From the Department of Cell Biology, Institute of
Molecular Biology, Austrian Academy of Sciences, Billrothstrasse
11, A-5020 Salzburg, Austria and the European Molecular
Biology Laboratory, Department of Structural Biology,
Mayerhofstrasse 1, D-69012 Heidelberg, Germany
Received for publication, October 19, 2000
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ABSTRACT |
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The Caenorhabditis elegans
unc-87 gene product is essential for the maintenance of the
nematode body wall muscle where it is found colocalized with actin in
the I band. The molecular domain structure of the protein reveals
similarity to the C-terminal repeat region of the smooth muscle
actin-binding protein calponin. In this study we investigated the
in vitro function of UNC-87 using both the full-length
recombinant molecule and several truncated mutants. According to
analytical ultracentrifugation UNC-87 occurs as a monomer in solution.
UNC-87 cosedimented with both smooth and skeletal muscle F-actin, but
not with monomeric G-actin, and exhibited potent actin filament
bundling activity. Actin binding was independent of the presence of
tropomyosin and the actin cross-linking proteins filamin and
The interaction of actin and myosin to produce force is an
essential prerequisite for a variety of cellular processes including muscle contraction (1), cell motility, and anchorage (2). The
organization of contractile and motile systems based on actin relies on
a large family of actin-associated proteins that regulate and define
the assembly of actin into filaments and then into filament arrays (3,
4). To date, more than 60 different proteins directly interacting with
actin have been identified, but the majority of F-actin-binding
proteins populate partially overlapping regions on the filament (5, 6).
Despite the large number of actin-binding proteins, functional
diversity is reflected by a limited number of basic structural modules
(7). Most actin cross-linking proteins exhibit two independent
actin-binding domains, each individual actin-binding domain
commonly composed of a tandem arrangement of the calponin homology
domain module (8) and other modular elements defining the distance
between and the relative orientation of the two actin-binding domains, often involving parallel or antiparallel dimerization (7).
We have shown recently that a unique sequence motif found in the
C-terminal third of the calponin
(CaP)1 molecule and other
members of the CaP family of actin-associated proteins (9), namely a
23-amino acid residue repeat, which we will refer to from now on as the
CLIK-23 repeat, forms an independent actin-binding site (10). This
finding was corroborated by Mino et al. (11) who
demonstrated the direct interaction of a peptide corresponding to the
first CaP repeat with actin in vitro. A survey of the
available data bases identified other proteins with CLIK-23 repeats, in
particular the Caenorhabditis elegans body wall muscle protein UNC-87 that exhibits seven tandem CLIK-23 repeats (12). A
protein with a similar molecular structure has also been described by
Irvine et al. (13) in the filarial worm Onchocerca
volvulus. Although the UNC-87 protein was identified as a key
molecule for maintenance of the structural integrity of the myoskeletal
apparatus in the nematode (12, 14), no further information on its
putative biological function or on the mode of interaction with actin
was obtained.
In this study we show that UNC-87 is an actin-bundling protein in
vitro and in vivo and present evidence for a binding
site of the CLIK motif on the actin filament different from that of other actin cross-linking proteins.
Construction of Plasmids--
The expression plasmids
UNC-87-pEGFP-C1 (for expression of a GFP-tagged full-length UNC-87
protein in mammalian cells) and UNC-87-pMW172 (for bacterial expression
of untagged full-length UNC-87) were constructed from the
unc-87 full-length cDNA clone pSG3 (accession number
U04711; see Ref. 14). This cDNA corresponds to the predominantly
transcribed smaller splice variant (UNC-87b) and contains exons A, C,
D, E, F, and G (14). Polymerase chain reaction on pSG3 was performed
using a forward primer that starts amplification at the first of two
potential start sites and introduces a BglII site
immediately 5' to the start codon. The reverse primer introduces an
EcoRI site immediately 3' of the original unc-87 stop codon. The polymerase chain reaction product was digested with
BglII and EcoRI and cloned into the corresponding
sites of pEGFP-C1 and the BamHI and EcoRI site of
pMW172 (15), respectively. All sequences were confirmed by dideoxy
sequencing using a LI-Cor model 400 automated sequencer (MWG
Biotech AG, Ebersberg, Germany).
