(Received for publication, November 21, 1995; and in revised form, January 24, 1996)
From the
Adducin is a membrane skeleton protein originally described in
human erythrocytes that promotes the binding of spectrin to actin and
also binds directly to actin and bundles actin filaments. Adducin is
associated with regions of cell-cell contact in nonerythroid cells,
where it is believed to play a role in regulating the assembly of the
spectrin-actin membrane skeleton. In this study we demonstrate a novel
function for adducin; it completely blocks elongation and
depolymerization at the barbed (fast growing) ends of actin filaments,
thus functioning as a barbed end capping protein (K
100 nM). This barbed end capping activity requires
the intact adducin molecule and is not provided by the
NH
-terminal globular head domains alone nor by the
COOH-terminal extended tail domains, which were previously shown to
contain the spectrin-actin binding, calmodulin binding, and
phosphorylation sites. A novel difference between adducin and other
previously described capping proteins is that it is down-regulated by
calmodulin in the presence of calcium. The association of
stoichiometric amounts of adducin with the short erythrocyte actin
filaments in the membrane skeleton indicates that adducin could be the
functional barbed end capper in erythrocytes and play a role in
restricting actin filament length. Our experiments also suggest novel
possibilities for calcium regulation of actin filament assembly by
adducin in erythrocytes and at cell-cell contact sites in nonerythroid
cells.
Precise control of actin filament length is an important functional consideration in a number of tissues including striated muscle (1) and the erythrocyte membrane skeleton(2) . These two examples are similar with respect to the uniform lengths of the filament population, although the actual lengths themselves are different, 1 µm in skeletal muscle (3) and 33-37 nm in erythrocytes (4, 5, 6) . These uniform length distributions suggest strict regulation of actin filament growth because pure actin filaments polymerize to an exponential length distribution at steady state in vitro(7) . In muscle, control of actin filament growth is achieved by capping the fast growing (barbed) ends with capZ (8, 9) and by capping the slow growing (pointed) ends with tropomodulin(10, 11, 12) . In erythrocytes, tropomodulin caps the pointed ends of the short actin filaments(11, 13, 14) , but a barbed end capping protein has not yet been identified.
A conserved mechanism
of actin filament length regulation between striated muscle and
erythrocytes would require a barbed end capping protein to be
identified in erythrocytes. There are five known actin binding proteins
that are associated with the short erythrocyte actin filaments:
spectrin, band 4.1, tropomyosin, tropomodulin, band 4.9, and adducin.
Most of these proteins can be ruled out because they bind along the
sides of actin filaments and have been shown directly not to block
actin polymerization from the barbed filament ends (for reviews see (15, 16, 17) ). However, several observations
raised the possibility that adducin, which was initially characterized
as a calmodulin-binding protein(18) , might be a candidate for
a novel erythrocyte barbed end capping protein. Adducin is associated
in stoichiometric amounts with the short erythrocyte actin filaments
(one ,
heterodimer per filament) (18) and has been
shown to bind to spectrin-actin complexes and promote spectrin binding
to actin (K
=
80
nM)(19, 20) , as well as to bind directly to
F-actin (K
=
280 nM)
and to bundle actin filaments(20, 21) . Curiously,
although the 39-kDa NH
-terminal globular ``head''
domains of both
and
adducin contain a region of sequence
homology to the actin-binding domain of the
-actinin family of
actin binding proteins(22) , the isolated head domains of
either
or
adducin do not appear to bind F-actin or
spectrin-actin complexes in cosedimentation assays (23) .
Indeed, the 33-kDa COOH-terminal extended tail domains of both the
and
adducin subunits were recently shown to be sufficient
for binding to spectrin-actin complexes and for recruitment of
additional spectrin molecules(24) . Thus, the functional
significance of the actin binding sequence homology in the head domain
of adducin remained an open question and suggested that adducin might
have additional actin-binding properties.
In this study, we show that whole adducin blocks elongation and depolymerization from the fast growing (barbed) ends of actin filaments. In contrast, neither the isolated head or tail domains have barbed end capping activity on their own. We further demonstrate that unlike other barbed end capping proteins, the capping activity of adducin is down-regulated by calmodulin in the presence of calcium. Sequencing of adducin cDNAs from erythrocytes and other tissues has demonstrated that adducins are a unique family of proteins associated with the spectrin-based membrane skeleton in erythroid and nonerythroid cells (for a review see (16) ). The combination of adducin's calcium/calmodulin-regulated barbed end capping, actin bundling, and enhancement of spectrin-actin binding activities indicates that adducins represent a new type of regulated actin filament binding and barbed end capping protein.
