Download Molecular cloning, over-expression, developmental regulation and

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Journal of Cell Science 110, 1215-1226 (1997)
Printed in Great Britain © The Company of Biologists Limited 1997
JCS9565
Molecular cloning, over-expression, developmental regulation and
immunolocalization of fragminP, a gelsolin-related actin-binding protein from
Physarum polycephalum plasmodia
Davy T’Jampens1, Kris Meerschaert1, Bruno Constantin1, Juliet Bailey2, Lynnette J. Cook2, Veerle De
Corte1, Hans De Mol1, Mark Goethals1, José Van Damme1, Joël Vandekerckhove1 and Jan Gettemans1,*
1Flanders
Interuniversity Institute of Biotechnology, Department of Biochemistry, Universiteit Gent, Ledeganckstraat 35, B-9000
Gent, Belgium
2University of Leicester, Department of Genetics, University Road, Leicester LE1 7RH, UK
*Author for correspondence (e-mail: [email protected])
SUMMARY
FragminP is a Ca2+-dependent actin-binding and microfilament regulatory protein of the gelsolin family. We
screened a Physarum polycephalum cDNA library with
polyclonal fragminP antibodies and isolated a cDNA clone
of 1,104 bp encoding 368 amino acids of fragminP,
revealing two consensus phosphatidylinositol 4,5 bisphosphate-binding motifs in the central part of the protein. The
first methionine is modified by an acetyl group, and three
amino acids were missing from the protein coded for by the
cDNA clone. Full-length recombinant fragminP was
generated by PCR, purified after over-expression from
Escherichia coli and displayed identical properties to native
Physarum fragminP. Northern blot analysis against RNA,
isolated from cultures at various stages of development,
indicated that fragminP is absent from amoebae and that
expression is initiated at an early stage during apogamic
development, in a similar way to that observed for the
profilin genes. In situ immunolocalization of fragminP in
Physarum microplasmodia revealed that the protein is
localized predominantly at the plasma membrane, suggesting a role in the regulation of the subcortical actin
meshwork. Our data indicate that we have isolated the
plasmodium-specific fragminP cDNA (frgP) and suggest
that, in each of its two vegetative cell types, P. polycephalum
uses a different fragmin isoform that performs different
functions.
INTRODUCTION
been elucidated (Uyeda et al., 1988). Fragmins A and P differ
in molecular mass (40 kDa and 42 kDa, respectively) and the
proteins are also immunologically distinct (Uyeda et al., 1988).
Plasmodial fragmin was originally isolated as a 42 kDa
actin-binding protein that severs F-actin filaments (Hasegawa
et al., 1980; Hinssen, 1981a,b). In addition it displays F-actin
(+) end-capping and actin nucleating activity (Maruta and
Isenberg, 1983; Gettemans et al., 1995). The actin subunit in
the EGTA-resistant 1:1 actin-fragmin heterodimer is phosphorylated by the P. polycephalum actin-fragmin kinase (AFK)
at residues Thr203 and Thr202 (Gettemans et al., 1992). AFK
represents a novel type of protein kinase (Eichinger et al.,
1996) and regulates the actin-binding properties of the actinfragmin complex in two ways: upon phosphorylation, (1) the
nucleating activity of the complex is inhibited, whereas (2) the
F-actin capping activity of actin-fragmin becomes dependent
on Ca2+ (Maruta et al., 1983; Gettemans et al., 1995).
FragminP, as a monomer or as a subunit of the actin-fragmin
heterodimer, is also a substrate of casein kinase II-type enzymes
from evolutionarily divergent species (De Corte et al., 1996a).
P. polycephalum contains two variants of this kinase with a
The acellular slime mould Physarum polycephalum is characterized by two distinctive growth phases: uninucleate amoebae
and multinucleate plasmodia (Dee, 1987). Development of
plasmodia by fusion of heterothallic amoebae (carrying compatible mating type) is a complex process that not only involves
changes in cell morphology but is also accompanied by activation of a set of genes (Bailey, 1995), the products of which
enable the plasmodium to display its characteristic behaviour.
Not surprisingly, a considerable number of these genes encode
cytoskeleton-associated proteins such as myosin (Kohama and
Ebashi, 1986; Kohama et al., 1986) and profilin (Binette et al.,
1990). Like higher eukaryotes, Physarum contains two genes
that code for different isoforms of profilin, but unlike higher
eukaryotes, these are expressed in a growth phase-dependent
manner: amoebae express only profilinA whereas plasmodia
express only profilinP (Binette et al., 1990). Interestingly, P.
polycephalum is thought to express an amoeba-specific
fragmin isoform (fragminA) and a plasmodial fragmin protein
(fragminP), but the genetic basis for this dimorphism has not
Key words: Expression cloning, Fragmin, Actin-binding protein,
Polyphosphoinositide, Physarum polycephalum development
1216 D. T’Jampens and others
molecular mass of 55 kDa and 150-160 kDa, respectively (De
Corte et al., 1996a). Like other members of the gelsolin family,
the actin nucleating and F-actin severing activities of plasmodial
fragmin are inhibited by phosphatidylinositol 4,5 bisphosphate
(PIP2) (Gettemans et al., 1995), thus pointing to a possible link
between polyphosphoinositide-mediated signal transduction
and microfilament organization, similar to that observed for
profilin-actin (Lassing and Lindberg, 1985; GoldschmidtClermont et al., 1990) and gelsolin-actin interactions (Hartwig
et al., 1995). Interestingly, De Corte et al. (1997) recently
showed that PIP2 dramatically enhanced the phosphorylation of
gelsolin, fragminP, CapG and profilin by pp60c-src, thus pointing
to a possible direct regulatory role of src kinase in the control
of various actin-binding proteins.
Using conventional protein chemical methods, Ampe and
Vandekerckhove (1987) determined approximately 88% of the
complete amino acid sequence of fragminP. However, to obtain
a clear understanding of its function (i.e. PIP2-binding,
enhancement of actin phosphorylation by the AFK, expression
during development and subcellular localization), it was
necessary to obtain the complete sequence of the fragminP
coding region. We therefore screened the ML8A P. polycephalum cDNA library (Bailey et al., 1992) with fragminP
antibodies and isolated a 1,104 bp fragminP cDNA clone.
Using northern blot analysis we studied the expression of
fragminP during development of amoebae into plasmodia, and
in situ immunolocalization analysis was performed to gain
insight into the subcellular distribution of fragminP in
Physarum cells. Our data shed new light on the role of
fragminP during Physarum development and strengthen the
idea that plasmodial fragmin functions as an important
regulator controlling the organization of the subcortical microfilament system of Physarum plasmodia.