The deletion mutants containing repeats 2-7 (amino acids 69-374),
3-7 (), 4-7 (), and 5-7 () were made by
polymerase chain reaction essentially the same way as described for the
full-length constructs and cloned into pEGCP-C1 and pMW172, respectively.
For construction of the deletion mutant containing repeats 1-3 (amino
acids 1-169) full-length UNC-87-pEGFP-C1 was digested with
AcyI followed by a complete fill-in of the overhang by
Klenow fragment. After digestion with BglII the UNC-87
fragment was cloned into the BglII and SmaI site
of pEGFP-C1. For bacterial expression this construct was again digested
with BglII and BclI and ligated into the
BamHI site of pMW172. The correct orientation was confirmed by sequencing.
Expression and Purification of UNC-87 Proteins--
The plasmid
UNC-87-pMW172 and the UNC-87 deletion mutants in pMW172, respectively,
were transformed into Escherichia coli BL21 DE3.
Ampicillin-resistant colonies were scraped off the plate and suspended
in 500 ml of LB with ampicillin. The cultures were grown to an
A600 = 0.6-0.8 and induced with 1 mM isopropyl-1-thio- Proteins--
Rabbit skeletal muscle actin or turkey gizzard
smooth muscle actin was prepared from acetone powder according to
Spudich and Watt (16) and Strzelecka-Golaszewska et al.
(17), respectively. Recombinant h1 and h2 CaP
were expressed and purified as described (10). Tropomyosin and
Transfection, Immunoprecipitation, and
Immunofluorescence--
Mouse melanoma cells (B16F1) or rat embryo
fibroblasts (REF 52) grown in Dulbecco's modified Eagle's medium + 10% fetal calf serum (PAA Laboratories, Linz, Austria) to 75%
confluency were transfected for 24 h using 2 µg of total DNA per
construct per 60-mm dish using 8.5 µl of Superfect (Qiagen,
Hilden, Germany) and prepared for immunofluorescence as described
elsewhere (10). GFP-tagged UNC-87 was visualized by direct fluorescence
using the excitation wavelength of fluorescein. F-actin was visualized by incubation with Alexa 568 Phalloidin (Molecular Probes,
Leiden, The Netherlands). Fluorescent images were photographed on a
Zeiss Axiophot using a × 63 oil immersion lens and Eastman Kodak
Co. P400 Tmax film. Immunoprecipitations using a polyclonal antibody to
recombinant EGFP were performed essentially as described earlier (20)
with minor modifications in the IP buffer (50 mM Tris, pH
7.5, 150 mM NaCl, 300 mM KCL, 5% (v/v)
glycerol, 0.5% (v/v) Triton X-100, 1 mM
phenylmethylsulfonyl fluoride, 1 mM NaN3, 1 mM EDTA, 1 mM EGTA). Proteins were
visualized by Western blotting using monoclonal antibodies to EGFP
(CLONTECH, Palo Alto, CA) and Actin Binding Assays--
Cosedimentation assays with smooth or
skeletal muscle F-actin were performed in F-actin buffer (20 mM imidazole, pH 7.0, 2 mM MgCl2,
50 mM NaCl, 100 KCl) or G-actin buffer (20 mM
imidazole, pH 7.0, 0.2 mM CaCl2, 0.5 mM ATP, 50 mM NaCl). Proteins were incubated at
25 °C for 30 min and pelleted either at 100,000 × g
for 30 min (high speed) using an air-driven ultracentrifuge (Beckman Instruments) or at 20,000 × g for 20 min (low speed)
using an Eppendorf model 5417 R centrifuge (Eppendorf-Netheler-Hinz
GmbH, Vienna, Austria). Pellets were resuspended in
the same buffer in the starting volume.
