(Received for publication, August 10, 1994; and in revised form, November 18, 1994)
From the
The actin-binding properties of the actin-fragmin complex from Physarum polycephalum microplasmodia were investigated with
respect to regulation by Ca, phospholipids, and
phosphorylation of the actin subunit by the endogenous actin-fragmin
kinase. Fragmin possesses two high affinity actin-binding sites and
probably also a third, low affinity site. Its nucleating and F-actin
severing activities are inhibited by phosphatidylinositol
4,5-bisphosphate (PIP
). Actin-fragmin specifically binds
PIP
which competes with actin for the
Ca
-sensitive site. However, PIP
cannot
dissociate the actin-fragmin complex nor the actin
-fragmin
trimer. Efficient F-actin nucleating activity by actin-fragmin is only
observed with unphosphorylated actin-fragmin, in the absence of
PIP
and at high Ca
(>µM)
concentrations. In the presence of PIP
, actin-fragmin only
caps actin filaments when unphosphorylated. The results suggest that in
the cell, hydrolysis of PIP
, concomitant with the increase
of cytosolic Ca
, could promote subcortical actin
polymerization.
The organization of the microfilament system is the result of specific interactions between actin and a number of actin-binding proteins(1, 2) . One class of proteins, of which gelsolin is a representative member, is thought to play an important role in the organization of the subcortical actin network in response to extracellular signals(3) . These actin-binding proteins are found throughout the eukaryotic world and display F-actin nucleating, severing, and capping activities in vitro(4) .
Gelsolin, originally isolated from macrophages(5) , is a
globular monomeric protein of M ± 90,000
and built up of six structurally similar segments, each consisting of
about 150 amino acids. The NH
terminally located domain
(segment 1) displays apparent
``Ca
-independent'' G-actin binding
properties(6) . The second segment binds F-actin and this
interaction is inhibited by phosphatidylinositol 4,5-bisphosphate
(PIP
)
; a third Ca
-dependent
actin binding site is located within the second half of the
molecule(7) .
The three-dimensional structure of the
actin-gelsolin segment 1 complex was recently described and reveals the
interface of the two proteins at atomic resolution(8) . Most
interacting side chains of gelsolin participate in a long -helix
and interact with residues belonging to subdomains 1 and 3 of actin.
Ca
forms a stable bridge between chelating residues
belonging to both proteins and is not removed upon subsequent treatment
with EGTA, explaining why this interaction was initially interpreted as
Ca
-independent. This actin binding region was
previously postulated on the basis of amino acid sequence homology
between fragmin, gelsolin, and Acanthamoeba profilin(9) .
The gelsolin-actin complex can be
dissociated by PIPin vitro(10) ,
suggesting a role for PIP
as a regulator of actin assembly
in the subcortical region of the plasma membrane. A similar actin
regulatory mechanism was previously postulated for profilin (11) and later confirmed(12) .
Fragmin is a
gelsolin-like actin-binding protein, isolated from Physarum
polycephalum plasmodia(13, 14) . Its sequence is
very similar to the NH-terminal half of gelsolin. Like
gelsolin, this 45-kDa protein nucleates polymerization of actin
monomers and caps and severs actin filaments in a
Ca
-dependent manner(15) . In the presence of
micromolar calcium concentrations, fragmin forms a stable 1:2 complex
with actin (A
F). Subsequent removal of calcium
converts the 1:2 complex into a 1:1 EGTA-resistant actin-fragmin
complex(9) . This dimer has no severing activity. The latter
(but not the 1:2 complex) is also a specific target for the endogenous
actin-fragmin kinase that phosphorylates the actin subunit at residues
Thr-203 (major site) and Thr-202 (minor
site)(16, 17) . These residues constitute part of the
actin-actin contact site in both proposed F-actin
structures(18, 19) .