MATERIALS AND METHODS
Materials and proteins
Pancreatic deoxyribonuclease I (DNAse I) was obtained from Worthington (New Jersey, USA). The T7 Sequencing kit and lysozyme
were from Pharmacia (Uppsala, Sweden). Bromo-chloro-indolyl
phosphate (BCIP), nitro-blue tetrazolium chloride (NBT) and
isopropyl β-D thiogalacto pyranoside were from Sigma (St Louis,
MO). The Wizard plasmid purification kit and the pUC/M13 forward
and reverse primers were from Promega (Madison, WI, USA). Nitrocellulose filters were purchased from Sartorius (Goettingen, FRG).
Restriction enzymes were from New England Biolabs, Inc. (Beverly,
MA, USA). Trypsin (sequencing grade) and T4 ligase were from
Boehringer Corp. (Mannheim, FRG). [α-35S]dATP was obtained from
ICN (Costa Mesa, CA, USA). Alkaline phosphatase-conjugated goat
anti-rabbit IgG was from Fluka (Buchs, Switzerland). The GeneClean
kit was from Bio101 (Vista, CA, USA). Taq polymerase and goat
serum were obtained from Gibco BRL (Gaithersburg, MD, USA).
ProBlott was from Applied Biosystems (Foster City, USA). Tetramethylrhodamine-isothiocyanate (TRITC)-conjugated goat anti-rabbit
IgG was from Nordic Immunological laboratories (Tilburg, Netherlands).
Cell culture
The apogamic strain CL (Cooke and Dee, 1974) was used in this
study. Amoebal stocks were cultured at 30°C as described by Blindt
et al. (1986); at this temperature plasmodium formation is inhibited.
Cultures of developing cells were incubated at 26°C, the permissive
temperature for development. For each RNA sample, 100 dilute semidefined medium (DSDM) plates were inoculated with 3-5×105
amoebal cysts and 0.1 ml SBS (standard bacterial suspension) and
incubated at 26°C prior to RNA isolation (Blindt et al., 1986). The
length of time at 26°C was different for each sample. The proportion
of multinucleate cells present at the time of harvest was determined
by counting the number of nuclei per cell in 500 cells by phasecontrast microscopy. The percentage of developing cells at the time
of harvest was determined by replating a sample of the harvested cells
on DSDM agar with SBS and incubating at 26°C. After 4 days, the
plateable plasmodia and amoebal colonies were counted, and the percentage of plateable plasmodia determined; only cells committed to
development at the time of replating give rise to plateable plasmodia.
CL microplasmodia were cultured axenically at 26°C in SDM
liquid plus hematin (Blindt et al., 1986). RNA was isolated from 50
ml cultures inoculated the previous day with 5 ml of dense microplasmodial suspension. CL macroplasmodia were obtained by placing
0.1 ml of concentrated microplasmodial suspension on a double layer
of sterile Oxoid Nuflow filters placed on a metal grid in a 9 cm Petri
dish filled with SDM. During the period of culture, the microplasmodia fused together and grew into a macroplasmodium. To avoid
artifacts due to the synchronous cell cycle of plasmodia, several
macroplasmodia were inoculated at different times and harvested
simultaneously 1-2 days later.
Growth and screening of the P. polycephalum cDNA
library
The ML8A P. polycephalum cDNA library (Bailey et al., 1992), was
grown on 9 cm diameter Petri dishes with LB medium containing 50
µg/ml ampicillin and 15 µg/ml tetracyclin. Expression screening of
the cDNA library was performed as described (Sambrook et al., 1989)
with slight modifications. Briefly, colonies were induced by applying
nitrocellulose filters soaked in 10 mM isopropyl β-D-thiogalactopyranoside (IPTG). After 3 hours further growing at 37°C, the
colonies were lysed overnight at room temperature on the nitrocellulose replicas in lysis buffer (100 mM Tris-HCl, pH 7.8, 150 mM NaCl,
5 mM MgCl2, 1.5% BSA, 1 µg/ml DNAse I and 40 µg/ml lysozyme).
The nitrocellulose membranes were quenched in TNT buffer (10 mM
Tris-HCl, pH 8.0, 150 mM NaCl and 0.05% NP40) with 3% milk
powder. Primary antibody (rabbit anti-fragmin antiserum) was
incubated with the membranes at a dilution of 1:1,000 in TNT buffer1% milk powder (overnight at 4°C). The secondary antibody (goatanti rabbit alkaline phosphatase-conjugated IgG) was used at a
dilution of 1:1,000. Colonies were visualized with bromo-chloroindolyl phosphate and nitro-blue tetrazolium chloride, dissolved in
100 mM NaHCO3, pH 9.8 and 2 mM MgCl2.
Plasmid isolation and DNA sequencing
Plasmid DNA was isolated with the Wizard MiniPreps Kit (Promega,
Madison WI, USA). The complete nucleotide sequence of the fragmin
clone was determined by the Sanger dideoxy chain termination
method (Sanger et al., 1977), using the T7 Sequencing Kit
(Pharmacia, Uppsala, Sweden) and by the method of primer-walking.
For sequencing the minus and plus strands, pUC/M13 forward and
reverse primers (Promega), respectively, were used in the first
reaction. New primers (obtained from Eurogentec, Belgium) were
designed for further sequencing until overlap between + and – strands
was obtained. High resolution DNA polyacrylamide gelelectrophoresis (6% acrylamide gels) was carried out with a Bio-Rad SequiGen
system according to the instructions of the manufacturer.
Construction of the full-length fragminP cDNA clone
The seven nucleotides that were missing at the 5′ end of the frgP cDNA
clone were deduced by reverse translation of the NH2-terminal amino
acid sequence of P. polycephalum fragminP and inserted in the cDNA
clone using the polymerase chain reaction (PCR). For this purpose we
made use of a unique NcoI restriction site (bp 186-191). The pBlue-
Cloning of Physarum fragminP 1217
script II KS-frgP vector was cut with EcoRI and NcoI and the fragment
was purified after agarose gelelectrophoresis. The following primers
were used for PCR: 5′ GAA GGA GCC ATG GTG TTT TTT GGG
GAC AGG 3′ (reverse primer, complementary to the + strand) and 5′
GGA ATT CCA TAT GCA GAA ACA GAA GGA GTA TAA CAT
TGC TG 3′ (forward primer, complementary to the − strand). The
forward primer contains a NdeI restriction site with the ATG start
codon. 25 cycles of PCR were performed with 50 pmol of both primers,
50 µM dNTP, 50 ng of template DNA and 2.5 units of Taq polymerase
in a total volume of 100 µl (denaturation: 94°C, 1 minute; annealing:
52°C, 30 seconds; extension: 72°C, 30 seconds, followed by a final
extension of 10 minutes at 72°C). The PCR product was digested with
EcoRI and NcoI, with an ethanol precipitation in between. This
fragment was ligated back into the pBluescript II KS vector containing
the truncated fragmin cDNA and electroporated in supercompetent XL1
Blue cells (Stratagene, CA, USA). The DNA sequence was confirmed
by sequencing one strand with the appropriate pUC/M13 primer. Next,
the complete fragmin cDNA was excised from pBluescript II KS with
NdeI and NotI and ligated into the pLT10T3 expression vector (Mertens
et al., 1995b) that was treated with the same restriction enzymes. The
fragminP ATG codon represents the authentic start codon. A sample of
the mixture was transformed into competent MC1061 λ cells, plated
onto LB medium supplied with 50 µg/ml ampicillin and grown at 28°C.