Electrophoresis and Western Blotting--
Analytical SDS gel
electrophoresis on 8-22% gradient polyacrylamide mini-slab gels and
Western blotting onto nitrocellulose (Amersham Pharmacia
Biotech) was performed as described (18). Transferred proteins
were visualized using horseradish peroxidase-coupled secondary
antibodies and the ECL chemiluminescence detection system (Amersham
Pharmacia Biotech). For detection of GFP fusion proteins we used a
monoclonal anti-GFP antibody (CLONTECH, Palo Alto, CA).
Protein Extraction--
Transfected cells in 60-mm Petri dishes
were washed twice in ice-cold phosphate-buffered saline and
subsequently extracted in 250 µl of IP buffer (50 mM
Tris, pH 7.5, 1 mM EDTA, 1 mM EGTA, 150 mM NaCl, 1% Triton X-100) supplemented with 200, 400, 600, or 800 mM KCl for 10 min. Extracts were collected with a
rubber policeman and centrifuged at 20,000 × g for 15 min. Extracts and Triton X-100-insoluble pellets were brought to
equal volumes and prepared for SDS gel electrophoresis and Western
blotting as described above.
Analytical Ultracentrifugation--
Sedimentation velocity
profiles of purified proteins (1 mg/ml in 140 mM KCl, 1 mM EDTA, 1 mM EGTA, 5 mM
KH2PO4, 5 mM
K2HPO4, pH 7.0) were collected in an analytical
ultracentrifuge (Optima XL-A, Beckman Instruments, Palo Alto, CA) at
60,000 rpm at 20 °C. The data were analyzed using the program
Ultrascan 4.1 (The University of Texas Health Center at San Antonio;
see Ref. 21) by using the van Holde-Weischet method (22).
Expression and Purification of Recombinant UNC-87--
The domain
structure of UNC-87 is similar to that of the C-terminal third
of the CaP molecule (Fig. 1A).
The UNC-87 sequence comprises 7 copies of a highly basic repeat of
23-26 amino acid residues, tentatively termed 23-residue
calponin-like repeat or CLIK-23 repeat,
interspersed by highly acidic "intervening" or linker sequences.
Whereas the repeats in UNC-87 show a striking sequence similarity to
those present in CaP, the intervening regions are unique in sequence
and variable in length but similar to the CaP intervening sequences in
their calculated isoelectric points.
We have cloned the coding region of the major 41-kDa UNC-87 isoform
(UNC-87b) and the truncation mutants depicted in Fig. 1B
into the prokaryotic expression vector pMW172 for the expression of
recombinant, nonfusion proteins. The proteins were purified under
native conditions from the bacterial cytosol by alternating ion
exchange and gel filtration chromatography (see "Experimental Procedures"). We routinely obtained between 15 and 25 mg of purified UNC-87 protein from a 1-liter bacterial culture in less than 48 h.
The final protein was more than 96% pure (Fig. 1C) and was stable in solution at 4 °C for more than 4 weeks.
UNC-87 Cross-links F-actin in Vitro--
High-speed
cosedimentation assays using purified smooth (Fig.
2A-C) or skeletal (not shown)
muscle F-actin showed that full-length UNC-87 bound tenaciously to
actin with a saturation of binding of UNC-87 to actin at a molar ratio
of 1:6-1:4 (Fig. 2A). No UNC-87 was pelletted in the
absence of F-actin at 100,000 × g. Addition of smooth
muscle TM to UNC-87-saturated F-actin filaments had no influence on the
binding of UNC-87 to actin, and binding of TM was likewise unaffected
in the reverse experiment using TM-saturated smooth muscle actin
filaments (Fig. 2, B and C).
It has been reported that CaP at high concentrations can induce bundles
of smooth muscle F-actin (23). When smooth or skeletal muscle F-actin
were incubated together with UNC-87 and centrifuged for 20 min at
18,000 × g, actin and UNC-87 were found together in
the low speed pellet, indicating the induction of actin bundles by
UNC-87 (Fig. 2D). This cross-linking effect was
concentration-dependent and ceased abruptly when the molar
ratio of actin:UNC-87 dropped below 6:1 (Fig. 2D). Thus,
UNC-87 functions as an actin cross-linking protein in
vitro.