To obtain better insight in
this complex regulatory mechanism, we studied the effects of
Ca and PIP
on the actin nucleating and
F-actin capping and severing properties of fragmin, the actin-fragmin
complex, and the phosphorylated actin-fragmin complex. The results
allow us to propose a more general model for the actin-regulatory
activities of gelsolin-like proteins.
The actin-fragmin complex and the actin-fragmin kinase were both isolated from P. polycephalum microplasmodia as described(17) .
Fragmin was obtained from the purified
actin-fragmin complex by dissociation with 6 M urea. The
dissociated complex was loaded onto a 1-ml DEAE-cellulose column
equilibrated in 50 mM Tris/HCl, pH 8.2, 1 mM dithiothreitol, and 0.02% NaN and the subunits were
eluted with a linear NaCl gradient (0-500 mM, 20 ml).
The first protein peak corresponded to fragmin and the second to actin
(as identified by SDS-polyacrylamide gel electrophoresis). Fractions
containing fragmin were pooled, 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 dithiothreitol, and 0.02% NaN
.
The inhibition of
actin-fragmin trimer formation by PIP
was
measured at a concentration of 150 nM pyrenyl-actin in
G-buffer to which the same concentration of actin-fragmin complex was
added. The latter was preincubated in G-buffer with increasing amounts
of PIP
(from 1 to 170 times molar excess over A
F).
The fluorescence of pyrenyl G-actin in the same concentration was set
at 0%.
The dissociation of the pyrenyl AF trimer
was measured at a concentration of 150 nM in G-buffer, adding
increasing concentrations of PIP
(ranging from 1 to 100
times molar excess) for 10 min at room temperature. The variations in
fluorescence were expressed as above.
Gel filtration was performed on a fast
protein liquid chromatography system (Pharmacia, Uppsala, Sweden),
using a Superose 12 HR 10/30 column, equilibrated in 20 mM MOPS, pH 7.4, 50 mM KCl, and 0.02% NaN. The
flow rate was kept at 0.5 ml/min. The eluate was monitored by
absorbance at 280 nm.
SDS-polyacrylamide gel electrophoresis (25) was performed on 10 or 15% polyacrylamide mini slab gels according to (26) . The gels were stained with 0.25% Coomassie Brilliant Blue in 10% acetic acid or by silver staining.
The villin
133-YNVQRLLHVKGKKNVV-147 PIP-binding peptide (27) was synthesized on an Applied Biosystems automated 431A
Peptide Synthesizer using the Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry procedure and
following the manufacturer's instructions. We have substituted
the cysteine residue at the end of the peptide in the original sequence
of villin (28) for a valine residue in order to prevent
artifactual dimerization due to oxidation and disulfide formation.
After cleavage with trifluoroacetic acid, the peptide was desalted in
water through Sephadex G-25 gel filtration (30
2.6 cm). The
eluate was monitored by UV absorbance at 254 nm. Finally, the peptide
was lyophilized and stored at -20 °C until further use.
Figure 1:
Actin nucleating activity of
actin-fragmin and its inhibition by phosphorylation. Induction of actin
polymerization by either fragmin (F; 140 nM) or the
actin-fragmin complex (AF; 140 nM) in the presence of
0.2 mM Ca
. C is the control and
represents the polymerization of actin (6 µM) in F buffer
but in the absence of nucleating agent (notice the lag phase).
A
F indicates the polymerization curve in the presence
of fully phosphorylated A
F (140
nM).
Figure 2:
Effect of PIP on the F-actin
nucleating activity of fragmin and actin-fragmin in the presence of
Ca
. Fragmin (A)
or actin-fragmin (B) were preincubated with the indicated
molar excesses of PIP
, prior to their addition to a
solution of pyrene-labeled G-actin. The polymerization was induced by
shifting to F buffer conditions. The control (C) consists of a
polymerization mixture without nucleating protein in the absence of
PIP
.