This pLT10T3-frgP plasmid was then transformed into E. coli MC1061
that already contained the pSCM26 plasmid (Mertens et al., 1995a;
Eichinger et al., 1996) and harbours the T7 polymerase gene. This strain
was used as a source for the production and purification of recombinant
fragminP.
Over-expression and purification of recombinant fragminP
from E. coli
Trial assays indicated that induction of fragminP with IPTG resulted
in very high expression levels, but a considerable amount of fragminP
was found in the pellet fraction. We therefore chose to grow the cells
at 28°C without induction.
The MC1061-pSCM26-pLT10T3-frgP strain was grown overnight
at 28°C in LB medium with 50 µg/ml ampicillin and 25 µg/ml
kanamycin. This culture was diluted 1:10-1:20 in the same medium
and further grown for 6-8 hours. Cells were harvested, resuspended
in 2 volumes TDA buffer (10 mM Tris-HCl, pH 7.5, 1 mM DTT,
0.02% NaN3) with 1 mM phenylmethylsulfonyl fluoride (PMSF) and
subsequently lysed with a French Press. The lysate was centrifuged
for 1 hour at 50,000 g and the clear supernatant was applied onto a
DEAE-cellulose ion exchange column (2.5 cm × 15 cm), equilibrated
with the same buffer. Adsorbed proteins were eluted with a 0-0.3 M
linear gradient of NaCl in TDA buffer and 4 ml fractions were
collected. Samples were analyzed on 10% SDS mini-slab gels and
fractions containing fragminP were pooled, dialyzed against TDA
buffer, pH 8.0 and applied onto a mono Q HR 5/5 column, connected
to an FPLC system (Pharmacia, Uppsala, Sweden). Adsorbed proteins
were eluted with the same NaCl gradient as for the DEAE column (20
ml total volume). FragminP eluted at 90 mM NaCl. The fractions were
concentrated in a Speed Vac Concentrator (Savant Instruments, Farmingdale, NY) and dialyzed against a solution containing 60% glycerol,
50 mM Tris-HCl, pH 7.5, 1 mM DTT and 0.02 % NaN3. This preparation was essentially free of contaminating proteins.
Southern and northern hybridization
RNA isolation
Cultures of amoebae and developing cells were washed off agar plates
with ice-cold water and rinsed twice with ice-cold water. Microplasmodia were pelleted and washed as above. Macroplasmodia were
grown as described above and scraped directly into the guanidine lysis
buffer. Total RNA was isolated from all cell types using the method
of Chomczynski and Sacchi (1987), with modifications by Puissant
and Houdebine (1990).
Northern blotting
Northern blots were run with 10 µg of total RNA in 1.1% agarose gels
using the method of Murray et al. (1994). Transfer to Hybond-N
membrane followed the method of Sambrook et al. (1989). The RNA
was fixed to the membranes by exposure to UV irradiation prior to
hybridization.
Isolation of genomic DNA
Genomic DNA was isolated using a method modified from that of
Burland and Pallotta (1985). Briefly, 3 ml packed volume of exponentially growing CL microplasmodia was spun down, washed once
in sterile water, and resuspended in 40 ml ice-cold NHS (nuclear
homogenization solution: 250 mM sucrose, 0.1% Triton X-100, 15
mM CaCl2, 10 mM Tris-HCl, pH 7.5; stored frozen in 100 ml
fractions). The microplasmodia were homogenized in a Waring blender
using ice-cold cups. A sample of the homogenized material was
examined microscopically to ensure that complete homogenization had
occurred without disruption of nuclei. Any poorly homogenized
material was removed by filtration through a milk filter. The volume
of the filtrate was made to 35 ml with NHS, 10 ml of Percoll was added
and mixed by inversion. This mixture was centrifuged at 4°C in a
Sorvall RT600 rotor at 2,500 rpm. The supernatant was removed, the
pellet resuspended in 45 ml NHS and centrifuged again. This washing
was repeated once more. The nuclear pellet was resuspended in the
residual liquid and kept on ice. Nuclear lysis buffer (100 mM EDTA,
0.5% Triton X-100, 25 mM Tris-HCl, pH 8.0) was mixed with one
twentieth volume of proteinase K stock (2 mg/ml in water, stored at −
20°C) and 4 ml of the mixture was added to the resuspended nuclei.
After incubation at 70°C for 15 minutes, the mixture was cooled to
37°C and, after addition of the same amount of proteinase K, incubated
at 37°C overnight. Next day, RNaseA was added to 1 mg/ml (stock
solution: 100 mg/ml in 10 mM potassium acetate, pH 5.2, heated to
80°C for 10 minutes and stored at −20°C) and incubated for a further
2 hours at 37°C. The DNA was extracted once each with equal volumes
of Tris-saturated phenol, phenol:chloroform:isoamyl alcohol (25:24:1)
and chloroform:isoamyl alcohol (24:1). It was then precipitated by
addition of one fifteenth volume 3 M sodium acetate and two volumes
of ethanol. The pellet was washed twice in 5 ml of 70% ethanol, air
dried and resuspended in 500 µl TE. The concentration was determined
by optical density reading using a spectrophotometer.
Southern blotting
DNA was digested to completion and electrophoresed in 0.8% agarose
gels prior to blotting using the method of Sambrook et al. (1989). The
DNA was transferred to Hybond-N membranes and fixed by exposure
to UV irradiation.
Synthesis of probes and hybridization (northern and Southern
blots)
The cDNA insert was digested from the vector with EcoRI and NotI,
purified and used as probe. Radiolabelled DNA probes were synthesized by the method of Feinberg and Vogelstein (1983). For these
probes, prehybridization and hybridization were carried out at 65°C
using the method of Church and Gilbert (1984). Stringency washes
were carried out at 65°C in 0.2-0.4 M Na2HPO4, pH 7.5 with 0.1%
SDS. Filters were exposed at −70°C for 5-14 days with an intensifying screen.
Protein chemical methods and mass spectrometry
Fragmin19 (De Corte et al., 1996b) was cleaved with trypsin for 4
hours at 37°C. The resulting peptides were separated on a microbore
reversed-phase HPLC system and peptide profiles were analyzed by
electrospray ionization-mass spectrometry as described elsewhere (De
Corte et al., 1996a). Amino acid sequence analysis was performed on
a 477A model pulsed liquid-phase sequenator equipped with a 120 A
phenylthiohydantoin amino acid analyzer (Applied Biosystems Inc.,
USA). Matrix-assisted laser desorption ionization reflection time of
1218 D. T’Jampens and others
flight-mass spectrometry (MALDI-RETOF-MS) and post-source
decay (PSD) mode analysis of peptides were performed as described
elsewhere (K. Gevaert et al., submitted for publication). Chemical
modification of the NH2-terminal fragminP peptide with 2-iminothiolane and iodoacetamide was performed as described by Bartlett and
Bush (1994).