To define the structure of the cross-linked actin assemblies in more
detail we analyzed them by electron microscopy. As seen in Fig.
3, A and B, UNC-87
caused the formation of dense, parallel actin bundles. The tight
packing of the individual filaments increased the stiffness of the
bundles, resulting in abrupt fractures (Fig. 3C). Identical
results were obtained with smooth muscle actin (not shown).
Because CaP has been shown to induce actin polymerization at low ionic
strength (24) we tested whether UNC-87 shared this ability. As seen in
Fig. 4, the UNC-87 protein failed to
induce the polymerization of G-actin as judged from both low speed
(Fig. 4C) or high speed (not shown) sedimentation. In
contrast, h1 CaP efficiently cosedimented with actin at low
speed under both F-actin (Fig. 4A) and G-actin conditions
(Fig. 4B), indicating the induction of actin polymerization
and bundling of the formed filaments. Binding of UNC-87 with actin was
unaffected by the presence of the actin-binding proteins UNC-87 Is a Monomer in Solution--
A prerequisite for the
cross-linking activity of actin-binding proteins is the presence of two
independent actin-binding sites, either on the single protein subunit
or as a result of a dimerization or oligomerization process. Secondary
structure predictions failed to identify two potential regions capable
of forming actin-binding sites in the UNC-87 molecule. To investigate
whether the UNC-87 molecule may form dimers in solution we used
analytical ultracentrifugation under native conditions. The van
Holde-Weischet extrapolation (22) from the sedimentation velocity data
showed that the full-length UNC-87 is monodisperse in solution and has
a sedimentation coefficient of 2.0 S (Fig.
6). The deletion mutants of UNC-87
containing the CLIK-23 modules 1-3, 3-7, or 5-7, respectively, all
had sedimentation coefficients GFP-tagged UNC-87 Induces Stress Fiber Bundling in Cultured
Cells--
The cosedimentation data demonstrated that UNC-87 is
capable of bundling actin in vitro. For studies of the
bundling activity in living cells we cloned the UNC-87 cDNA into
the pEGFP C1 vector to generate a mutant protein fused to GFP at its
amino terminus. Various cell lines were transfected with this
construct, and the cellular localization was determined by fluorescence
microscopy. In REF 52 fibroblasts UNC-87 localized to the actin stress
fibers, and the ectopic expression of the UNC-87 protein caused a
significant increase in stress fiber bundling (Fig.
7, A and B).
Moreover, stress fiber formation was significantly enhanced in the
mouse melanoma cell line B16F1 (Fig. 7, C and D),
consistent with the strong bundling effects observed in our in
vitro experiments.
To assay for the strength of association with the actin cytoskeleton
in vivo we analyzed the extractability of the UNC-87 protein. The amount of soluble protein was analyzed by Western blotting
using a monoclonal antibody to GFP. For comparison, we used extracts of
cells transfected with GFP-tagged Deletion Mutants Pinpoint the First Three Repeats as the Region
Essential for Actin Binding--
To further delineate the region(s)
involved in the interaction with the actin filament we performed high
and low speed cosedimentation assays as above but using the truncated,
purified proteins shown in Fig. 1C. As summarized in Table
I, deletion of more than the first three
repeats (mutant 4-7) completely abolished both binding and bundling
activities in vitro, whereas a mutant comprising the first
three repeats (mutant 1-3) retained a weak actin association and actin
bundling activity. A similar effect was observed in REF 52 cells
transiently transfected with the mutant UNC-87 constructs fused to GFP
at their amino terminus (Fig. 9). The
progressive deletion of repeat sequences was mirrored by a reduction in
stress fiber localization. Weak stress fiber localization was still
observed for the amino-terminal mutant 1-3.
Finally, we confirmed the loss of binding and bundling activity for the
respective UNC-87 mutants by coimmunoprecipitation from cell lysates of
transiently transfected REF 52 cells using a polyclonal anti-GFP
antibody (Fig. 10). Only those
constructs displaying bundling activity in vitro (compare
with Table I) coprecipitated actin in this assay.