In
view of these conflicting data, and particularly with the intention to
measure the influence of PIP on the capping activity (see
below), we have readdressed this issue by a more quantitative
assay(22) . When an F-actin (+) end capping protein is
added to a mixture of unlabeled short actin filaments and
pyrene-labeled G-actin monomers, it will slow down the overall
polymerization rate as compared to the control (no capping protein),
since elongation of filaments can only proceed from the pointed ends.
From Fig. 3it can be inferred that, as expected, F-actin
capping by A
F results in a drastic reduction of the
polymerization rate. In the presence of EGTA, the fully phosphorylated
A
F did not display any capping activity (Fig. 3A). Conversely, when 0.2 mM Ca
is present, phosphorylated A
F capped
actin filaments in a way similar to that observed for the unmodified
complex (Fig. 3B). It is worth noting that
two-dimensional gel analysis of the phosphorylated complex demonstrated
complete phosphorylation of the actin subunit (data not shown),
implying that the remaining capping activity due to unphosphorylated
A
F can be excluded. Thus phosphorylation of the A
F complex
in actin imposes Ca
dependence on the F-actin capping
activity.
Figure 3:
Effect of actin-fragmin phosphorylation on
the F-actin capping activity. Panel A shows the polymerization
kinetics in the presence of EGTA; panel B shows the
experiments carried out in the presence of Ca.
A
F (14 nM) indicates the actin-fragmin complex.
A
F, the phosphorylated complex (14 nM).
Since F-actin nuclei were added to G-actin (3 µM) to
induce the polymerization, no lag phase appears. C, control,
actin alone.
In the
presence of Ca, PIP
inhibits the capping
activity of A
F. A 500-fold molar excess of PIP
over
A
F results in ±50% inhibition (Fig. 4A).
Identical PIP
concentrations exert an almost total
inhibition on the activity of the phosphorylated complex (Fig. 4B). Thus, both A
F and its phosphorylated
form are inhibited by PIP
in their F-actin capping
activities in the presence of Ca
. However, the effect
is much more pronounced with the phosphorylated form. As demonstrated
above, a similar PIP
-dependent differential effect was also
noticed when the nucleation activities of both fragmin and A
F
were compared (see Fig. 2, A and B). These
findings can be explained by assuming a third actin binding site in
fragmin. This site is active only under conditions where excess G-actin
is present, such as nucleation, but not capping (see also
``Discussion''). Furthermore, this third site is probably
PIP
-insensitive.
Figure 4:
Effect of PIP and other
phospholipids on the F-actin capping activity of actin-fragmin and the
phosphorylated complex. F-actin capping activities were
measured as in Fig. 3. A, activity of A
F in
increasing concentrations of PIP
. The molar excess of
PIP
over A
F is shown on the right; B, the
same PIP
concentrations as in A but now with
phosphorylated A
F (A
F); C, protein
ratios as in B but A
F capping activity was measured in
the presence of 2 mM EGTA and the indicated PIP
molar excess; D, effect of various phospholipids: PIP,
PI, and PC on the activity of the A
F complex. Concentrations of
phospholipids are the same as the PIP
concentration
resulting in 50% inhibition (500
molar excess). E,
inhibition of the activity of the phosphorylated complex
(A
F) by various phospholipids including PS and PE.
The effect of IP
(500
molar excess over
A
F) is also shown. The control (C) is as
described in the legend to Fig. 3.
In the presence of EGTA (Fig. 4C), the inhibitory effect of PIP on
A
F hardly differs from that observed in Ca
(Fig. 4A). These studies were not carried out for
phosphorylated A
F, since this form does not cap F-actin in EGTA
(see Fig. 3A).
We have also studied inhibition of
the F-actin capping activity on AF by other phospholipids (Fig. 4D). This was done using concentrations of
phospholipids similar to those of PIP
that resulted in
±50% inhibition (see Fig. 4A). Neither PIP, PI,
nor PC displayed significant inhibitory effects.
Similar effects
were measured with the phosphorylated form of the AF complex at
high Ca
concentrations (Fig. 4E).
