From the Departments of Cell Biology and Anatomy and
Cellular and Molecular Biology, University of Arizona,
Tucson, Arizona 85724, the § European Molecular Biology
Laboratory, Heidelberg 69012, Germany, and the
¶ Department of Cell Biology, The Scripps Research Institute,
La Jolla, California 92037
Received for publication, June 28, 2000, and in revised form, September 6, 2000
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ABSTRACT |
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Strict regulation of actin thin filament length
is critical for the proper functioning of sarcomeres, the basic
contractile units of myofibrils. It has been hypothesized that a
molecular template works with actin filament capping proteins to
regulate thin filament lengths. Nebulin is a giant protein (~800 kDa)
in skeletal muscle that has been proposed to act as a molecular ruler to specify the thin filament lengths characteristic of different muscles. Tropomodulin (Tmod), a pointed end thin filament capping protein, has been shown to maintain the final length of the thin filaments. Immunofluorescence microscopy revealed that the N-terminal end of nebulin colocalizes with Tmod at the pointed ends of thin filaments. The three extreme N-terminal modules (M1-M2-M3) of nebulin
bind specifically to Tmod as demonstrated by blot overlay, bead
binding, and solid phase binding assays. These data demonstrate that
the N terminus of the nebulin molecule extends to the extreme end of
the thin filament and also establish a novel biochemical function for
this end. Two Tmod isoforms, erythrocyte Tmod (E-Tmod), expressed in
embryonic and slow skeletal muscle, and skeletal Tmod (Sk-Tmod),
expressed late in fast skeletal muscle differentiation, bind on
overlapping sites to recombinant N-terminal nebulin fragments. Sk-Tmod
binds nebulin with higher affinity than E-Tmod does, suggesting that
the Tmod/nebulin interaction exhibits isoform specificity. These data
provide evidence that Tmod and nebulin may work together as a linked
mechanism to control thin filament lengths in skeletal muscle.
Sarcomeres, the basic contractile units of myofibrils, are complex
structures composed of numerous structural and regulatory proteins
organized in an exquisitely precise manner. These structures are
composed of alternating parallel arrays of thin and thick filaments.
Helical, polar actin polymers are the principal component of the thin
filaments. In vitro, actin monomers polymerize at both the
fast growing (barbed) and the slow growing (pointed) ends of the actin
filaments until the critical concentration is reached. Once this is
attained, the length distribution of the filaments becomes exponential
(for further discussion, see Refs. 1 and 2). In sharp contrast to this,
skeletal muscle cells exhibit a strikingly narrow thin filament length
distribution (e.g. 1.1 ± 0.03 µm in rabbit psoas
muscle) (3). Additionally, although cardiac muscle has a wider range of
thin filament lengths compared with skeletal muscle (e.g.
0.6-1.1 µm in rat heart) (4), the distribution is much more uniform
than that of pure actin filaments in vitro. Thus, regulatory
mechanism(s) must exist in striated muscle to tightly control thin
filament length. Although these mechanisms remain unknown, one highly
favored hypothesis that has existed for years is that a "molecular
ruler" protein works together with actin filament capping proteins to
regulate thin filament length (reviewed in Refs. 1 and 5).
The giant, actin-binding protein, nebulin, is an excellent candidate
for a molecular ruler that functions to specify thin filament length in
skeletal muscle (reviewed in Refs. 1 and 6-9). Strikingly, the
molecular mass of nebulin (~600-800 kDa) correlates with variations
in thin filament lengths observed in different types of skeletal muscle
(10). Additionally, a great deal of evidence indicates that single
molecules of nebulin associate with the thin filaments along their
entire length. The extreme C-terminal region of nebulin is inserted in
the Z disc (11-14). This region contains a unique 20-kDa domain and an
Src homology 3 domain (13). More is known about this region of nebulin
in comparison with other parts of the molecule, since a significant amount of recent work has focused on deciphering the molecular interactions and targeting domains responsible for anchoring nebulin in
the Z disc (e.g. 15-17). The vast majority of nebulin,
however, is composed of ~185 modular repeats that are each ~35
amino acids in size. Groups of seven of these modules are classified as
"super repeats" (13, 18, 19). Biochemical, biophysical, and
structural studies suggest that a single nebulin module interacts with
a single actin monomer and that each super repeat interacts with each
regulatory unit of the thin filament (comprised of a troponin complex
and a tropomyosin molecule for every seven actin monomers) (13, 18,
19). Last, the N-terminal region of nebulin is predicted to be located
at or near the pointed ends of the thin filaments (12, 13, 20). This
region contains eight unique "linker" modules (M1-M8) as well as a
unique, acidic 8-kDa domain (13). The function of these N-terminal
domains is unknown, but it is intriguing to speculate that they are
specialized for interacting with other sarcomeric proteins and have
distinct functions.
Nebulin's periodic, modular structure probably enables it to dictate
the number of actin monomers to be polymerized into the thin filaments
in skeletal muscle. What is the mechanism, then, that prevents the thin
filaments from elongating once their specified length is attained? In
this regard, actin filament capping proteins that bind to the ends of
actin filaments prevent actin monomer addition and filament growth (for
a review, see Ref. 21). In striated muscle, CapZ has been shown to cap
the barbed ends of the thin filaments at the Z line, while the pointed
ends of the filaments are capped by tropomodulin
(Tmod)1 (22-24).
Investigations of CapZ and Tmod in cultured myocytes suggest
that CapZ plays a role in nucleating the initial assembly of actin
filaments into sarcomeres, while Tmod functions later to maintain thin
filament lengths (25-27). For example, microinjection of an antibody
to Tmod that disrupts its capping activity in in vitro
assays results in abnormal elongation of the thin filaments from their
pointed ends in chick cardiac myocytes (26). Additionally, cells in
which Tmod function is inhibited no longer beat, and overexpression of
Tmod in the myocardium of transgenic mice causes myofibrillar disarray
and dilated cardiomyopathy (26-28). These studies indicate that Tmod
regulation of thin filament length is critically important for muscle function.