Our findings identify the UNC-87 protein as an actin-bundling
molecule, which uses seven copies of an archetypal protein module (the
CLIK-23 repeat) to bind to and cross-link F-actin. The overall molecular structure of UNC-87 is similar to that of the C-terminal third of CaP, featuring alternating basic and acidic amino acid stretches (Fig. 1A). The CaP family of actin-binding
proteins exhibiting CLIK-23 repeat motifs likely evolved from an
ancestral molecule by gene duplication and subsequent diversification.
Three copies of the CLIK-23 repeat are found in all calponins flanking the strong actin-binding site and the myosin ATPase inhibitory region
responsible for the inhibition of the actin-activated myosin ATPase
activity (25, 26). Thus, the CLIK-23 repeats, which constitute the
second autonomous actin-binding site (ABS2) in CaP, may serve to attach
this molecule to a second site on the filament not directly involved in
the regulation of actomyosin interactions.
The sharp breaks seen in the filament bundles suggest that UNC-87
confers rigidity to the actin filaments. A similar observation has been
reported earlier for the actin cross-linking protein fimbrin (27).
Thus, UNC-87 may act as a structural component of the nematode muscle
by cross-linking actin filaments into stable bundles. This hypothesis
is consistent with the previous observations that UNC-87 knock-out
animals display distorted myofilaments (12, 14). A structural role has
also been postulated for CaP (10, 28), which bundles filaments at low
ionic strength (29). Thus, we hypothesize that the
cytoskeleton-stabilizing function of CaP is contributed by the
C-terminal repeats.
In their original work, Goetinck and Waterston (12) mapped UNC-87
to the myofilament system of the invertebrate body wall muscle, and
immunocytochemical analyses pointed toward an association with the
actin-containing thin filaments. Here we have demonstrated that UNC-87
binds directly to F-actin in vitro and that the protein possesses potent bundling activity both in vitro and in
living cells. This result is concordant with the documented importance of the molecule for the maintenance of muscle integrity. The
localization of UNC-87 in C. elegans was unaffected in the
TM null-mutants, suggesting that the two proteins are capable of
coassociating with the thin filament (12), which we now confirmed by
in vitro analyses. UNC-87 binding to actin was also
unaffected by saturating concentrations of either Our deletion studies demonstrate that the individual repeats contribute
differently to the overall actin binding activity of UNC-87. Whereas
the three N-terminal repeats still show detectable binding activity
in vitro and in transfected cells, the C-terminal four
repeats are not sufficient for actin binding. Thus, despite the
sequence similarity of the CLIK-23 modules, the acidic intervening sequences may also be relevant for the overall structural integrity of
the molecule and in particular the actin binding interface(s). Notably,
all CLIK-23 modules identified thus far are interspersed by these
acidic linker regions and are found exclusively in odd numbers
of copies (1, 3, 5, or 7). Detailed structural analyses of the UNC-87
molecule will shed more light on this question in the future.
The exclusive localization of GFP-UNC-87 (and also that of Myc-tagged
UNC-87; data not shown) along actin stress fibers is similar to the
subcellular localization seen with HA- or GFP-tagged smooth muscle CaP
and the C-terminally truncated versions of all three CaP isoforms (30).
GFP-tagged UNC-87 caused the condensation of actin stress fibers into
thick bundles in transfected cultured REF 52 cells and induced the
formation of prominent stress fibers in the mouse melanoma cell line
B16F1. Together with our data from the in vitro binding
assays and the coimmunoprecipitations this illustrates that UNC-87,
like CaP, is capable of interacting with both muscle- and
nonmuscle-type vertebrate actins. The interaction of UNC-87 with
filamentous actin was essentially insensitive to ionic strength. Actin
cosedimentation under both low and high speed conditions was unchanged
at salt concentrations ranging from 50-300 mM KCl (data
not shown). More significantly, the majority of the UNC-87 protein
remained associated with the Triton X-100-insoluble cytoskeletal
fraction even at 800 mM KCl, whereas We conclude that the basic CLIK-23 repeats and not the acidic
intervening sequences are responsible for contacting the actin filament. First, the intervening sequences of CaP and UNC-87 share no
apparent similarities other than the predicted acidic pI (see also Fig.