Again PIP
displays the highest inhibition, whereas PIP only
shows a marginal effect. The other polyphosphoinositides PI, PC, PE,
PS, and IP
were not inhibitory at all.
Figure 5:
Inhibition of fragmin severing activity by
various phospholipids. F-actin filaments were precapped with AF
and then diluted below the critical concentration of the(-)-end.
Severing by fragmin was measured as a decrease in fluorescence due to
rapid depolymerization at the newly created pointed ends. The decrease
in fluorescence per min (during the linear phase of the curve) in the
presence of fragmin was set at 100% severing activity. Depolymerization
in the absence of fragmin was taken as 0% severing activity. Lane
1, fragmin (25 nM); lane 2, fragmin +
PIP
(1:300 molar ratio); lane 3, fragmin +
PIP
(1:150 molar ratio); lane 4, fragmin +
PIP
(1:60 molar ratio); lane 5, mixture of
PIP
(1:150 molar ratio) and PC; lane 6, PIP
(1:150)/PS; lane 7, PIP
/IP
(1:150)). The following phospholipids were added in a 300-fold
molar excess over fragmin: PIP, PI, PS, PC, PE, and the
phosphoinositide IP
(lanes 8-13). Notice
that inhibition of F-actin severing activity is very specific for
PIP
and less so for PIP.
When other polyphosphoinositides were
employed, only PIP displayed a significant inhibitory effect (Fig. 5, lane 8) while PI, PS, PC, PE, and IP were inactive. Mixed polyphosphoinositides, containing PC or PS
in addition to PIP
, reduced the inhibitory effect of
PIP
on the severing activity of fragmin (Fig. 5, lanes 5-7). A summary of the activities of fragmin,
actin-fragmin, and the phosphorylated actin-fragmin complex described
above is given in Table 1.
When AF was incubated with pyrene-labeled G-actin under
nonpolymerizing conditions, an increase in fluorescence intensity was
observed, due to complex formation between A
F and pyrenyl
G-actin. At a molar ratio of 1 or higher, a plateau was seen (Fig. 6A). When this experiment was carried out in the
presence of EGTA, no change in the fluorescence was measured. These
results are in agreement with the existence of a second actin-binding
site which is Ca
-dependent, confirming previous
studies(9) . The same experiment with fully phosphorylated
A
F resulted in nearly identical plots as for A
F, both in
high or low calcium (Fig. 6A). When fragmin was used
instead of A
F, maximal fluorescence values were obtained at a
fragmin/pyrenyl-actin molar ratio of 0.5, in accordance with the
binding of two actin molecules to fragmin (Fig. 6A).
Figure 6:
Stoichiometry of actin-fragmin
interactions: modulation by phosphorylation, Ca, and
PIP
. A, pyrenyl G-actin
was incubated with increasing concentrations of actin-fragmin in
Ca
(
) or EGTA (
), phosphorylated
actin-fragmin in Ca
(
) or EGTA (
), or
with fragmin and Ca
(
). The molar ratios of the
proteins are given on the abscissa. The ordinate shows the relative fluorescence enhancement due to binding of
pyrene-labeled actin. B: upper panel, the inhibitory effect of
increasing amounts of PIP
on the formation of the trimer
(A
F) consisting of Physarum actin-fragmin
(A
F) and pyrenyl-muscle actin (150 nM each). The first bar shows the fluorescence enhancement in the absence of
PIP
. The abscissa shows the molar ratios of
PIP
relative to the actin-fragmin complex. Lower
panel, inability of PIP
to dissociate the
actin
-fragmin trimer once it is formed. The abscissa now shows the molar ratios of the A
F trimer and
PIP
. The results are mean ± S.D. of triplicate
determinations.