Tmod, first identified as a tropomyosin-binding protein in the
erythrocyte membrane cytoskeleton (E-Tmod; Ref. 29), is present at thin
filament pointed ends in chicken slow skeletal muscle fibers, in
embryonic skeletal muscle, and in heart (30, 31). A second isoform,
skeletal Tmod (Sk-Tmod), is present at thin filament pointed ends late
in chicken fast skeletal muscle differentiation (32). In
vitro assays indicate that both E-Tmod and Sk-Tmod bind to
tropomyosin (Kd ~150-200 nM) (29,
32). In the presence of tropomyosin, Tmod completely blocks the
polymerization and depolymerization of actin filaments at their pointed
ends (Kd ~50 pM), while in the absence
of tropomyosin, Tmod has a lower affinity for the pointed ends
(Kd ~0.3-0.4 µM) (24, 33). This
suggests that the tight capping of tropomyosin-coated actin filaments
is due to Tmod's ability to bind both tropomyosin and actin (reviewed
in Ref. 34). To date, actin and tropomyosin are the only identified
binding partners for Tmod.
While investigations into the properties of Tmod described above
demonstrated that Tmod is a critical component for maintaining thin
filament length by blocking actin polymerization at the pointed end,
there is no evidence that Tmod is involved in specifying the
lengths (for a review, see Ref. 35). In fact, it is difficult to
envision how a capping protein, which is present only at the end of the
filament, could alone determine the characteristic thin filament
lengths that differ among various types of skeletal muscle. Thus, we
hypothesized that the N-terminal region of nebulin, the part of the
molecule predicted to be at or near the pointed ends of the thin
filaments, interacts directly with Tmod as part of a linked regulatory
mechanism in skeletal muscle to determine and maintain the lengths of
the thin filaments. To test this idea, we first established by
immunofluorescence microscopy that Tmod and the N-terminal end of
nebulin colocalize in skeletal tissue. Next, we determined that Tmod
specifically binds to a 13.5-kDa recombinant fragment from the extreme
N-terminal modules of nebulin, as demonstrated by blot overlay, bead
binding, and solid phase binding assays. Interestingly, the
Sk-Tmod/nebulin interaction is of higher affinity than the
E-Tmod/nebulin interaction. This isoform specificity of nebulin binding
suggests that thin filament length regulation by nebulin and Tmod may
differ in some respects in fast and slow skeletal muscle fibers and/or
during skeletal muscle differentiation. The data presented here
demonstrate that the N terminus of the nebulin molecule extends to the
extreme end of the thin filament and also establish a novel biochemical function for this end. This study provides direct evidence to support
the proposed model that the interaction of the capping protein, Tmod,
with the molecular ruler, nebulin, may be pivotal for regulating actin
filament assembly and length in skeletal muscle.
Purified Proteins and Antibodies--
Antibodies against human
E-Tmod were generated in rabbits and affinity-purified as described
(36). Antibodies to recombinant fragments from the N-terminal region of
nebulin (see below for details on expression and purification of
recombinant nebulin fragments) were generated in rabbits (RR Research
and Development, Stanwood, WA) and affinity-purified. Monoclonal
anti-E-Tmod antibody (monoclonal antibody 95) was purified from
hybridoma supernatants on a protein G column (Amersham Pharmacia
Biotech) (37). Recombinant chicken Sk-Tmod and recombinant chicken
E-Tmod were prepared and purified as described previously (32, 38).
Tropomyosin was prepared from rabbit skeletal muscle, and human E-Tmod
was purified from erythrocytes as described (36, 39).
Biotinylation of Tmod--
Purified recombinant chicken E- and
Sk-Tmods were biotinylated using aminohexanoyl-biotin
N-hydroxysuccinimide according to the manufacturer's
instructions (Zymed Laboratories Inc., San Francisco,
CA). Briefly, after dialysis against 0.1 M bicarbonate buffer, pH 8.4, Tmod was incubated at a molar ratio of 1:10 with aminohexanoyl-biotin N-hydroxysuccinimide (dissolved in
N,N-dimethylformamide) at room temperature for
1 h. The biotinylated Tmod was then immediately dialyzed against
0.1 M KCl, 10 mM Hepes, pH 7.5, aliquoted,
frozen in liquid nitrogen, and stored at Indirect Immunofluorescence Microscopy--
Primary cultures of
embryonic chicken skeletal myotubes were prepared as described (37),
and indirect immunofluorescent staining to localize Tmod and the
N-terminal region of nebulin was performed (40). Briefly,
paraformaldehyde-fixed cultures were permeabilized in 0.2% Triton
X-100/phosphate-buffered saline for 15 min and immediately blocked in
2% bovine serum albumin/phosphate-buffered saline for 1 h. The
myotubes were stained with monoclonal antibodies to Tmod (monoclonal
antibody 95) at 2 µg/ml, followed by rhodamine-conjugated sheep
anti-mouse IgG (1:200) (Roche Molecular Biochemicals). Next, the cells
were incubated with affinity-purified polyclonal rabbit anti-neb N-term + M1 (see below for nomenclature of nebulin fragments) antibodies (10 µg/ml), followed by fluorescein-conjugated goat anti-rabbit IgG
(1:200) (Roche Molecular Biochemicals). The myotubes were analyzed on a
confocal microscope (Bio-Rad MRC600), and digital images were processed
and merged using Adobe Photoshop software (San Jose, CA).
Blot Overlays--
Nebulin was solubilized from rat psoas muscle
fibers and resolved from other myofibrillar proteins using a large
pore, 3.3-12% linear gradient SDS-gel electrophoresis system, under
conditions optimized to resolve giant myofibrillar proteins (41, 42). After transfer to nitrocellulose (0.2 µm; Schleicher and
Schuell), blocking, and incubation with 1 µg/ml purified human E-Tmod
in binding buffer (80 mM KCl, 2 mM
MgCl2, 10 mM Hepes, pH 7.3, 2% Triton X-100,
20 mg/ml bovine serum albumin, 1 mM dithiothreitol), Tmod
binding to nebulin was detected by incubation with affinity-purified rabbit anti-human E-Tmod antibodies followed by
125I-protein A and autoradiography. Nebulin was detected on
a parallel nitrocellulose strip by immunoblotting with goat
anti-nebulin antibodies (generously provided by Dr. Kuan Wang,
University of Texas at Austin) followed by rabbit anti-goat antibodies
and 125I-protein A.