1A), but the isolated repeat region of CaP colocalized with
actin stress fibers in transfected cells
(10).2 Secondly, Mino
et al. (11) have shown the direct interaction of a peptide
corresponding to the first repeat of CaP with actin. Emerging
structural information suggests that nature operates with a limited
number of actin binding motifs (31). The CLIK-23 repeats share no
apparent similarity with any of the currently known actin binding
sequences. An actin-binding protein containing six copies of the Kelch
motif and consisting predominantly of The UNC-87 protein appears as a monomer in solution according to the
data from the analytical ultracentrifugation. Similarly, the deletion
mutants 3-7, 5-7, and 1-3 also sedimented at values indicating a
monomeric molecule corresponding to the calculated molecular mass.
Chemical cross-linking, using the zero length cross-linker
1-ethyl-3-[3-(dimethyl amino) propyl] carbodiimide (EDC), but also
[Bis sulfosuccinimidyl) suberate] (BS3) or
m-Meleimidobenzoyl-N-hydroxysuccinimide ester
(MBS), confirmed these results (data not shown). Thus, UNC-87 is likely
to function as a monomer in vivo. Because cross-linking
activities depend on the presence of at least two binding interfaces
these results indicate that the seven CLIK 23 repeats form a minimum of
two actin-binding sites. The fact that the binding and bundling
capabilities were lost simultaneously in the deletion mutants argues
for a structural requirement that involves both N- and C-terminal
repeat regions. Notably, under the same buffer conditions used for the cross-linking and sedimentation assays both full-length UNC-87 and the
deletion mutants migrated significantly faster on analytical gel
filtration columns, and the proteins eluted at positions corresponding to 1.5-2.3 times the calculated molecular mass, indicating that the
UNC-87 molecule may form an extended, rigid rod
structure.3
The expression of CaP isoforms is not restricted to muscle tissue, and
different members of the CaP family have been identified in a wide
range of vertebrate cells. CaP variants have thus been implicated in
the regulation of a variety of actomyosin-based processes, including
neurite outgrowth (33) and the organization of the nonmuscle cell
cytoskeleton (34). The CLIK-23 module may have acquired a specialized
function in the course of evolution from invertebrates to vertebrates
and the concomitant specialization of different muscle types (see also
Ref. 35). It is, however, worth noting that the C. elegans
genome contains proteins closely related to the SM22-like members of
the CaP family (consisting of a single copy of both a CLIK-23 repeat
and a single N-terminal calponin homology domain) but also Vav and
IQGAP-like proteins harboring a single calponin homology domain (8, 9)
and no CLIK-23 module. Thus, UNC-87 may be a CaP ortholog specialized for actin filament assembly processes in invertebrate obliquely striated muscle.
Conclusion--
In summary, we have demonstrated that UNC-87
interacts directly with F-, but not G-actin, and causes the formation
of rigid actin bundles. We suggest a physiological role for this
protein as a "rectifying" component of the actomyosin system in
nematodes and propose a similar structural role, performed by the three homologous CLIK-23 repeats in the smooth muscle protein calponin in
vertebrate muscle.
-actinin. Consistent with its actin bundling activity in
vitro, UNC-87 tagged with green fluorescent protein associated
with and promoted the formation of actin stress fiber bundles in living
cells. These data identify UNC-87 as an actin-bundling protein and
highlight the calponin-like repeats as a novel actin-binding module.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-galactopyranoside. 3-4 h after induction the culture was centrifuged at 1250 × g for 15 min, and the pellet was resuspended in Buffer E (5 mM KH2PO4, 5 mM
K2HPO4, 10 mM NaCl, 1 mM EDTA, 1 mM EGTA). The bacteria were lysed in
a French® pressure cell press (Spectronic Unicam, Cambridge, United Kingdom) and centrifuged at 25,000 × g for 15 min. The supernatant was applied immediately onto a 5-ml HiTrap SP
cation exchange column (Amersham Pharmacia Biotech) equilibrated in
Buffer E. Proteins were eluted with a linear gradient, ranging from
0-350 mM NaCl in Buffer E. Peak fractions were pooled and
further purified on a Sephacryl S100HR (500 ml) equilibrated in Buffer
E. Fractions containing >96% pure UNC-87 were concentrated on a 1-ml
HiTrap SP and eluted again with a gradient of 0-350 mM NaCl.