Immunofluorescence of microplasmodia
Microplasmodia of P. polycephalum grown in axenic cultures were pretreated and fixed as described previously (Kukulies et al., 1985), with
some modifications. Microplasmodia were first repeatedly washed by
gentle agitation in 1 mM Ca(H2PO4)2 solution containing 10 mM
glucose, pH 6.3, in order to remove the thick extracellular slime layer.
Freshly washed microplasmodia were then allowed to settle for 1 hour
onto coverslips coated with 0.1% poly-L-lysine. Various fixation and
extraction solutions were tested to improve the staining conditions.
After washing three times in Tris-buffered saline (TBS, 10 mM TrisHCl, pH 7.4, 170 mM NaCl), the samples were fixed at room temperature for 20 minutes in a solution of 3% paraformaldehyde in TBS.
The fixed cells were then washed in TBS and permeabilized by 1%
Triton X-100 in TBS for 30 minutes at room temperature. After
washing in TBS (3× 5 minutes), cells were incubated for 30 minutes
at 37°C in the presence of affinity-purified rabbit anti-fragminP polyclonal antibodies (Gettemans et al., 1995), diluted 1:100 in TBS and
supplemented with 1% BSA and 2% goat serum (Gibco BRL). After
washing 3 × 10 minutes in TBS, cells were subsequently incubated for
30 minutes at 37°C with tetramethylrhodamine-isothiocyanate
(TRITC)-conjugated goat anti-rabbit IgG, diluted 1:200 in TBS/1%
BSA/2% goat serum. For F-actin staining, FITC-phalloidin was used
at a final concentration of 1 µg/ml. After washing, samples were
mounted on a glass slide with Vectashield mounting medium (Vector
Laboratories). Slides were observed with an inverted microscope
(Axiovert 135 M, Zeiss), equipped with 20× and 40× Plan-Neofluar
objectives, a HBO 50W mercury short-arc lamp, and a 560/590 nm
filter combination. The fragmin and F-actin distribution were studied
using a system for laser scanning confocal microscopy (Zeiss LSM
410), based on an inverted microscope (Zeiss axiovert 100), equipped
with 40× and 100× Neofluar oil immersion objectives. Samples were
excited with the visible lines (488 nm and 543 nm) of an argon laser
beam, and emissions were collected via a photomultiplier tube through
a band pass filter (515-525 nm) for green FITC fluorescence or a band
pass filter (590 nm-610 nm) for red TRITC fluorescence.
Miscellaneous
SDS-polyacrylamide gel electrophoresis (Laemmli, 1970) was
performed on 10% or 15% polyacrylamide mini slab gels according
to Matsudaira and Burgess (1978). Protein concentration was determined by the method of Bradford (1976) using bovine serum albumin
as standard. Protein sequence alignments were done using the GCG
program.
Labelling of actin with pyrene-iodoacetamide (Brenner and Korn,
1983) and F-actin severing assays were performed as described
(Gettemans et al., 1995). Recombinant actin-fragmin kinase was
purified from E. coli as described (Eichinger et al., 1996).
RESULTS
Isolation and sequence of the fragminP cDNA clone
Approximately 13,000 colonies of the P. polycephalum cDNA
library were screened with polyclonal anti-fragminP antibodies
for expression of recombinant fragminP. Using this approach
we specifically selected for fragminP cDNAs that were cloned
in the correct reading frame. The library was originally
designed to identify genes involved in the transition from
amoeba to plasmodium, and was therefore constructed from a
developing cell population, consisting of 45% uninucleate
committed cells, 5% multinucleate cells and 50% amoebae
(Bailey et al., 1992).
We isolated three positive colonies putatively encoding
fragminP. One of these contained an insert of 1.17 kb following
EcoRI/NotI digestion of the pBluescript II KS vector and agarose
gelelectrophoresis (data not shown). Since fragminP was
thought to contain approximately 400 amino acid residues
(Ampe and Vandekerckhove, 1987), we estimated the size of the
cDNA coding region at 1.2 kb. The insert was sequenced by the
method of primer walking. The nucleotide and deduced amino
acid sequences are presented in Fig. 1. The cDNA insert
comprises a 1,171 bp open reading frame with 1,104 bp coding
for 368 amino acids of fragminP. Between the TAA stopcodon
and the poly(A)+ tail there is a 46 nucleotides long 3′ untranslated sequence. This clone is not full length, as can be deduced
from the lack of an ATG initiation codon. However, when the
molecular mass of this 368-residue segment was calculated
(40,632.5 Da) and compared with the experimentally determined
mass of native plasmodial fragmin (41,080 Da; De Corte et al.,
1996a), it was obvious that only a few amino acids were missing
at the NH2 terminus (Fig. 1). At this stage it appeared therefore
more convenient to determine the amino acid sequence at the
extreme NH2 terminus of fragminP by protein chemical means.
Determination of the amino acid sequence of the
blocked NH2 terminus of fragminP by MALDIRETOF-mass spectrometry
The sequencing strategy starts from the knowledge that conventional sequencing of a CNBr-cleavage fragment (Ampe and
Vandekerckhove, 1987), covering the NH2-terminal region of
fragminP, overlaps with the deduced cDNA sequence, but
contains a NH2-terminal Gln-Lys extension. Taking into
account the specificity of the CNBr cleavage procedure used,
we could thus postulate a Met-Gln-Lys sequence at the amino
terminus of fragminP. The overall mass of fragminP, including
this tripeptide, differed by only 43 Da from the experimentally
determined value. The difference corresponds exactly with the
mass of an acetyl group, suggesting the sequence Ac-M-Q-K
at the extreme NH2-terminal primary structure of fragminP.