Bacterial Expression and Purification of Cloned Nebulin
Fragments--
Nebulin cDNA fragments encoding various domains
were amplified from total human skeletal muscle cDNA by polymerase
chain reaction, using a matchmaker cDNA library
(CLONTECH, Palo Alto, CA). The primer sequences
used are listed in the Appendix. The obtained fragments were subcloned
into the T7 promoter-regulated pET vectors, which express their inserts
C-terminally fused to 6× histidine tags (Novagen, Madison, WI). The
expressed nebulin fragments corresponded to neb N-term (11.3 kDa); neb
N-term + M1 (13.6 kDa); neb N-term + M1-M2-M3 (21.8 kDa); M1-M2-M3
(13.5 kDa); M1-M8 (33.7 kDa); and M9-M15 (32.8 kDa). Additionally, a
fragment corresponding to the unique acidic N-terminal domain plus the
first three N-terminal modular repeats (nette + R1-R2-R3) of the
cardiac nebulin-like protein, nebulette, was also expressed as a
control (24.3 kDa). To exclude potential artifacts from the histidine
tags, the M1-M2-M3 nebulin fragment also was expressed in the pETM-11
vector, which allows histidine tag removal after cleavage by the TEV
protease (Life Technologies, Inc.). The inserts were confirmed by DNA
sequencing, and the expression vectors were transformed into Epicurian
Coli BL21-Codon Plus (DE3)-RIL competent cells (Stratagene, La Jolla, CA). Large scale preparations of the nebulin fragments and the nebulette fragment were purified from the cell lysates according to
instructions from Qiagen (Valencia, CA). Briefly, a 100-ml overnight
culture of the transformed bacterial cells was used to inoculate 1 liter of Luria broth containing 35 µg/ml kanamycin. The cells were
grown to an A600 of 0.6-0.8, induced with
0.2-0.4 mM isopropyl
Solid Phase Binding Assays--
Solid phase binding assays were
performed in duplicates using flat-bottom Costar EasyWash, High
Binding, 96-well microtiter plates (Corning, Acton, MA). The volume of
all solutions used was 100 µl/well. Wells were coated overnight at
4 °C with 100 nM nebulin fragments or the N-terminal
nebulette fragment or with 100 nM skeletal muscle
tropomyosin (as a positive control), diluted in 0.1 M
carbonate buffer, pH 9.6. The wells were washed three times with
binding buffer (20 mM Hepes, pH 7.4, 80 mM KCl,
2 mM MgCl2, 0.002% NaN3, 0.05%
Tween 20, 0.2% bovine serum albumin) and then blocked in the same
buffer for 1 h at 4 °C. The wells were then incubated with
binding buffer only or with 2.5 nM (or otherwise noted
concentrations) biotinylated recombinant E or Sk-Tmod for 1 h at
4 °C. After three additional washes, alkaline phosphatase-conjugated
ImmunoPure streptavidin (Pierce) was diluted 1:10,000 in binding buffer
and incubated at 4 °C for 1 h, the washes were repeated, and 1 mg/ml p-nitrophenyl phosphate (pNPP) (Sigma) (diluted into
0.1 M glycine, 1 mM MgCl2, 1 mM ZnCl2, pH 10.4) was added to the wells and
incubated for 15 min at 37 °C. Binding of Tmod to nebulin was
determined by a colorimetric reaction at A405 on
a Tecan plate reader (Phenix; Hayward, CA) using Tecan Winselect
software (Phenix). Data were analyzed using Microsoft Excel.
For determining the dissociation constant (Kd) for
the Tmod/nebulin interaction, a direct calibration ELISA (dcELISA) study was performed (43). The dcELISA utilizes a series of linearized plots to accurately quantify association constants. Briefly, in a
series of ELISA experiments within the linear range, the absorbance is
related by a constant, unknown calibration factor (c) to the amount of immobilized "receptor" (i.e. the M1-M2-M3
nebulin fragment) bound to its soluble "ligand" (i.e.
the biotinylated Tmod). This calibration factor can be determined
assuming that a defined amount of ligand in solution becomes completely
bound to an immobilized receptor upon repeated transfer from one
receptor-coated well to another in a series of "transfer assays."
For the dcELISA, three sets of experiments were required, using the
solid phase binding assay conditions described above. First, the
incubation time of the receptor with the ligand was varied in a time
course to determine the rate constant, kc. Second, a
series of "transfer assays" (where unbound ligand from one
receptor-coated well is transferred to the next, identically coated
well) was performed at various times of incubation (10, 15, 20, and 30 min) in order to determine the transfer factor, Ft
as well as c. Finally, a saturation curve under equilibrium
conditions was generated to determine the amount of immobilized
receptor available to bind to the ligand. The obtained results allowed for the calculation of the association constant, Ka, the inverse of which is equal to the Kd value.
Bead Binding Assay--
An assay to confirm binding of Tmod to
nebulin under native conditions was adapted from Watakabe et
al. (44). 1 µg of biotinylated Tmod was incubated with 1 µg
(or otherwise noted amount) of the recombinant nebulin fragment,
M1-M2-M3, in 20 µl of binding buffer (20 mM Hepes, pH
7.4, 80 mM KCl, 2 mM MgCl2, 0.002%
NaN3) at room temperature for 1 h. 20 µl of a 1:1
slurry of streptavidin-conjugated Sepharose 4B beads (Amersham
Pharmacia Biotech) in binding buffer were added to the protein solution
and incubated for 1 h at room temperature with gentle rocking. 20 µl of the supernatant, containing unbound proteins, were recovered
and boiled in SDS sample buffer. The Sepharose slurry was washed five
times with 400 µl of binding buffer, and bound proteins were eluted
by incubating the slurry with 20 µl of 0.1 M glycine, pH
2.8, for 1 h at room temperature. The eluted proteins were
neutralized with 1 µl of 1 M Tris-HCl, pH 9.0, and boiled
in SDS sample buffer. The same volume of unbound and eluted proteins
was separated on 18% SDS-polyacrylamide gel electrophoresis and
visualized by Coomassie Blue staining.