Figure 1:
Effect of adducin on
the rate of actin polymerization from the barbed ends of spectrin-actin
seeds. A, raw data. Elongation was initiated by the addition
of spectrin-actin seeds and salts to a 5 µM G-actin
solution (5% pyrenyl-actin) containing increasing concentrations of
adducin as indicated. B, percentage of inhibition of
elongation (percent capping) plotted against increasing adducin
concentrations. (The polymerization rate is calculated as a percentage
of the initial rate of polymerization in the absence of adducin and is
inversely proportional to capping.) The concentration of adducin
required to produce 50% inhibition of polymerization (100 nM)
was taken as a measure of the K.
Not only can adducin completely inhibit elongation from the
barbed ends of actin filaments, but saturating amounts of adducin (700
nM) also greatly reduce the depolymerization rate of F-actin
that has been diluted below its critical concentration (Fig. 2).
This effect is primarily due to inhibition of depolymerization from the
barbed end because under the conditions of our assays the higher off
rate of the barbed ends dominates the depolymerization kinetics; for
example, even complete inhibition of depolymerization at the pointed
end in these assays would have been expected to lead to only about a
10% reduction in the rate of depolymerization(35) .
Furthermore, we observe a similar reduction in the initial rate of
actin depolymerization in the presence of saturating amounts (30
nM) of nonmuscle capping protein isolated from erythrocytes (Fig. 2), a known barbed end capping protein (30) . The initial rates of depolymerization for
actin in the presence of adducin or capping protein were 53 and 56
arbitrary units, respectively, as compared with a rate of 754 arbitrary
units for actin alone. Interestingly, after the initial period, the
rate of actin depolymerization in the presence of adducin was slower
than in the presence of capping protein. We attribute this difference
to an effect of adducin on the pointed filament end because adducin
reduced (but did not block) the rate of actin depolymerization from the
pointed ends of gelsolin capped actin filaments (data not shown). The
inhibitory effect of adducin on the rate of actin depolymerization from
the barbed filament ends also indicates that adducin does not function
by exclusively sequestering actin monomers; monomer sequestering would
have been expected to lead to an increase in the rate of actin
disassembly, which was also not observed (see below). Finally, these
data also rule out possible artifacts in the nucleated actin
polymerization assay in Fig. 1from fluorescence signal
quenching due to light scattering and demonstrate that protein
aggregation does not occur in these assays. Taken together, the ability
of adducin to inhibit both actin polymerization and depolymerization
indicates that adducin is a barbed end capping protein with a capping
affinity of 100 nM.
Figure 2:
Effect of adducin on the rate of actin
depolymerization. Depolymerization of pyrene-labeled F-actin was
initiated by dilution of a 20 µM stock to a final
concentration of 1 µM into buffer A with or without
adducin or nonmuscle capping protein as indicated. All measurements
utilized a single actin stock to maintain a constant filament number in
the assay. Final concentrations in the assay of intact adducin were 700
nM, and that of nonmuscle capping protein was 30 nM.
Under the low salt conditions used in this assay, the final actin
concentration after dilution (1 µM) is less than the actin
critical concentration under these conditions (120
µM). This leads to the complete disassembly of the actin
filaments into monomers, as indicated by the decrease in the pyrene
fluorescence signal for the pure actin curve almost to the baseline. CP, nonmuscle capping protein.
Complete capping of barbed filament ends by previously described barbed end capping proteins results in an increase of the actin monomer concentration at steady state with the filaments, reflecting the higher critical concentration for the pointed ends(35, 36) . Fig. 3A shows that saturating concentrations of nonmuscle capping protein isolated from erythrocytes resulted in about a 3-fold increase in the amount of actin monomer remaining in the supernatant at steady state from 0.4 to 1.3 µM. In contrast, increasing concentrations of adducin (from 100 to 700 nM) led to a considerably smaller increase in the amount of actin monomer in the supernatant from 0.3 to 0.6 µM (Fig. 3B). This result suggests that adducin may reduce the critical concentration at the pointed end of the filament in addition to blocking monomer addition and loss at the barbed filament end. This interpretation is consistent with the observation that adducin reduces the rate of depolymerization while increasing the rate of elongation at the pointed filament end (data not shown). This may be a consequence of adducin reducing the actin monomer off rate for the pointed filament end by its binding along the sides of actin filaments. Finally, the observation that increasing amounts of adducin do not lead to continuously increasing amounts of nonpolymerizable actin also demonstrates that the effects of adducin on polymerization kinetics at the barbed filament end are not solely due to monomer sequestration.