This was verified by isolating the corresponding tryptic tripeptide by reversed-phase HPLC in combination with on-line
electrospray ionization-mass spectrometry (ESI-MS; results not
shown). Since Gln and Lys have a nearly identical mass, their
exact order in this peptide could not be ascertained. We therefore
determined the amino acid sequence of the peptide by a combination of MALDI-TOF and MALDI-RETOF mass spectrometry (K. Gevaert et al., submitted for publication) following modification of the lysine residue with 2-iminothiolane and
iodoacetamide, which results in a mass increase of 158 Da. The
Ac-M-Q-K M + H+ parent ion has a monoisotopic mass of 448.6
Da (Fig. 2A) and amino acid sequence analysis confirmed that
it was blocked (not shown). The modified M′ + H+ parent ion
displays a monoisotopic mass of 606.7 Da (Fig. 2B). Both the
unmodified and modified parent peptides were subsequently
fragmented by laser-induced ionization and analyzed by
MALDI-reflection time of flight mass spectrometry. A variety of
ions are thus generated, each corresponding to a particular
ionized peptide fragment. The mass spectra obtained are shown
in Fig. 2C-D. The peaks with masses of 258.1 and 275.1 Da
Cloning of Physarum fragminP 1219
Fig. 1. Nucleotide and deduced amino acid sequence of the frgP cDNA clone. The 1,171 bp insert is flanked by the EcoRI linker (5′ end) and a
NotI restriction site at the 3′ end (underlined). The fragminP cDNA starts at the arrow (these nucleotides are designated A8A9). Nucleotides at
the 5′ end represented in bold were deduced by reverse translation after determination of the NH2-terminal sequence of fragminP by mass
spectrometry. The TAA stopcodon is indicated by an asterisk and the putative polyadenylation signal is boxed. Amino acids are represented in
the three letter code. This sequence is available from GenBank under accession number U70047.
represent the Gln-Lys b- and Gln-Lys c-ions, respectively,
whereas fragments of 130.1 and 174.1 Da match with the Lys zand Ac-Met b-ions (Fig. 2C). Significantly, both spectra contain
a peak with a monoisotopic mass of 302 Da. This single positively charged peptide fragment can only correspond with the
sequence Ac-Met-Gln, because the mass of this peptide remains
the same before and after modification. In the putative sequence
Ac-Met-Lys, the mass would increase with 158 Da after modification, and this was not observed. The peak with a monoisotopic mass of 305 Da in Fig. 2D represents the modified Lys
residue (147+158 Da). In the unmodified peptide, this amino
acid is identified as a peak with a monoisotopic mass of 147 Da
(Fig. 2C). We therefore concluded that the NH2-terminal
sequence of fragminP corresponds to Ac-Met-Gln-Lys and that
only three amino acids were missing from the cDNA clone.
The amino acid sequence of fragminP and
delineation of fragmin19, a proteolytic fragment that
covers the NH2-terminal half of fragminP
The predicted molecular mass of the cloned fragminP,
including the missing start Met, Gln and Lys residues, was
41,080 Da and this value is in agreement with the value that
was previously determined experimentally by ESI-mass spectrometry using native fragminP (De Corte et al., 1996a). Thus
fragminP consists of 371 amino acids. An alignment of the
deduced amino acid sequence of fragminP with D. discoideum severin is shown in Fig. 3. The overall homology
with severin (66% identity) and the NH2-terminal half of
gelsolin (57% identity) is rather high, pointing to similar
functions.
The fragminP amino acid sequence deduced from the cDNA
clone differs from the previously determined amino acid
sequence (Ampe and Vandekerckhove, 1987) at three positions
(Fig. 3): two substitutions (Ser64 to Gly and Thr76 to Ser) and
one insertion (Pro at position 141). These differences were
investigated by amino acid sequencing of Physarum fragmin19,
a degradation product of native fragminP, covering the NH2terminal half (see below). The amino acid sequences of tryptic
and chymotryptic fragmin19 peptides (data not shown)
indicated that the cDNA deduced sequence was correct. The
insertion of proline at position 141 is more enigmatic than the
substitutions, which could be explained as the result of genetic
drift. We therefore speculate that this residue may have been
overlooked previously.
1220 D. T’Jampens and others
The deduced amino acid sequence also connects the NH2terminal half with the COOH-terminal half by filling a gap of
11 residues, TRLLHLKGKKH (Fig. 3, boxed region) that were
previously not encompassed (Ampe and Vandekerckhove,
1987). It constitutes a highly basic and hydrophobic region.
Interestingly, this new sequence revealed a PIP2-binding motif
(RLLHLKGKK), very similar to the consensus sequences of
other actin-binding proteins of the gelsolin family (Yu et al.,
1992; Janmey et al., 1992). The identification of a second PIP2binding site that conforms more closely to the canonical PIP2binding motif explains the observed inhibition of the actinbinding properties of fragminP by PIP2 (Gettemans et al.,
1995).
In some preparations of the Physarum EGTA-resistant 1:1
actin-fragmin complex we noticed the presence of a dimer consisting of actin and a 20 kDa polypeptide, immunologically
related to fragminP (De Corte et al., 1996b). Amino acid
sequencing and molecular mass measurements identified the
20 kDa component as the NH2-terminal half of fragminP,
covering amino acids 1-168. Its exact mass of 19,123 Da was
determined by ESI-MS and we therefore call it fragmin19. The
COOH terminus of fragmin19 is located within the internal
peptide of 11 amino acids that also contains the PIP2 binding
consensus motif, and illustrates why it was previously not
possible to correctly delineate the size of fragmin19. Because
fragmin19 is not detected in the crude cytosol of Physarum
microplasmodia we assume that it originates by proteolysis in
the course of the isolation of the actin-fragmin complex.
Over-expression and characterization of intact
recombinant fragminP
To obtain full-length recombinant fragminP, the three missing
amino acids at the NH2 terminus (M-Q-K) were added by PCR
(see Materials and Methods). We purified recombinant
fragminP from E. coli MC1061, transformed with pSCM26
(Mertens et al., 1995a) and the pLT10T3-frgP expression
vector, to homogeneity by a two-step procedure. The purity of
the final preparation is shown in Fig. 4A and the identity of the
protein was confirmed after determination of the NH2-terminal
amino acid sequence by automated Edman degradation (data
not shown). Approximately 1 mg of fragminP was purified
from 62 mg of crude bacterial extract.
The biological activities of recombinant fragminP were
assayed by two criteria: F-actin severing and its ability to
Fig. 2. Determination of the fragminP amino-terminal sequence by MALDI-RETOF analysis. Mass/charge spectrum of the blocked NH2terminal fragminP peptide before (A) and after (B) modification with 2-iminothiolane and iodoacetamide. Notice the relative increase of 158
Da after modification. (C,D) Post-source decay spectra of the unmodified (C) and modified (D) fragminP peptide. The peak with a
monoisotopic mass of 302 Da (small arrow) is present in both C and D and is consequently unaffected by modification. The mass of this peak
corresponds to the sequence Ac-Met-Gln. The peak with a monoisotopic mass of 147 Da (arrowhead, C) represents the lysine residue (amino
acid 3) and shifts to 305 Da (arrowhead, D) after modification. m/z, mass/charge. a.i., absolute intensity.
Cloning of Physarum fragminP 1221
Fig. 3. Alignment between the deduced
fragminP sequence, the previously
determined partial sequence of fragminP
and severin. The differences between
the predicted sequence of fragminP (this
paper, FragminP1996) and the amino
acid sequence determined previously by
Ampe and Vandekerckhove (1987;
fragminP1987) are marked by an
asterisk. The PIP2-binding motif
(italicized), identified after sequencing
of the cDNA clone, links the two halves
and complies with the PIP2-binding
K/RX4K/RXK/RK/R consensus
sequence (X represents any amino acid).