To study the effect of PIP on A
F
trimer formation and dissociation, we preincubated the A
F complex
with pyrenyl-actin in the presence of Ca
and measured
the change in fluorescence intensity after mixing the trimer with
increasing concentrations of PIP
. The phospholipid had no
effect on the fluorescence intensity of the actin
-fragmin
trimer (Fig. 6B, lower panel), suggesting it is unable
to dissociate the trimer. Conversely, PIP
was able to
inhibit the formation of a trimeric complex: when increasing amounts of
PIP
were incubated with A
F, we noticed a decrease in
fluorescence that leveled out to zero at a 100-fold molar excess
PIP
(Fig. 6B, upper panel). Thus PIP
and the second actin molecule mutually exclude each other.
The
stability of the AF complex in the presence of PIP
was also assayed by gel filtration and immunoprecipitation. When
A
F was incubated with PIP
and then passed over a
Superose 12 gel filtration column, we noticed a significant increase in
apparent M
but no dissociation into fragmin and
actin (results not shown). Prolonged incubation, up to 24 h, showed no
significant dissociating effect. The binding of PIP
to
A
F could be inhibited when the phospholipid was first incubated
with a synthetic PIP
-binding peptide derived from
villin(27) : a 2-fold molar excess of peptide over PIP
resulted in a shift to the original apparent M
for 75% of actin-fragmin (data not shown). The remaining 25% of
the actin-fragmin complex eluted from the column at the position where
the PIP
-fragmin-actin complex elutes. In a second
experiment we followed a similar approach as Janmey et
al.(10) : polyclonal anti-fragmin IgGs, immobilized on
Sepharose beads, were used to immunoprecipitate the actin-fragmin
complex preincubated with various concentrations of PIP
. No
enrichment for actin in the supernatant was found after
SDS-polyacrylamide gel electrophoresis (data not shown), consistent
with the existence of a stable A
F complex in PIP
.
In conclusion, these results indicate that PIP binds to
fragmin and to the actin-fragmin complex and inhibits further addition
of actin to these proteins. However, once the complex is formed,
neither the A
F dimer nor the A
F trimer can be
dissociated by PIP
.
The primary structure of fragmin reveals a triple repeat of a segment of approximately 150 amino acids. Because of the high degree of sequence homology between fragmin and members of the gelsolin family (9) , we predict that these repeats fold into domain structures, each similar to the three-dimensional structure of gelsolin S1 (8) or villin 14T(32) . All three domains of fragmin can therefore be considered as potential actin-binding domains.
Apart from structural homology, fragmin shares a number of
functional properties with gelsolin. For instance its ability to bind
two actin molecules in the presence of Ca, thus
forming an A
F trimer. In this paper we demonstrated
that A
F is not converted into the A
F
heterodimer upon addition of excess of fragmin (Fig. 6A). Thus the second high affinity
Ca
-dependent actin binding site is involved in a
stable association. Similar results were previously reported for
gelsolin (33) but contrast those described for
severin(23) , where the actin
-severin complex is
converted into a stable dimer upon addition of excess severin.
Fragmin also displays properties distinct from those of other
members of the gelsolin family. For instance, it is only after its
association with actin that the actin moiety of the complex becomes
amenable for phosphorylation by the actin-fragmin kinase(17) .
A similar phosphorylated gelsolin-actin complex has not been described
so far (34) , nor have we been able to phosphorylate the
gelsolin-actin complex with the Physarum actin-fragmin
kinase(16) . This important functional difference suggests
altered microfilament regulatory properties for fragmin. Therefore, we
carried out F-actin nucleation, capping, and severing experiments using
Ca, PIP
, and phosphorylation as
modulators. Two unexpected findings emerged from our experiments with
PIP
. First, nucleation by unphosphorylated A
F cannot
be fully inhibited by PIP
(Fig. 2B). This
can be explained by supposing that PIP
has a low affinity
for the second actin binding site or by assuming the existence of a
third actin binding site (see also below). The second unexpected result
illustrates an important difference between the actin-gelsolin complex
and A
F: whereas the former is dissociated by PIP
, the
latter is not.