Nebulin Colocalizes with Tmod at the Pointed Ends of the Thin
Filament--
Previous immunolocalization studies have revealed that
the N-terminal region of nebulin is located at or in close proximity to
the pointed ends of the thin filaments (10, 12, 20), which is where
Tmod is located. To determine if the N-terminal region of nebulin
colocalizes with Tmod at the pointed ends of the thin filaments,
indirect immunofluorescence microscopy studies were performed.
Polyclonal antibodies were generated against a recombinant fragment
comprising nebulin's extreme N-terminal unique 8-kDa domain (which we
refer to as neb N-term) plus the first unique module, M1. These
anti-nebulin (neb N-term + M1) antibodies were shown to be specific by
Western blotting, since they detected a single band corresponding to
the molecular weight of nebulin in muscle lysates (data not shown).
Double staining of primary cultures of embryonic chicken skeletal
myotubes with the anti-nebulin (neb N-term + M1) (Fig.
1A) and Tmod antibodies (Fig.
1B) revealed that these two proteins colocalize (Fig.
1C). This demonstrated that the extreme N-terminal region of
nebulin is in the correct location in the sarcomere to interact with
Tmod.
Tmod Binds to Nebulin in Blot Overlay Studies--
As an initial
approach to determine whether Tmod binds to nebulin, we performed blot
overlay studies with isolated rat myofibrils. A large pore, SDS
gradient polyacrylamide gel system was used to resolve giant proteins
(41, 42). Coomassie Blue staining of the proteins demonstrated that a
band corresponding to the molecular mass of nebulin (~800 kDa)
was resolved using this system (Fig. 2,
lane 1). The proteins were transferred onto
nitrocellulose membranes, and the strips were then incubated with
purified human E-Tmod. Following incubation with affinity-purified
anti-Tmod antibodies and 125I-protein A (Fig. 2,
lane 3), Tmod was found to bind to a protein with
the same mobility as nebulin. Western blot analysis confirmed that the
band detected as a Tmod-binding protein in lane 3 comigrates with nebulin (Fig. 2, lane 2). The
results of these blot overlay studies suggested that nebulin is a
Tmod-binding protein.
Tmod Binds to the M1-M2-M3 Modules in the N-terminal Region of
Nebulin--
If the binding of nebulin to Tmod is physiologically
relevant, the Tmod-binding site on nebulin must be present in
nebulin's N-terminal region, which is located at the pointed ends of
the thin filaments. The extreme N-terminal region of nebulin,
consisting of an 8-kDa domain (neb N-term), is adjacent to eight unique
modules (M1 through M8) (13). These modules are each approximately 35 residues in size and are similar to one another but only distantly related to the other classes of nebulin modules (13). To determine the
location of the Tmod-binding site on nebulin, we generated recombinant
fragments, including various combinations of these N-terminal modules
with and without the neb N-term domain, for use in solid phase
Tmod-binding assays (Fig. 3).
The recombinant nebulin fragments, as well as control fragments and
proteins (see below), were adsorbed onto microtiter plates at equal
molar concentrations to obtain equal numbers of putative Tmod binding
sites per well. Biotinylated recombinant E- and Sk-Tmods were incubated
with the immobilized fragments or with the known Tmod binding partner
protein, skeletal tryopomyosin, as a positive control (Fig.
4A). As expected, Sk-Tmod
bound to skeletal tropomyosin, verifying the utility of this binding
assay. Comparison of Tmod binding to the N-terminal nebulin fragments
showed first that no binding of Sk-Tmod to neb N-term (the unique 8-kDa
acidic domain at the extreme N-terminal end of nebulin) was detected.
Occasionally, Sk-Tmod binding to neb N-term + M1 (the 8-kDa domain
linked to the first unique module) was detected, albeit weakly,
suggesting that the M1 module may contain a partial binding site for
Sk-Tmod. In contrast, Sk-Tmod was observed to bind strongly and
consistently to neb N-term + M1-M2-M3 (the 8-kDa domain fused to the
first three N-terminal unique modules), to M1-M2-M3 alone, and to the modules M2-M8. To exclude potential artifacts that may have resulted from the presence of the histidine tag fused to the recombinant nebulin
fragments, the tag was cleaved from the M1-M2-M3 fragment, and no
difference in binding to Tmod was detectable (data not shown). Further
mapping of the Tmod binding site using smaller recombinant fragments
was hindered by the insolubility of these fragments (data not
shown).
Interestingly, in comparing the binding of Sk-Tmod to neb N-term + M1-M2-M3 with the binding of Sk-Tmod to M1-M2-M3, it was found that
Sk-Tmod bound more strongly to M1-M2-M3 alone. Why the larger neb
N-term + M1-M2-M3 nebulin fragment, which also contains the Tmod
binding site, was not able to bind to Tmod as strongly as M1-M2-M3
alone is not known, although one possibility is that the neb N-term
domain sterically hinders the complete Sk-Tmod binding site. The
results from these studies indicate that the first three unique
N-terminal modules of nebulin, the M1-M2-M3 region (representing 13.5 kDa of the ~800-kDa molecule), are sufficient for binding to
Tmod.
In order for binding of Sk-Tmod to the N-terminal region of nebulin to
be specific, Tmod should bind only to this region and not elsewhere
along the length of the nebulin molecule. In fact, binding of Sk-Tmod
to a series of modules comprising the first nebulin super repeat (SR1),
M9-M15, which is located adjacent to (toward the C-terminal end of)
the unique M1-M8 modules, was not detected (Fig. 4A). As an
additional control, a recombinant fragment comprising the N-terminal
domain and first three modular repeats of nebulette, nette + R1-R2-R3,
was used. Nebulette is a small (107 kDa), Z-line-associated protein
found in cardiac muscle that shares sequence homology and a similar
domain layout with nebulin (14, 45, 46). No binding of Sk-Tmod to this nebulette fragment was detected. This was expected, since the location
of the nebulette N terminus is predicted to be located only ~0.2 µm
from the Z line and not at the pointed ends of the filaments where Tmod
is found (45). We conclude from these data that the binding of Tmod to
nebulin is specific for the M1-M2-M3 modules at the N-terminal end of
nebulin. Furthermore, this binding of Tmod to the unique M1-M2-M3
modules strongly indicates that the N-terminal end of nebulin does, in
fact, extend to the extreme end of the thin filaments.