The previously identified PIP2-binding
motif (K/RX3K/RXK/RK/R) is
underlined. The PIP2-binding motifs of
gelsolin are shown for comparison.
Identical amino acids are noted as
letters, homologous residues are
indicated by vertical lines. The severin
sequence is taken from André et al.
(1988).
enhance actin phosphorylation in the 1:1 EGTA-resistant actinfragmin complex by the AFK. F-actin severing proteins
introduce new free (−) ends resulting in the depolymerization
of F-actin in a manner proportional to the number of free
filament ends. At a concentration of 25 nM, recombinant
fragminP severed F-actin filaments as efficiently as native
fragminP, shown by the decrease in fluorescence intensity of a
pyrene-labeled F-actin solution (Fig. 4B). This activity was
abolished by preincubation of the protein with PIP2 (Fig. 4B).
As a second proof for biological activity, recombinant
fragminP was preincubated with muscle actin in the presence
of micromolar amounts of Ca2+ and the mixture was subsequently added to the actin-fragmin kinase in the presence of
[γ-32P]ATP/Mg2+ and EGTA. A strong phosphorylation of
actin was observed (Fig. 4C, lanes 2-5), indicating that
fragminP is able to stimulate actin phosphorylation by the
Fig. 4. Recombinant fragminP severs F-actin and enhances
phosphorylation of actin by the AFK in the actin-fragmin
complex. (A) Purification of fragminP from an E. coli
crude extract analyzed by SDS-polyacrylamide
gelelectrophoresis on a 10% acrylamide gel. Lane 1,
proteins from a high speed supernatant of an E. coli
MC1061-pL10T3-frgP strain. Lane 2, sample of the
fractions that were pooled after DEAE chromatography of
the E. coli crude extract. Lane 3, mono Q ion exchange
chromatography of the sample obtained after DEAE-ion
exchange chromatography. Lane 4, 0.5 µg actin-fragmin.
Molecular mass markers are shown on the left. The gel was
stained with Coomassie Brilliant Blue. (B) F-actin severing
activity of recombinant fragminP. 8 µM of an F-actin
solution was diluted to 400 nM, resulting in a slow
disassembly of actin monomers at their (−) end (0 nM). In
the presence of 5 or 25 nM recombinant fragminP or 25
nM native fragminP, a pronounced increase in the
depolymerization rate is observed, typical for an F-actin
severing protein. A 250× molar excess of PIP2 over
fragminP abolishes this activity (+ PIP2). The higher initial fluorescence level in the presence of PIP2 is due to its light scattering activity.
(C) Ca2+-dependent formation of a complex between fragminP and actin and concomitant phosphorylation of actin by the AFK. 6 µM muscle
actin was preincubated with increasing concentrations of fragminP (indicated on top) in the presence of 0.2 mM Ca2+. Phosphorylation with
recombinant AFK and [γ-32P]ATP/Mg2+ was allowed to proceed for 10 minutes in the presence of 2 mM EGTA, thus giving rise to the EGTAresistant 1:1 actin-fragmin complex, the true substrate of the AFK. Samples were analyzed by SDS-PAGE followed by autoradiography.
Exposure time was overnight at room temperature. Note that G-actin alone is not phosphorylated.
1222 D. T’Jampens and others
Fig. 5. Developmental regulation of
fragminP. (A) The figure shows a
northern blot of total RNA isolated
from the following cultures of the
apogamic strain CL: A, amoebae;
Mi, microplasmodia; Ma,
macroplasmodia; 1-56%, cultures
containing cells that were
developing into plasmodia. The
figure indicates the percentage of
developing cells in each sample.
frgP is a 1.2 kb mRNA encoding
fragminP that is expressed in
plasmodia but not amoebae (top
panel). mRNA size markers
(Promega) are shown on the left.
The middle and lower panels show
the same RNA samples probed with
two constitutively expressed genes:
actin and a cDNA D6/16A, coding
for an unknown gene. The slight
variations in the signals are due to
differences in expression levels and
transfer efficiency onto the
membrane rather than to differences
in the amount of RNA loaded (10 µg
per lane). (B) FragminP is not
present in amœbae. A western blot is
shown with cytosolic proteins from
amœbae (a) or microplasmodia (Mi), probed with fragminP antiserum (left panel) or affinity-purified fragminP antibodies (right panel). 5
µg crude extract was applied. Lane c, purified recombinant fragminP (control). Molecular mass markers are indicated.
kinase through formation of a stoichiometric complex with
actin. No actin phosphorylation was noticed in the absence of
fragminP (Fig. 4C, lane 1).
Transcriptional regulation of the fragminP gene
during development of P. polycephalum amoebae
into plasmodia
Genomic DNA from the haploid apogamic strain CL was
digested, Southern blotted and hybridized against a 32Plabelled fragminP cDNA probe. HindIII and KpnI digestion
both resulted in two major hybridizing fragments of 7.0/1.6 kb
and 9.4/1.8 kb, respectively, whereas digestion with PstI
resulted in two hybridizing fragments of 3.5 and 2.0 kb (data
not shown). HindIII and PstI both cut at a unique site in the
coding region of the fragminP cDNA insert, in agreement with
Fig. 6. Phase-contrast and epifluorescence analysis
of fragminP in microplasmodia. (A) Phase-contrast
image of a microplasmodial segment.
(B) Epifluorescence staining pattern of fragminP
from the same segment as in A. Note the intense
staining along the plasma membrane and diffuse
cytoplasmic staining. Circular structures are visible
in the center of the segment. Scale bar applies to A
and B.
two hybridizing fragments. Since KpnI does not cut within the
known cDNA but results in two hybridizing fragments, there
must be at least one intron in the coding region of fragminP.
Additional faint bands were also visible: a 6.6 kb band for
HindIII and a 15 kb band for KpnI. The origin of these bands
was clarified by the results of the northern hybridization.
For northern analysis we screened RNA that was isolated
from the apogamic P. polycephalum CL strain. The cellular and
molecular events of apogamic development have been well
characterized (Bailey et al., 1987, 1990; Bailey, 1995) and
these strains therefore provide a good source of material for
the study of genes involved in plasmodium development in P.
polycephalum. The fragminP cDNA hybridized to a mRNA
with a size of approximately 1.2 kb (Fig. 5A, upper panel).
Interestingly, no signal was detected in amoebae (Fig. 5A,
Cloning of Physarum fragminP 1223
upper panel, lane 1). As the percentage of developing cells in
the population increased, so did the amount of fragminP
mRNA (Fig. 5A, upper panel, lanes 2-10). The highest level of
expression was detected in microplasmodia; macroplasmodia
also expressed fragminP, but at a somewhat lower level (Fig.
5A, upper panel, lanes 11-12, respectively). Two constitutively
expressed genes, actin and a clone D6/16A coding for an
unknown gene (Bailey et al., unpublished observations), were
used as controls. The expression levels of the corresponding
mRNAs remained essentially the same throughout development (Fig. 5A, lower panels). Western blots on crude extracts
of amœbae or microplasmodia using fragminP antiserum or
affinity-purified antibodies confirmed the data from the
northern blot: no fragminP was detected in amœbae (Fig. 5B).