A model incorporating these new findings is presented
in Fig. 7and is based on the existence of two high affinity
actin-binding domains and a third putative actin-binding site in
fragmin. These domains are each differently regulated and display
different affinities for actin. They are designated domains I, II, and
III in Fig. 7A. This organization is reminiscent of the
one described previously for severin(23) , where two lipid
binding domains, two Ca binding domains, and three
actin-binding sites were demonstrated.
Figure 7:
Domain organization of fragmin and
schematic representation of the complexes that can be formed between
actin and fragmin. A, the organization and properties of the
domains of fragmin. B, the different complexes that can be
formed between actin and fragmin in the presence or absence of
PIP and Ca
. Complexes 6 and 7 describe
the properties of the phosphorylated forms. Severing, capping, and
nucleating activities are allocated to each of these complexes. For
detailed information see
``Discussion.''
In the presence of
Ca, both site I and II bind actin, forming an
A
F trimer (Fig. 7B, 1). PIP
interferes with the formation of this complex (Fig. 7B, 2). Upon Ca
depletion, only
the actin on site II is released and this A
F heterodimer (Fig. 7B, 3) has capping activity (through the residual
actin moiety) but surprisingly also possesses weak nucleating activity
in EGTA (data not shown). Since nucleation requires binding of two
actin molecules, two possibilities arise. Either the
Ca
-sensitive high affinity site II is turned into a
low affinity site in the absence of Ca
, or a
different low affinity site exists (site III). The first possibility
can be ruled out from our experiments with PIP
. Indeed,
this nucleating activity is not abolished in the presence of PIP
(Fig. 2B), whereas binding of actin to site II is
inhibited by PIP
(Fig. 7B, 4). Therefore, a
third actin binding site probably exists to account for the nucleating
activity by A
F in the presence of this phospholipid (Fig. 7B, 5, hatched). Because of its low affinity,
site III would be occupied only at high actin concentrations, typically
found in F-actin nucleation conditions, but not in capping conditions.
We further postulate that this site is only accessible when site II is
free, thus at low Ca
concentrations or when site II
is occupied by PIP
. Phosphorylation of actin residing on
site I converts the EGTA-resistant heterodimer into a fully inactive
complex with respect to nucleating and capping activity (the latter in
EGTA) (Fig. 7B, 6). This phosphorylated complex
displays full capping activity in Ca
via site II. In
the presence of PIP
, site II is blocked and consequently no
capping activity is observed (Fig. 7B, 7).
In free
fragmin, site I and II are inhibited by PIP (Fig. 7B, 2). Thus under nucleating conditions,
we expect no activity (Fig. 2A). The higher nucleating
activity of actin-fragmin as compared to fragmin (Fig. 2A) is probably due to binding of fragmin to the
first actin monomer, a reaction which in the case of gelsolin, has been
found to be slow(35, 36, 37) . Nor did we
measure F-actin severing activity by fragmin in the presence of
PIP
(Fig. 5), since this assumes the presence of at
least two free, high affinity, actin-binding sites. Finally, there will
be no capping activity because at low actin concentrations no site will
be occupied.
We showed that unlike complexes of actin with
gelsolin(31) , profilin (11) , severin(23) , or
Cap 100(22) , the actin-fragmin complex is not dissociated by
PIP (Fig. 7). This property is also shared by the
45-kDa sea urchin fragmin-like protein(38) . No physiologically
relevant agent that dissociates the actin-fragmin complex has been
identified so far, although Ohnuma and Mabuchi (39) have
mentioned a factor that dissociates the PIP
-resistant
45-kDa protein-actin complex.
In previous studies, the different
domains and corresponding functions of gelsolin (40, 41, 42) and severin (23) were
allocated. Based on the sequence homology between fragmin and gelsolin,
it is reasonable to predict that the NH-terminal segment of
fragmin corresponds with gelsolin S1. This segment in gelsolin is
involved in PIP
binding, since a mutant lacking the 15
COOH-terminal amino acids no longer binds PIP
(43) .