Using this assay, we also found that Sk-Tmod appeared to bind more
strongly to nebulin compared with E-Tmod binding to nebulin (Fig.
4A). E-Tmod was observed to bind consistently only to
M1-M2-M3, although it sometimes bound weakly to neb N-term + M1-M2-M3
and to M1-M8. Since both Sk-Tmod and E-Tmod bound to the recombinant nebulin fragment M1-M2-M3 to a greater extent than to other nebulin fragments (Fig. 4A), M1-M2-M3 was used in studies designed
to further characterize the Tmod/nebulin interaction. First, the interaction between Sk-Tmod and M1-M2-M3 was compared with the interaction of E-Tmod with M1-M2-M3. We performed competition assays
using our solid phase binding system, where biotinylated Tmods were
incubated with excess unlabeled Tmods (Fig. 4, B and C). The results show that the binding of both biotinylated
Tmod isoforms to M1-M2-M3 was inhibited by the addition of excess
amounts of unlabeled Tmods. Thus, the binding of the biotinylated Tmods to M1-M2-M3 in the assay is not due to the biotin label (on Tmod) binding nonspecifically to nebulin. Additionally, unlabeled Sk-Tmod was
more effective than unlabeled E-Tmod at inhibiting the binding of
biotinylated Sk-Tmod or E-Tmods to the immobilized M1-M2-M3 (Fig. 4,
B and C). These results indicate that, first,
both Tmod isoforms probably bind to similar or overlapping sites on
nebulin and, second, that Sk-Tmod binds to nebulin with a higher
affinity than does E-Tmod, which is consistent with the results
described above (Fig. 4A).
Tmod Binds to the N-terminal End of Nebulin in Solution--
The
solid phase binding assays described above require that the nebulin
fragments be adsorbed onto the wells of a microtiter plate, which can
result in partial denaturation of the peptides. In order to determine
whether Sk-Tmod binds to nebulin under native conditions, we performed
bead binding studies in which the proteins were incubated together in a
soluble state. The recombinant nebulin fragment M1-M2-M3 was incubated
with biotinylated Sk-Tmod, and the protein mixture was then incubated
with streptavidin-conjugated Sepharose beads. The bound
(lanes B) and unbound (lanes
U) fractions were separated by SDS-polyacrylamide gel
electrophoresis and stained with Coomassie Blue (Fig.
5). When M1-M2-M3 was incubated with the
streptavidin beads alone, all of the fragment was recovered in the
unbound fraction; i.e. M1-M2-M3 did not bind alone to the beads (Fig. 5, lanes 1 and 2).
Consistently, however, when we added biotinylated Sk-Tmod to the
M1-M2-M3 fragment, a portion of M1-M2-M3 (ranging from 20 to 50% of
the total) was recovered in the bound fraction (Fig. 5, lane
4). The presence of unbound M1-M2-M3 in the supernatant may
be due to the saturation of all of the potential binding sites
(Fig. 5, lane 3). Similar results were obtained
when the M1-M2-M3 fragment was incubated with biotinylated E-Tmod (data
not shown). The results of the bead binding assays indicate that Tmod
also binds to nebulin when both proteins are present under native
conditions.
Binding of Tmod to the Nebulin M1-M2-M3 Modules Is Saturable and of
High Affinity--
The next series of experiments was designed to
determine whether Tmod binding to the nebulin fragment, M1-M2-M3, was
saturable and to obtain an approximate value for the disassociation
constant (Kd) for the binding of Tmod to M1-M2-M3.
Immobilized M1-M2-M3 was incubated with increasing amounts of either
biotinylated Sk-Tmod (Fig. 6A)
or E-Tmod (Fig. 6B) for 1 h in saturation binding studies. Determination of the concentration of Tmod corresponding to
the half-maximal A405 value yields a
semiquantitative estimate of the Kd value for Tmod
binding to M1-M2-M3. These experiments showed that the binding of Tmod
to M1-M-M3 is saturable. Sk-Tmod bound to M1-M2-M3 with an apparent
Kd of approximately 15 nM, while E-Tmod
bound to M1-M2-M3 with an apparent Kd of
approximately 30 nM. The results from these saturation
studies are consistent with the previous finding (Fig. 4) that more
Sk-Tmod binding to M1-M2-M3 was detected compared with E-Tmod. We also analyzed the interaction of E-Tmod with immobilized skeletal muscle tropomyosin (Fig. 6B). In these assays, E-Tmod bound to
tropomyosin with a much weaker apparent affinity than it bound to
M1-M2-M3, with a Kd in the range of 100-150
nM (Fig. 6B); this Kd value
is comparable with that obtained from previous binding studies (~200
nM) (38).
To more accurately determine Kd values for the
Tmod/M1-M2-M3 interaction, a series of equilibrium and kinetic binding experiments in a dcELISA was performed (43). The dcELISA
utilizes a series of linearized plots to accurately quantify
association constants. Three sets of experiments are required. First,
the incubation time of the receptor with the ligand is varied in a time
course to determine the binding complex formation rate constant, kc. Second, a series of transfer assays at various
binding times is performed to determine the transfer factor,
Ft, and the calibration factor, c.
Finally, a saturation curve is used to determine the amount of
immobilized M1-M2-M3, R0. The results obtained
allow for the calculation of the association constant,
Ka, the inverse of which is equal to the
Kd value.
