent a novel mechanism for nucleating actin filaments (see
also Pantaloni et al., 2000). Resolution of these and other
important questions, such as how the stimulation of Arp2/
3-dependent actin nucleation by CARMIL is regulated,
should go a long way towards answering the more general
question of why this complex binds both the principal nu-
cleator and terminator of actin filament assembly.
Finally, CARMIL binds myosin I. Given that type I my-
osins are bona fide, barbed end–directed motors, the myo-
sin I bound to CARMIL may function to translocate the
complex towards the barbed end of existing actin fila-
ments (Fig. 12), thereby serving to concentrate CARMIL
and the Arp2/3 complex at the interface between the ac-
tin-rich cortex and the plasma membrane. Actin poly-
merization in this zone would most effectively drive pro-
trusion. The added ability of type I myosins to move
membranes relative to actin (Zot et al., 1992) might allow
them, in conjunction with their ability to target machinery
regulating actin assembly, to facilitate the growth of fila-
ments directly abutting the plasma membrane. However,
for all of these mechanisms of action, the fact that myosin I
cannot function at low density as a processive motor (Os-
tap and Pollard, 1996b) must be taken into consideration
(see Machesky, 2000).
Generality of Our Results
Several recent papers have revealed important physical
and functional connections between the two type I myo-
sins in yeast and the Arp2/3 complex. First, Anderson et
al. (1998) reported that the SH3 domains of Myo3p and
Myo5p bind to verprolin. The subsequent demonstra-
tion that verprolin interacts physically and genetically
with Las17p/Bee1p (Naqvi et al., 1998), together with the
fact that Las17p/Bee1p, like its mammalian homologue
WASp, binds to and activates the Arp2/3 complex (Ma-
chesky and Insall, 1998; Winter et al., 1999), provided an
indirect connection between the yeast type I myosins and
the Arp2/3 complex. This connection has been strength-
ened by the recent work of Evangelista et al. (2000) and
Lechler et al. (2000), both of whom showed that the SH3
domains of Myo3p and Myo5p interact with Las17p/Bee1p
as well with verprolin, and that an acidic sequence at the
COOH terminus of Myo3p and Myo5p, which is homolo-
gous to the acidic, Arp2/3-binding A domain of Las17p/
Bee1p and other WASp/SCAR proteins, binds the Arp2/3
complex. Loss of the acidic motifs of either Las17p/Bee1p
or the type I myosins did not cause a dramatic cellular
phenotype, whereas loss of both led to striking defects in
growth, actin organization, and F-actin content (Evange-
lista et al., 2000). Similarly, the ability of actin to polymer-
ize in the cortex of permeabilized yeast cells was normal
when supplemental extracts contain either acidic motif,
but dramatically impaired when extracts lacked both mo-
tifs (Lechler et al., 2000). These results suggest that the A
domains of myosin I and Las17p/Bee1p function redun-
dantly in the activation of the Arp2/3 complex (see also
the recent results for the fission yeast myosin I, Myo1p;
Lee et al. 2000). Given that the mechanism of this activa-
tion appears to require a WH2/V domain–dependent
G-actin binding activity as well as an acidic domain (Ma-
chesky and Insall, 1998; Egile et al., 1999; Higgs et al.,
1999), and that the yeast type I myosins possess only the
latter, it seems likely that the activation pathway used by
these myosins requires their SH3 domain–dependent in-
teraction with verprolin. In this way the WH2/V domain of
verprolin could act in trans with the acidic domain of the
type I myosins to activate the Arp2/3 complex.
To date, the only type I myosins that have been found to
possess a COOH-terminal acidic domain are those from
fungi (Evangelista et al., 2000), suggesting that the direct
interaction between type I myosins and the Arp2/3 com-
plex is fungal-specific. Furthermore, connections between
type I myosins and proteins that interact with the Arp2/3
complex either directly or indirectly have been made only
in yeast, raising the question of how general these interac-
tions are. Here we show that type I myosins in Dictyostel-
ium are also connected to the Arp2/3 complex, albeit by a
different mechanism that involves the CARMIL protein.
Moreover, the Dictyostelium complex contains capping
protein as well as Arp2/3. Interestingly, these two compo-
nents, together with cofilin, have recently been shown to
be sufficient to drive actin-based motility (Loisel et al.,
The Journal of Cell Biology, Volume 153, 2001 1496
1999). We note that CARMIL represents the first cyto-
skeletal protein other than actin that has been shown to
bind capping protein. We also note that CARMIL repre-
sents a novel Arp2/3 activator since it exhibits no sequence
similarity with ActA (Welch et al., 1998), cortactin (Uruno
et al., 2001; Weaver et al., 2001), or WASp/SCAR family
proteins (Welch, 1999) beyond a short sequence shared by
many proteins that bind G-actin. Finally, our efforts to
identify metazoan homologues of CARMIL confirmed
and extended those of Xu et al. (1997) regarding a C. ele-
gans homologue, identified a D. melanogaster homologue,
and, using this latter sequence for searches, identified
CARMIL homologues in mouse and human. These re-
sults, and the fact that metazoans also contain type I myo-
sins with SH3 domains (Sellers, 1999), suggest that the
complex we have identified may be present in many eu-
karyotic organisms.
We thank John Cooper, Kathy Gould, and Eugenio De Hostos for antibod-
ies, Blair Bowers for help with microscopy, and Edward D. Korn for com-
ments on the manuscript.
Submitted: 25 April 2001
Accepted: 11 May 2001
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