The consensus PIP
-binding motif in this segment has been
identified as KXXXKXKK. Fragmin, however, displays
only partial homology with this region (KXXXRXLD).
One of the two remaining domains (here presented as site II) contains
the Ca
-dependent site. Inhibition of site II by
PIP
may be analogous to the PIP
-dependent
F-actin binding site, involved in F-actin severing, located in the
second segment of gelsolin(7) . The consensus sequence for this
PIP
-binding motif is
KXXXXKXKK(43) , but the amino acid sequence
of this region in fragmin remains unknown (9) . Using deletion
mutagenesis, Sun et al.(44) recently showed that the
third segment of gelsolin may contain an additional
polyphosphoinositide-binding site and that it does not contain a high
affinity G-actin binding site. The corresponding segment in fragmin on
the other hand probably binds actin monomers with low affinity, but
regulation by PIP
is unlikely.
So far, we have little
indication of how much fragmin in Physarum microplasmodia
exists in the free form. Immunoprecipitation experiments with
antifragmin antibodies in the presence of EGTA reveal that most fragmin
is bound to actin in crude extracts. ()This raises the
hypothesis that under these growth conditions, the actin-fragmin
complex shown in Fig. 7B, 2, is predominant. Binding of
A-F to PIP
suggests a direct physical link with the
phosphoinositide signaling pathway. Based on these observations, the
following models for A
F function in vivo could be
envisaged. Although cytosolic A
F caps actin filaments
independently of the Ca
concentration, there is a
difference between filaments that are capped in the presence of high or
low Ca
. Indeed, the former will have both site I and
II on fragmin occupied with actin and therefore cannot anchor actin
filaments on PIP
-containing membranes. However, when
Ca
is low, A
F capped filaments could attach via
the unoccupied site II of fragmin. When PIP
is hydrolyzed,
these filaments are released into the subcortical area and site II will
now be occupied by actin because Ca
is high. These
filaments may attach again to the membrane when Ca
is
decreased and PIP
formed.
One could also consider the
situation in which the AF complex is associated with the membrane
where it could function as an anchoring site for uncapped actin
filaments and here attachment of filaments is independent of the
Ca
status of the cell. When PIP
is
hydrolyzed and Ca
increased, A
F will be
released from the membrane and converted into an F-actin nucleating
agent, inducing actin filaments to grow rapidly from the subcortical
region. At this moment, however, we have no decisive evidence to
support our hypothesis. The proposed mechanism is different from the
model described previously for gelsolin activity in
vivo(3, 45, 46) . There PIP
dissociates the gelsolin-actin complex, allowing actin to
polymerize. When PIP
is hydrolyzed, and Ca
increases, gelsolin acts as an F-actin severing protein, leading
to a fast destruction of the subcortical actin network.
What is the
role of AF phosphorylation? At this stage it cannot be excluded
that phosphorylation is a signal for intracellular translocation;
alternatively, it could be the first step leading to dissociation of
the complex. Its major role, however, is undoubtedly correlated with
the regulation of actin binding to the A
F complex as is evident
from the location of the phosphorylated amino acid in the actin-actin
contact site(16) .
So far it is not clear if the degree of
phosphorylation is altered in response to various stimuli or is
correlated with stages of development of Physarum cells.
Obviously, more detailed information related to these questions, as
well as to the mechanism of dephosphorylation, will tell us more about
the role of AF phosphorylation.
In conclusion, important
elements of our model will depend on the fate of the phosphorylated
complex once bound to the membrane. Indeed, if PIP association could function as a trigger for dephosphorylation,
then this step would lead to a membrane located reactivation of
A
F. Consecutive PIP
hydrolysis and Ca
increase would release a fully active nucleating species.
In
these models, we have not considered the interplay between AF and
other actin-binding proteins, that could regulate the stability of the
actin filaments. Their activity will be important for the ideas which
have been proposed here and future experiments will have to address
these questions.