Fig. 7 shows results obtained from the
dcELISA study. In Fig. 7A, we determined the M1-M2-M3-Tmod
complex formation rate constant, kc, by varying the
time that biotinylated Tmod was incubated with the immobilized
recombinant M1-M2-M3 fragment. Following linear regression analysis on
the data plotted as
ln(At
Finally, to determine the Ka values, saturation
curves were generated under equilibrium conditions, where Tmod was incubated with nebulin for 4 h at 4 °C (Fig. 7, C
for Sk-Tmod and D for E-Tmod). Using the saturation
absorbance obtained by nonlinear regression analysis (data not shown),
as well as the values obtained for c and F in the
previous series of experiments, we calculated the Ka
values to be 0.227 × 109 liters/mol for Sk-Tmod
binding to M1-M2-M3 and 0.067 × 109 liters/mol for
E-Tmod binding to M1-M2-M3. The inverse of these values yields a
Kd value of 4.4 nM for Sk-Tmod binding to M1-M2-M3 and a Kd value of 15 nM for
E-Tmod binding to M1-M2-M3. These values are in close agreement with
those estimated from the half-maximal A405
values from the saturation curves performed under equilibrium
conditions in C and D (3.2 nM for
Sk-Tmod binding to M1-M2-M3 and 15.8 nM for E-Tmod binding
to M1-M2-M3). These Kd values are also comparable
with those values approximated from the initial saturation curves
performed under nonequilibrium conditions (incubation of M1-M2-M3 with
Tmod for 1 h) (Fig. 6, A and B). Therefore,
quantitative analysis revealed an approximate 4-fold greater affinity
of Sk-Tmod binding to M1-M2-M3 than E-Tmod binding to M1-M2-M3.
Saturable and high affinity binding of Tmod to M1-M2-M3 provides
additional evidence that this interaction is specific.
Strikingly, the interaction of Tmod with the N-terminal region of
nebulin (Kd ~4-16 nM) is considerably
tighter compared with previously reported interactions of nebulin with
other proteins. Although these previous investigations were performed
using different in vitro binding assays, it is nevertheless
of interest to compare these interactions with the Tmod/nebulin
interaction. For example, nebulin has been reported to bind F-actin
with a Kd ranging from ~0.1 to ~400
µM (depending on the nebulin fragment utilized), troponin
with a Kd of ~100-200 nM, tropomyosin
with a Kd of ~500 nM, calmodulin with
a Kd of ~100 nM, and myosin with a
Kd of ~160 nM (19, 47-51). The affinity of the Tmod/M1-M2-M3 interaction is more similar, however, to
the interaction of Tmod with the pointed ends of tropomyosin-coated actin filaments (Kd < 1 nM) (24, 33).
The tight binding of Tmod to both the N-terminal end of nebulin and to
the pointed ends of the thin filaments suggests that together these
interactions have a critical role in the sarcomere.
Tropomyosin Does Not Affect Binding of Tmod to M1-M2-M3--
It
has previously been established that the interaction of tropomyosin
with Tmod is important for modulating the interaction of Tmod with
actin and for Tmod's actin filament capping activity (24, 36). Thus,
we next determined if tropomyosin affects the interaction of Tmod with
nebulin. Biotinylated Tmod was incubated with immobilized M1-M2-M3 in
the presence of increasing amounts of purified rabbit skeletal muscle
tropomyosin (Fig. 8). Since the affinity
of Tmod for tropomyosin is 2 orders of magnitude weaker than the
affinity of Tmod for M1-M2-M3 (150-200 nM
versus 4-16 nM; see above and Ref. 38), we
included a 200-fold excess of tropomyosin over Tmod in these assays.
Tropomyosin had no detectable effect on the interaction of either
Sk-Tmod or E-Tmod with M1-M2-M3. This suggests that skeletal
tropomyosin and M1-M2-M3 have distinct, or nonoverlapping, binding
sites on Tmod. Since tropomyosin binds to Tmod within the Tmod residues
1-130 (38, 52), it is expected that the primary M1-M2-M3 binding site
is located outside of these Tmod residues.
The length distributions of the thin filaments in skeletal muscle
are strikingly narrow and uniform, indicating that an intricate regulatory mechanism exists for precisely controlling their lengths. It
has been proposed that this mechanism must involve at least two types
of proteins, namely template molecules to specify thin filament length (e.g. nebulin) and capping proteins to
maintain the specified length (e.g. CapZ at the
barbed ends and Tmod at the pointed ends) (1, 5). In this study, we
report that the actin-capping and tropomyosin-binding protein, Tmod,
has a third binding partner, nebulin. Tmod binds specifically to the extreme N-terminal modules, M1-M2-M3 (13.5 kDa), of the giant nebulin
molecule (~600-800 kDa) as demonstrated by blot overlay studies,
solid phase binding assays, and bead binding assays. To our knowledge,
this is the first report of a biochemical function for the N-terminal
region of nebulin. The establishment of the Tmod/nebulin interaction
demonstrates that nebulin's final N-terminal modules indeed extend out
to, and interact with, the extreme ends of the thin filaments, a
requirement necessary to function as a molecular ruler for thin
filament length specification. These data provide evidence that
nebulin's N-terminal end, together with Tmod, probably function as
part of a linked regulatory mechanism for thin filament length
regulation in skeletal muscle.
Based on our results and the investigations of others, we propose a
molecular model that demonstrates how the interaction of nebulin with
Tmod could regulate thin filament length at the pointed ends in various
types of skeletal tissues (Fig. 9).
First, the association of each actin subunit in the thin filament with an individual nebulin module specifies the length of the thin filament
(10, 13, 19). Specific thin filament lengths are precisely defined by
different nebulin isoforms containing varying numbers of modules. It
should be noted that the exact mode of association of nebulin along the
length of the actin/thin filament has not yet been resolved. Structural
studies and microscopic investigations indicate that nebulin is likely
to bind in the central cleft of the actin filament, i.e. in
the phalloidin binding site (53-55). However, in vitro
binding studies have led to the alternative proposal that nebulin
associates with the outer edges of the actin filament, forming a
composite regulatory complex with tropomyosin and the troponins (19,
47, 48, 56). Interestingly, recombinant nebulin fragments have also
been reported to inhibit actomyosin ATPase activity (49). Therefore,
nebulin may serve additional regulatory roles in the sarcomere. Second,
a tropomyosin polymer is associated with each of the strands in the
actin filament in a head-to-tail manner, stabilizing the filament and
helping to prevent actin disassembly from the pointed ends
(e.g. see Ref. 57). Third, once the filaments attain their
mature lengths, nebulin's extreme N-terminal modules target Tmod to
the pointed ends of the thin filaments that have polymerized to the
length of the nebulin ruler. Fourth, Tmod caps the actin filament
pointed ends and perhaps the tropomyosin polymers, thus maintaining the correct lengths of the filaments (26). Finally, targeting of Tmod to
the pointed ends of thin filaments of correct lengths, via binding to
nebulin, is likely to be a dynamic process. This hypothesis is based on
recent studies indicating that the thin filaments are not irreversibly
capped by Tmod and can still exchange actin subunits with the monomer
pool.2 Therefore, a
nebulin-mediated mechanism may regulate and/or stabilize Tmod's
capping activity at the pointed ends. Future studies will be directed
at investigating the effect of the N-terminal region of nebulin on the
capping activity of Tmod in actin filament polymerization assays.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
APPENDIX
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
APPENDIX
REFERENCES
70 °C until use.