Figure 3: The effect of increasing concentrations of nonmuscle capping protein (A) or adducin (B) on actin critical concentration. The extent of actin polymerization was determined after a 24-h incubation by centrifugation followed by SDS-polyacrylamide gel electrophoresis of supernatants and pellets as described under ``Experimental Procedures.'' The amount of actin remaining in the supernatant was taken to be the actin monomer concentration (actin critical concentration) and plotted versus the concentration of nonmuscle capping protein and adducin using the same y axis to facilitate comparison. The actin critical concentration in the absence of adducin or capping protein varied from about 0.3-0.4 µM (this experiment) down to about 0.05 µM in other experiments; however, the relative increases in the actin critical concentration in the presence of adducin or nonmuscle capping protein were similar in all experiments.
In an effort to determine whether the
actin filament capping activity of adducin could be localized to either
the NH-terminal globular head or to the COOH-terminal
extended tail domains, we tested bacterially expressed tails (24) and a head-neck domain prepared by trypsin digestion of
human erythrocyte adducin(23) . Neither the head nor the tail
domains (tested up to final concentrations of 1 and 5 µM,
respectively) had any significant effects on actin polymerization or
depolymerization in our assays (data not shown). Therefore, the barbed
end capping activity of adducin appears to be an attribute of the
entire molecule requiring both the heads and tails.
Figure 4:
Effect of calmodulin on the ability of
adducin to inhibit actin polymerization (A) and
depolymerization (B) in the presence of calcium. A, percentage
of capping calculated as described in the legend to Fig. 1B plotted against increasing concentrations of calmodulin. The
ability of 200 nM adducin to inhibit polymerization from
spectrin-actin seeds was tested as described above except with the
inclusion of increasing concentrations of calmodulin as indicated. The
standard assay conditions contained 0.2 mM CaCl (see ``Experimental Procedures''). B, effect
of calmodulin on the ability of adducin to inhibit the rate of actin
depolymerization in the presence of calcium. This assay was performed
as described above in Fig. 2but with diluting the F-actin into
a solution containing adducin and calmodulin to give final
concentrations of 700 nM and 10 µM, respectively.
Calmodulin had no effect on actin polymerization in the absence of
adducin (not shown).
Using both a nucleated actin polymerization assay (8) and an F-actin depolymerization assay, we have shown that
adducin caps the barbed ends of actin filaments with a K
100 nM. The association of
stoichiometric amounts of adducin with the short actin filaments in the
erythrocyte membrane skeleton (18) implies that adducin may
indeed be the functional barbed end cap in erythrocytes and supports
our hypothesis that the mechanism of restriction of actin filament
length in erythrocytes is based on capping actin filaments at both ends
to prevent growth or shrinking, similar to striated muscle. Although it
has been assumed that the barbed ends of the actin filaments in the
erythrocyte membrane skeleton are uncapped, this idea stems mainly from
the use of purified membranes or isolated spectrin-actin oligomeric
complexes to nucleate actin
polymerization(32, 37, 38, 39) . Our
results raise the possibility that the capping activity of adducin
might have been inactivated during hemolysis and/or the extraction
procedures used to prepare membranes and purify the spectrin-actin
complexes used in these experiments.
Calmodulin has been demonstrated to regulate the mechanical properties of the erythrocyte membrane in the presence of calcium(40) . The inhibitory effect of calcium/calmodulin on the ability of adducin to cap the barbed ends of actin filaments suggests that the effect of calmodulin on membrane mechanics may be partly mediated via changes in actin filament polymerization. Calcium/calmodulin also inhibits adducin binding to F-actin and to spectrin-actin complexes(19, 20) , as well as inhibiting spectrin-protein 4.1 cross-linking of actin filaments(41, 42) . These observations emphasize that calmodulin is likely to regulate membrane mechanical properties via multiple effects on membrane skeleton organization.
The capping
affinity of adducin is considerably lower than has been described for
other barbed end capping proteins (e.g. gelsolin = 1
pM; capping protein = 1 nM; for reviews see (9) and (36) ) and is similar to the affinity of
adducin for binding to the sides of actin filaments and bundling them (K =
280 nM)(20) . It
is also striking that the inhibitory effect of calmodulin on the barbed
end capping activity of adducin has a similar dependence on calmodulin
concentration as that of calmodulin's inhibition of adducin side
binding to F-actin and to spectrin-actin
complexes(19, 20) . Furthermore, the calmodulin
binding site on adducin has been proposed to be located on the
midregion of the COOH-terminal tail domains(43) ; yet
dissection of adducin into NH
-terminal head and
COOH-terminal tail domains shows that the entire molecule is required
for barbed end capping activity. Therefore, the barbed end capping
activity of adducin may be functionally linked to its ability to bind
along the sides of actin filaments.