A second mRNA of ±2.5 kb was also detected by the
fragminP probe and this RNA is present only in fully developed
micro- and macroplasmodia (Fig. 5B, lanes 11 and 12). It is
unlikely that the cross-reaction was due to non-specific hybridization because this particular RNA seems also to be developmentally regulated. Furthermore, several weaker bands were
also visible in Southern blots of genomic DNA (data not
shown) and may be derived from the gene coding for this particular protein. Because fragminP is expressed predominantly
in P. polycephalum plasmodia we term the gene coding for this
protein as frgP and we propose to call the as yet uncloned
amoebal fragmin gene frgA. The gene coding for the unknown
fragminP-related protein (mRNA of 2.5 kb) is designated frgR.
FragminP is located at the plasma membrane of
microplasmodia
We have previously demonstrated that fragminP, like other
members of the gelsolin family, specifically interacts with the
phospholipid PIP2 (Gettemans et al., 1995). To investigate the
role of fragminP in P. polycephalum plasmodia, we studied its
subcellular localization by staining with fragminP antibodies
and analyzed the resulting pattern by epifluorescence and
confocal microscopy. Although microplasmodia are morphologically heterogenous, a consistent staining pattern was found.
Epifluorescence showed that fragminP stains predominantly
along the rim of the cells, whilst a diffuse cytoplasmic staining
was also observed (Fig. 6B). However, in view of the irregular
and complex morphology of microplasmodia, the cytoplasmic
staining of fragminP could reflect peripheral staining but in a
different focal plane. This possibility was investigated by
confocal microscopy. Fig. 7A illustrates that fragminP
localizes almost exclusively at the plasma membrane in
confocal sections of plasmodia (stage II microplasmodia, 100300 µm). This is particularly visible in a transverse section
(Fig. 7B). In control experiments where either preimmune
serum or the secondary antibody was used, we did not observe
staining. Nor was staining observed in the nuclei, arguing
against phosphorylation-mediated shuttling of fragminP to and
from the nucleus as was found for CapG (Onada and Yin,
1993). This suggests that fragminP phosphorylation by casein
kinase II (De Corte et al., 1996a) is probably related to another,
as yet unidentified, mechanism. In larger microplasmodia
(stage III, 300-1,000 µm) that were double-stained for F-actin
and fragminP, fragminP did not co-localize with cytoplasmic
fibrillar F-actin (Fig. 7D). FragminP staining was observed predominantly in small surface extensions resembling lobopodia.
These structures are visible in epifluorescence images as
Fig. 7. Immunolocalization of fragminP in microplasmodia analyzed
by confocal microscopy. (A) Segment of a multi-lobed
microplasmodium, illustrating the highly localized plasmalemma
staining of fragminP. The dotted line indicates where a transverse
section was made. (B) Transverse section in the z-direction of the
plasmodial fragment from A, showing the uniform distribution of
fragminP at the ventral, dorsal and lateral areas of the plasma
membrane. (C) F-actin staining of a microplasmodial segment
similar to that in A. A typical pattern is shown, consisting of a
central, dense patch of F-actin, from where fibrillar microfilaments
emanate. (D) Double staining of F-actin and fragminP in a large
microplasmodium of stage III. Note that fragminP does not colocalize with the fibrillar actin network, but is restricted to surface
projections characterized by a relatively low content of F-actin.
These projections are seen as diffuse, circular structures in Fig. 6B.
circular spots, seemingly present in the cytoplasm but actually
rising out of the focal plane. A similar overall plasma
membrane staining pattern was found in small, spherical (stage
I) microplasmodia (not shown). Taking into consideration the
interaction between fragmin and PIP2 in vitro, the general
staining pattern may suggest that fragminP is anchored to the
plasma membrane lipid bilayer through interaction with PIP2.
Alternatively, fragminP may be associated with the cortical
microfilament system, but it certainly does not seem to interact
with cytoplasmic fibrillar actin filaments.
DISCUSSION
We have isolated a cDNA clone encoding fragminP, an actinbinding protein from Physarum that is structurally and functionally homologous to vertebrate gelsolin, a well-known
regulator of cytoskeletal organization in cells (Weeds and
Maciver, 1993). Only seven nucleotides of the cDNA clone
were lacking at the 5′ end, and this enabled us to predict the 5′
end by reverse translation of the Met-Gln-Lys sequence. The
reverse translated nucleotide sequence at the 5′ end of the
1224 D. T’Jampens and others
fragminP cDNA clone is represented in bold in Fig. 1. This
prediction was used for construction of the full-length frgP
cDNA clone by PCR. Purified recombinant fragminP displayed
actin-binding properties identical to native fragminP.
Three amino acid differences were identified between the
deduced sequence and the partial amino acid sequence determined previously (Ampe and Vandekerckhove, 1987). The
substitutions were verified by peptide sequences of fragmin19,
a truncated form of native fragminP. This showed that the
cDNA deduced sequence was correct. We therefore ascribe the
three amino acid changes to allelic differences between strains
of different genetic background. Such changes are not exceptional, as allelic actin and tubulin restriction-length polymorphisms have been demonstrated previously in P. polycephalum
(Schedl et al., 1984; Schedl and Dove, 1982). Apart from these
observations, Southern blotting with genomic DNA indicated
that fragminP is a single copy gene.
No cross-hybridization was observed with the mRNA of
fragminA (expected size approximately 1.0-1.1 kb), even at
low stringency conditions. FragminA and fragminP probably
share amino-acid sequence homologies that are not reflected at
the DNA level. Similarly, profilinA and profilinP from
Physarum do not cross-hybridize at the DNA level, although
both proteins are 66% homologous (Binette et al., 1990). Our
results therefore indicate that fragminA and fragminP are
coded by different genes.
The finding that P. polycephalum expresses two isoforms of
fragmin is significant but not a singular observation in this
organism. Physarum contains five actin genes at four loci,
ardA-E (Hamelin et al., 1988; Gonzales-y-Merchand and Cox,
1988; Nader et al., 1986; Adam et al., 1991). Only ardB and
ardC are abundant and constitutively expressed at all stages of
development (Monteiro and Cox, 1986; Hamelin et al., 1988).
Although microfilaments in amoebae and plasmodia are
assembled from the same actin protomers, these Physarum cell
types differ profoundly in organization of microfilaments (i.e.
amoebae do not contain thick cytoplasmic actin fibrils), mode
of locomotion and use of microfilaments (i.e. cytokinesis – or
lack of it in plasmodia). Consequently, it is reasonable to conjecture that the (re)organization of microfilaments during
Physarum development must be the result of the interaction
with actin-binding proteins. This hypothesis is strengthened by
the observation that several other actin-binding proteins from
Physarum are developmentally regulated, including profilin
(Binette et al., 1990) and the LAV3-4 and LAV3-5 cDNAs,
encoding alfa-actinin and an actin-bundling protein, respectively (St-Pierre et al., 1993). Based on our and previous observations (Uyeda et al., 1988), it is very likely that fragminA and
fragminP also play a distinct role in the remodeling of microfilaments. Furthermore, myosin heavy and light chains and
tubulin are also expressed in a cell-specific manner (Burland
et al., 1983; Kohama and Ebashi, 1986; Kohama et al., 1986).