-D-thiogalactopyranoside (Sigma), and 3-4 h later were
collected by centrifugation. The bacterial pellets were resuspended in
15 ml of ice-cold lysis buffer (20 mM Tris-HCl, pH 8.0, 10 mM imidazole, pH 7.5, 200 mM NaCl, 0.2%
Nonidet P-40, and 2 mM
-mercaptoethanol plus 100 µg/ml
of tosyl-L-lysyl chloromethyl ketone, 100 µg/ml
phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 10 µg/ml
pepstatin A, 1 µg/ml aprotinin, and 0.5 mM PefaBloc) and
sonicated to lyse cells and reduce viscosity of the lysate. The lysate
was ultracentrifuged for 30 min at 50,000 × g, and the
supernatant was loaded onto a column containing 300 µl of Ni2+-nitrilotriacetic acid-agarose (Qiagen). The column was
washed sequentially with 30 ml of lysis buffer, 30 ml of lysis buffer without Nonidet P-40, 1 ml of 1 M NaCl, and 50 ml of lysis
buffer without Nonidet P-40. The His6-tagged protein was
eluted from the column with 10 ml of 300 mM imidazole, pH
7.4, and peak fractions were pooled. Some nebulin fragment preparations
(containing contaminating bacterial proteins) were further purified
using a fast protein liquid chromatography system on a Mono Q anion
exchange column or Superose 6 gel filtration column (Amersham Pharmacia
Biotech). The pooled peak fractions were concentrated using Centriplus
centrifugal filter devices (molecular weight cut-off of 3,000)
(Millipore Corp.), and dialyzed against binding buffer for the solid
phase binding assays (20 mM Hepes, pH 7.4, 80 mM KCl, 2 mM MgCl2, 0.002% NaN3) using Slide-A-Lyzers (molecular weight cut-off of
3,000) (Pierce). Protein concentrations were determined by the BCA
assay, according to the manufacturer's instructions (Pierce). All
proteins were aliquoted, frozen in liquid nitrogen, and stored at
70 °C until use.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
APPENDIX
REFERENCES
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Fig. 1.
The extreme N-terminal modules of nebulin
colocalize with Tmod. Double immunofluorescence confocal
micrographs of nebulin neb N-term + M1 (A) and E-Tmod
(B) localization in primary cultures of embryonic chicken
skeletal myotubes. A merged image is shown in
C. Bar, 10 µm.
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Fig. 2.
Tmod binds to nebulin on blot overlays of
myofibrillar proteins isolated from rat psoas skeletal muscle
fibers. Nebulin was resolved from other myofibrillar proteins on a
large pore, 3.3-12% SDS-polyacrylamide gel electrophoresis (Coomassie
Blue-stained gel; lane 1). After transfer to
nitrocellulose, blocking, and incubation with 1 µg/ml purified human
E-Tmod, Tmod binding to nebulin was detected by incubation with
affinity-purified rabbit anti-human E-Tmod antibodies followed by
125I-protein A and autoradiography (lane
3). As expected, the antibody labels endogenous Tmod present
in the rat muscle (lanes 3 and 4,
arrowhead). Nebulin was detected on a parallel
nitrocellulose strip by immunoblotting with goat anti-nebulin
antibodies, followed by rabbit anti-goat antibodies and
125I-protein A (lane 2).
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Fig. 3.
Schematic of skeletal muscle nebulin
(modified from Ref. 13). The nebulin modules are classified as
unique domains (black), linker repeats (dark
gray), super repeats (light gray), or
simple repeats (white). The recombinant nebulin fragments
generated in this study are shown below to the
representative regions of the molecule. Asterisks designate
the fragments exhibiting detectable binding to Tmod as well as the
qualitative degree of binding that was observed from several solid
phase binding assays (relative scale, where */ denotes weak,
inconsistent binding to Tmod, and *** denotes strong binding to
Tmod).
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Fig. 4.
Tmod specifically binds to recombinant
fragments from the N-terminal end of nebulin in solid phase binding
assays. A, 100 nM of purified rabbit
skeletal muscle tropomyosin, recombinant nebulin fragments neb N-term
(the unique 8-kDa domain from the extreme N terminus), neb N term + M1
(the unique N terminus plus the first unique module), neb N term + M1-M2-M3 (the unique N terminus plus the first three unique modules),
M1-M2-M3 (the first three unique modules), M2-M8 (seven of the eight
unique N-terminal modules), M9-M15 (internal modules comprising the
first super repeat), as well as a recombinant nebulette fragment,
nette-R1-R2-R3 (the nebulette N-terminal domain plus the first three
unique modules), were adsorbed onto microtiter plates. After washing
and blocking, the wells were incubated with 2.5 nM
biotinylated E-Tmod (open bars) or biotinylated
Sk-Tmod (solid bars). Following washes and
incubation with alkaline phosphatase-conjugated streptavidin, bound
Tmod was detected by the addition of the substrate pNPP, followed by
colorimetric development at A405. The
A405 readings of wells coated with buffer alone
were subtracted out as background values from all other wells. The
graph is representative of three experiments. Values are the
mean of duplicates ± S.D. B and C, binding
of biotinylated Sk-Tmod and E-Tmod to the immobilized recombinant
nebulin fragment M1-M2-M3 in the presence of unlabeled competitor. 100 nM of recombinant M1-M2-M3 was adsorbed onto microtiter
plates and incubated with 25 nM biotinylated Sk-Tmod
(B) or biotinylated E-Tmod (C) in the presence of
increasing concentrations of unlabeled E-Tmod (white
bars) or unlabeled Sk-Tmod (black
bars). The plates were washed and incubated with alkaline
phosphatase-conjugated streptavidin, followed by pNPP substrate. Bound
biotinylated Tmod was determined by a colorimetric reaction at
A405 and was plotted as the percentage of the
control (the amount bound in the absence of competitor). The
graphs are representative of three experiments. Values are
the mean of duplicates ± S.D.