The actin-binding sequence
homology in the NH-terminal head domain of the adducin
and
subunits diverges significantly from the consensus
actin-binding domain found in the functional members of the
-actinin protein family (similarity:
-adducin 47.9%,
-adducin 40.7%; identity:
-adducin 21.6%,
-adducin
12.8%). (
)In addition, both the
and
subunits of
adducin deviate from the sequence found in
-actinin by the
presence of an extra portion of sequence; in the case of the
subunit, this is in one of the regions identified as the actin-binding
site(44) . The divergence from the consensus actin-binding
sequence has been suggested to explain the lack of binding to actin
filaments by the head domain of adducin(22) . However, we would
propose that this actin-binding site in the NH
-terminal
head domain of adducin may have, through divergent evolution, developed
the capability to bind to actin so as to cap the barbed end of the
filament. Actin monomers in the filament are oriented such that
subdomains 1 and 3 are exposed at the barbed end of the actin
filament(45, 46) . It may be significant that a
portion of the
-actinin actin binding site has been demonstrated
to be on subdomain 1 of actin(47, 48, 49) .
Based on these considerations, we propose that the barbed end
capping activity of the NH-terminal head domain is reliant
on the side binding of the adducin COOH-terminal tails to actin
filaments. It is likely that the COOH-terminal tail domains of the
and
subunits possess an F-actin binding site based on their
ability to bind to spectrin-actin complexes and to promote spectrin
binding to actin (24) . Binding of the tails along each strand
of the actin filament in a polarized fashion (21) could
position the head domains to bind to a site in subdomain 1 of the
terminal actin monomer at the barbed filament end, hence interfering
with association of new actin monomers as well as stabilizing the
filament and preventing monomer dissociation. A model depicting binding
of adducin tails to actin and capping the barbed end of the actin
filament by adducin heads is shown in Fig. 5. In addition to
promoting spectrin binding to actin, binding of adducin tails along the
sides of the erythrocyte actin filaments may also play a role in
stabilizing them at their pointed filament ends, as suggested by the
actin critical concentration experiment (Fig. 3). The
multifunctional actin binding properties of adducin may not be unique;
tensin is another protein that has been shown to cap actin filament
barbed ends as well as to bind along the sides of actin filaments and
bundle them(50, 51) .
Figure 5:
A schematic model for actin capping by
adducin. The barbed end of an erythrocyte actin filament is shown with
six associated spectrin molecules(4, 6) . For clarity
only the NH-terminal actin-binding region of
-spectrin
is depicted, and
-spectrin, protein 4.9, protein 4.1, and the
tropomodulin and tropomyosin at the pointed end of the actin filament
have been omitted. The NH
-terminal adducin head domains
form a tetramer (24) at the barbed end of the filament blocking
the exchange of actin monomers. The extended COOH-terminal adducin
tails lie along each strand of the actin filament forming contacts
between the actin and spectrin molecules, enhancing their interaction.
We propose that this lateral actin binding by the adducin tails is
responsible for maintenance of the cap at the end of the filament.
Recruitment of extra spectrin molecules is either due to increased
affinity of spectrin for the complex or the reorganization of the bound
spectrin to accommodate more molecules. Hence adducin plays a crucial
role in the organization of the molecular interactions at the barbed
end of the actin filaments in erythrocytes.
The actin capping properties of adducin suggest that adducin could provide a link between membranes and the barbed ends of spectrin-associated actin filaments. In erythrocytes, adducin has been shown to bind directly to stomatin(52) , a membrane protein that is associated with ion channels and has been genetically linked to hereditary stomatocytosis(2) . In nonerythroid cells, adducin is associated with the spectrin-based membrane skeleton at cell-cell contact sites on the lateral borders of the plasma membrane of epithelial cells(53) . Protein kinase C phosphorylation of adducin in cultured epithelial cells induces redistribution of adducin away from cell-cell contact sites(53) . Functional linkage of adducin's barbed end capping activity to the filament side binding activity of the tails may imply that phosphorylation of tails by protein kinase C (23) could also regulate adducin's capping function, as we have demonstrated here for calcium/calmodulin. It is tempting to speculate that adducin could be a regulatory target for signal transduction pathways that lead to remodeling of the actin cytoskeleton at cell-cell contact sites.