The tubulin isoforms participate in the different mitotic
spindles that are formed in amoebae versus plasmodia
(Solnica-Krezel et al., 1991).
FragminP expression starts early in development, at a time
when apogamic amoebae acquire characteristics that are
typical for plasmodia such as switching from open to closed
mitosis. During development the cells undergo an extended cell
cycle that takes about two and a half times as long to complete
as a normal cell cycle. During this cell cycle, the amoebae
reach a ‘point of no return’, at which stage they become
committed to development. It is in the second half of this
extended cell cycle that fragminP (this paper), profilinP
(Binette et al., 1990; J. Bailey, unpublished observations) and
the myosin heavy and light chain proteins (Kohama and
Ebashi, 1986; Uyeda and Kohama, 1987) are first expressed.
Several lines of evidence indicate that the initiation of
fragminP expression runs parallel with the developmental
switch from amoeba to plasmodium. For instance, tubulin
genes alter their pattern of expression during the second half
of the extended cell cycle (Solnica-Krezel et al., 1990; 1991;
Bailey et al., 1990). In addition, normal developing cells
acquire the ability to ingest amoebae (a trait characteristic of
plasmodia) in the second half of the extended cell cycle and
also change their mode of locomotion. Finally, during the long
cell cycle many other cell-type specific genes modify their
pattern of expression (Sweeney et al., 1987). These observations further indicate that the timing of frgP expression
coincides with the time when the cell begins to modify its
structural organization and behaviour and gradually gains characteristics typical for plasmodia. Since the fragminP and
profilinP mRNA are first detectable at the same time, we
predict that the fragminA mRNA would disappear at the same
time as profilinA mRNA, which starts to decline in abundance
at the 20% developing cells level (J. Bailey, unpublished observations).
Physarum plasmodia contain an extensive cortical actin
cytoskeleton, resembling the erythrocyte membrane actin
skeleton. This membrane-associated network differs in its
three-dimensional organization from the cytoplasmic actin
scaffold that is more fibrillar in nature. In amoebae, F-actin
stains mostly in patch-like structures, coinciding with relatively
large pseudopods and finger-like small filopodia (Pagh and
Adelman, 1988). In microplasmodia, F-actin typically stains
the many invaginations that result in a two- to fivefold increase
in cell surface, allowing efficient and quick response to variations in the external environment. FragminP did not co-localize
with the fibrillar cytoplasmic actin network, in contrast to what
was found for macroplasmodia (Naib-Majani et al., 1983;
Osborn et al., 1983). Based on our in situ localization studies,
fragminP could control assembly-disassembly of microfilaments in the cortical area of the cell and since this system is
thought to be the basis for motive force generation (Brix et al.,
1987) it could be involved in cell motility.
FragminP is the most recent member of the gelsolin family
for which the cDNA sequence and amino acid sequence have
been determined. At micromolar [Ca2+] it forms an actin2fragmin trimer, indicating that fragminP contains two actinbinding sites. In contrast, fragmin19 only makes up a 1:1
complex with actin (De Corte et al., 1996b), suggesting that
the second Ca2+-dependent actin-binding site is located beyond
Leu169 in the COOH-terminal half of the molecule. FragminP
also contains two PIP2-binding motifs. The first motif was previously postulated at residues 142 through 149 (Gettemans et
al., 1995). As in severin (Eichinger et al., 1991; Eichinger and
Schleicher, 1992) and CapG (Yu et al., 1990), this motif is less
tightly conserved than that of gelsolin. Residues
167RLLHLKGKK175, deduced from the frgP cDNA clone,
constitute the second PIP2-binding consensus motif of
fragminP and this sequence is in fact more similar to villin than
to gelsolin (Arpin et al., 1988).
Cloning of Physarum fragminP 1225
FragminP, fragmin19 and fragmin 60 (Furuhashi and Hatano,
1989) can each form a dimeric complex with actin that is phosphorylated in the actin subunit by the P. polycephalum actinfragmin kinase (AFK) (Gettemans et al., 1992; De Corte et al.,
1996b; Furuhashi et al., 1992). The interaction between
fragminA and actin may also give rise to a substrate for the
AFK, but this remains an open question. The actin-fragmin
kinase is remarkable because of its unusual structure
(Eichinger et al., 1996). It does not contain conserved residues
commonly found in the majority of kinases and the COOHterminal half of the AFK is made up almost entirely of six socalled kelch repeats (Xue and Cooley, 1993), a structural motif
found in proteins from diverse species such as Limulus scruin
(Way et al., 1995) and the human Host Cell Factor (Wilson et
al., 1993). Apart from this, the function of fragminP in phosphorylation of actin by the AFK can now be studied at the
molecular level by designing truncation mutants of the frgP
cDNA clone and analyzing the ability of the recombinant
deletion mutants to activate phosphorylation of actin after
complex formation. This approach should lead to a better
understanding of the structural requirements necessary to make
up a protein complex that is recognized by the AFK. A more
thorough assessment of the function of fragminP in vivo can
now be performed by disrupting the gene in apogamic amoebae
using established methods (Burland and Pallotta, 1995) and
examining whether the cells develop normally or which structures are being affected during development. Since, other genes
and proteins from amoebae and plasmodia were found to crossreact with frgP at the mRNA or protein level, the frgP cDNA
clone may therefore serve as a stepping stone towards the
isolation and characterization of these genes and determining
their role in modelling the actin cytoskeleton in Physarum
polycephalum.
We thank Prof. Frans Van Roy and Kurt De Vos for the use of the
confocal microscope and Stefaan Rosseneu for help with the DNA
sequencing. This work was supported by grants from the Flanders
Institute for Science and Technology (VLAB-COT), the Concerted
Research Actions of the Flemish Community (GOA), the Belgian
National Fund for Scientific Research (N.F.W.O.) and the EU contract
CHRX-CT94-0652 to J.V.. J.B. would like to thank Drs T. Burland
and L. Solnica-Krezel and Prof. W. F. Dove for helpful advice and
comments during the early part of this work. Thanks are also due to
Dr Burland for providing the DNA isolation protocol. J.B. wishes to
acknowledge the support of the University of Wisconsin Graduate
School, Programme Project grant CA23076 and Core grant CA07175
from the National Cancer Institute, and Wellcome Trust grant 034879.
J.B. and L.C. are currently supported by the Wellcome Trust (grant
042524).
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(Received 24 October 1996 – Accepted 13 March 1997)