-actinin were purified from turkey gizzard smooth muscle as
described (18, 19).
-actin (Sigma), respectively.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
A, molecular domain structure of UNC-87.
The molecule contains seven copies of the CLIK-23 module. Similar to
the situation in smooth muscle h1 CaP, the basic repeats
alternate with acidic intervening sequences. Calculated pH values of
the individual domains are indicated. CHD, calponin homology
domain; ABS 1, actin-binding site 1. B, schematic
representation of the molecular domain structure and residues of the
UNC-87 constructs used in this study. Identical constructs, but
carrying an EGFP fusion at their respective amino termini, were used
for the transfection of cultured cells. C, expression and
purification of recombinant UNC-87 proteins. Coomassie-stained gel of
purified recombinant proteins. Mutants and the positions of molecular
mass markers are indicated. f.l., full-length.
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Fig. 2.
Actin binding of UNC-87. A-C,
high speed cosedimentation assay with 3 µM smooth muscle
F-actin and increasing amounts of UNC-87 (A and
B) or tropomyosin (B) from molar ratios of 1:10
to 1:1. Note the saturation of the filaments indicated by the presence
of UNC-87 and TM, respectively, in the supernatant. D, low
speed cosedimentation assay. Assay conditions were as in A.
Note the sharp onset of actin cross-linking at molar ratios between 1:6
and 1:7.
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Fig. 3.
Negative stain electron micrographs of
UNC-87-induced skeletal muscle F-actin bundles. UNC-87 causes the
formation of tight actin bundles (A and B). The
increased stiffness of the bundles causes the bundles to break
frequently (C). Bar is 0.05 µm.
-actinin
(Fig. 5) and filamin (not shown)
indicating that
-actinin and UNC-87 occupy different sites on the
actin filament.
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Fig. 4.
UNC-87 associates primarily with filamentous
actin. Despite the high cross-linking activity UNC-87 fails to
induce actin polymerization and bundling in a low speed cosedimentation
assay using smooth muscle G-actin (C). In contrast, smooth
muscle h1 CaP causes the formation of F-actin bundles under
both F-actin (A) and G-actin conditions
(B).
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Fig. 5.
UNC-87 and -actinin
bind simultaneously to F-actin. Low speed cosedimentation assay
under the same conditions as in Fig. 2 using decreasing amounts of
-actinin and UNC-87, respectively, is shown. Note that the actin
filaments can be simultaneously saturated with both proteins.
1.6 S (not shown). Thus, the
multiple CLIK-23 modules may serve to form at least two independent
actin binding interfaces.
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Fig. 6.
Analytical ultracentrifugation reveals that
UNC-87 is a monomer in solution. The van Holde-Weischet plot from
a sedimentation velocity experiment shows that the extrapolated
sedimentation speed from various sedimentation boundary positions
correspond to a sedimentation coefficient of 2.0 S.
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Fig. 7.
Localization of GFP-tagged UNC-87 in
transiently transfected REF 52 (A and
B) and B16F1 cells (C and
D). UNC-87 associates strongly with the actin
stress fibers in REF 52 fibroblasts and causes the formation of
prominent, thick bundles (A and B). Transfection
into B16F1 melanoma cells induces the formation of stress fibers
(C and D). Note the absence of actin stress
fibers in the surrounding nontransfected cells in C. A and C, F-actin visualized by incubation with
Alexa 568 Phalloidin; B and D: GFP fluorescence.
Bar is 10 µm.
-actinin, extracted in the same
way. As demonstrated in Fig. 8, UNC-87
was strongly retained in the Triton X-100-insoluble cytoskeletal
fraction even at 800 mM KCl, an ionic strength at
which
-actinin was readily solubilized.
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Fig. 8.
Western blot probed with a monoclonal
anti-GFP antibody. Extracts and insoluble cytoskeletal residues of
REF 52 cells transiently transfected with GFP UNC-87 or GFP -actinin
and extracted at the indicated KCl concentrations in the presence of
1% Triton X-100 are shown. UNC-87 remains associated with the
insoluble cytoskeletal fraction at 800 mM KCl, whereas
-actinin is almost quantitatively extracted under these
conditions.
Summary of activities from high speed (binding) and low speed
(bundling) actin cosedimentation assays using the indicated UNC-87
deletion mutants. The data are representative of at least four
independent experiments. Note the concomitant loss of binding and
bundling for the mutant constructs 4-7 and 5-7, whereas the amino
terminal mutant 1-3 retains weak binding activity.
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Fig. 9.
Subcellular localization of GFP-tagged UNC-87
mutants in transiently transfected REF 52 fibroblasts. Note the
gradual decrease in stress fiber association with progressing deletion
of repeats. F-actin was visualized by incubation with Alexa 568 Phalloidin. Bar is 10 µm.
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Fig. 10.
Coimmunoprecipitation of cytoplasmic
-actin by the bundling-competent mutants of
UNC-87. Western blot of precipitated proteins is shown. Antibodies
used for precipitation and Western blotting are indicated
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-actinin or
filamin, indicating the occupation of nonoverlapping sites on the actin
filament. In support of this Chalovich and colleagues (28) reported
that
-actinin, filamin, and calponin show only little displacement
and are capable of binding the actin filament simultaneously in almost
stoichiometrical amounts. Thus, the second actin-binding site of CaP
(ABS2) and that of UNC-87 are likely to interact with a similar region
along the filament. Studies specifically addressing the question of how
and where UNC-87 and CaP bind to actin using three-dimensional helical
image reconstructions of cryoelectron micrographs are currently underway.
-actinin was readily extracted from the cells under these conditions. Thus, electrostatic interactions may play a subordinate role in the binding
of UNC-87 to actin. Taken together, the binding and extraction data
point toward a high affinity interaction of UNC-87 with actin.
-sheet structures is found in
the Limulus sperm protein scruin (5), which also bundles actin in
vitro (32). Computer-assisted secondary structure analysis (data
not shown) of UNC-87 or the corresponding repeat region in CaP predict
for both sequences a high content (70%) of unstructured turns with
only 17% helical and 13%
-sheet folds, clearly different from
scruin. Thus, the actin-binding site(s) formed by the multiple CLIK-23
repeats in CaP and UNC-87 appears to constitute a novel structural
actin binding motif, which awaits further analysis.
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ACKNOWLEDGEMENTS |
---|
We are grateful to Sue Goetinck, Pam Hoppe,
and Bob Waterston (Washington University) for generously supplying
UNC-87 cDNA constructs, Matthias Krause and Jürgen Wehland
(Gesellschaft für Biotechnologische Forschung,
Braunschweig) for the GFP--actinin construct, and Günter
Lepperdinger for help with the analytical gel filtration. We
thank Vic Small for support, comments on the manuscript, and help with
the electron microscope, Michael Schleicher (University of
Munich) and all members of the Gimona laboratory for
stimulating discussions, and Ulrike Müller and
Maria Schmittner for expert technical assistance and photography.
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FOOTNOTES |
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* This work was supported in part by grants from the Austrian Science Foundation (Fonds zur Förderung der wissenschaftlichen Forschung).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed. Tel.: 43-662- 63961-19; Fax: 43-662-63961-40; E-mail: mgimona@server1.imolbio. oeaw.ac.at
Published, JBC Papers in Press, November 28, 2000, DOI 10.1074/jbc.M009561200
2 R. Mital, C. Danninger, and M. Gimona, unpublished data.
3 W. Kranewitter, G. Lepperdinger, and M. Gimona, unpublished data.
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ABBREVIATIONS |
---|
The abbreviations used are: CaP, calponin; TM, tropomyosin; GFP, green fluorescent protein; EGFP, enhanced GFP; REF, rat embryo fibroblasts.
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REFERENCES |
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