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Fig. 5.
Sk-Tmod binds to M1-M2-M3 under native
conditions. 1 µg of recombinant M1-M2-M3 was incubated in 20 µl of binding buffer for 1 h at room temperature in the absence
(lanes 1 and 2) or presence
(lanes 3 and 4) of 1 µg of
biotinylated Sk-Tmod. Streptavidin-conjugated Sepharose beads were then
added (1:1) to the protein solution, and the unbound (lanes
U) and bound (lanes B) fractions were
separated by 18% SDS-polyacrylamide gel electrophoresis and visualized
by Coomassie Blue staining. Note that under elution conditions using
0.1 M glycine, pH 2.8, the biotinylated Tmod remained bound
to the streptavidin beads and therefore was not recovered in either
bound or unbound fractions. The gel is representative of 10 experiments.
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Fig. 6.
The binding of Tmod to M1-M2-M3 is saturable
in solid phase binding assays. 100 nM M1-M2-M3
(A and B, squares) or 100 nM purified rabbit skeletal tropomyosin (B,
diamonds) was adsorbed onto microtiter plates and incubated
with increasing concentrations of biotinylated Sk-Tmod (A)
or biotinylated E-Tmod (B) for 1 h at 4 °C.
Following washes and incubation with alkaline phosphatase-conjugated
streptavidin, wells were incubated with pNPP substrate. Binding of Tmod
was determined by a colorimetric reaction at
A405. Background values of wells coated with
buffer alone were subtracted from the values obtained from all other
wells. A405 values were plotted
versus the concentration of biotinylated Tmod and fitted to
a moving average trendline with a period of 2, using Microsoft Excel.
The graphs are representative of three experiments. Values are the mean
of duplicates ± S.D. The approximate Kd is
estimated by determining the concentration of Tmod at half-maximal
A405 for the fitted curve. Note that the
saturation curves for the three proteins, Sk-Tmod, E-Tmod, and
tropomyosin, were performed in various combinations, and similar
results were obtained.
At) versus time (Fig.
7A), we determined that the kc for
Sk-Tmod binding to M1-M2-M3 is 4.3 × 10
4 s
1 and the
kc for E-Tmod binding to M1-M2-M3 is 2.5 × 10
4 s
1. In the next
experiments, transfer assays were performed where immobilized M1-M2-M3
was incubated with biotinylated Tmod for various amounts of time (Fig.
7B). Unbound Tmod remaining in solution was then removed
from the well and transferred to the next well, also containing
immobilized M1-M2-M3. The results from one of a total of four transfer
assays are shown, where Tmod was incubated with M1-M2-M3 for 30 min/well (Fig. 7B) before unbound Tmod was transferred to
the next, identically coated well. After generating semilogarithmic
plots of ln 405 versus the total number of transfers performed at each incubation time, linear regression was performed to
obtain the Ft values (data not shown). These
experiments also were used to calculate the calibration constant,
c, according to the equation c = L0/At × (1
F) (data not shown).
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Fig. 7.
Quantitative analysis reveals that Tmod binds
to M1-M2-M3 with high affinity. A, 100 nM
M1-M2-M3 was adsorbed onto microtiter plates and incubated with 2.5 nM biotinylated Sk-Tmod (squares) or E-Tmod
(diamonds) for various times, washed, and incubated with
alkaline phosphatase-conjugated streptavidin. Following additional
washes and incubation with pNPP substrate, binding of Tmod was
determined by a colorimetric reaction at A405.
The A405 values were plotted versus
time, and nonlinear regression analysis was performed to determine
At (data not
shown). The data points were then plotted as
ln(At
At) versus time and represent the
mean of duplicates (y =
0.027x + 0.03889 and R2 = 0.9175 for Sk-Tmod; y =
0.0145x
0.2738 and R2 = 0.99 for E-Tmod). A linear regression analysis leads to a
kc = 4.3 × 10
4
s
1 for Sk-Tmod binding to M1-M2-M3, and a
kc value of 2.5 × 10
4 s
1 for E-Tmod
binding to M1-M2-M3. B, 100 nM M1-M2-M3 was
adsorbed onto microtiter plates and incubated with 2.5 nM
biotinylated Sk-Tmod (squares) or E-Tmod
(diamonds) for various times. Next, the Tmod solution was
transferred to an adjacent well for the same amount of time. Between
four and seven transfers were performed for each experiment, for either
10, 15, or 20 min (data not shown) or 30 min (B) of
incubation for each well. Each well was washed and then incubated with
alkaline phosphatase-conjugated streptavidin, followed by incubation
with pNPP substrate. The data were plotted as ln405 versus
the transfer number (y =
0.337x + 0.6929 and R2 = 0.981 for Sk-Tmod; y =
0.2718x
0.7459 and R2 = 0.9331 for E-Tmod). Using the absorbances of the first wells as
A1 and the initial concentration of M1-M2-M3
(L0), calibration constant (c) values
were calculated for each transfer plot and averaged to
c = 0.32 nM for Sk-Tmod and 0.30 nM for E-Tmod. C and D, to determine
Ka values, saturation curves for biotinylated
Sk-Tmod and E-Tmod were calculated under equilibrium conditions. 100 nM M1-M2-M3 was adsorbed onto microtiter plates and
incubated with various concentra tions of biotinlyated Sk-Tmod (C) or E-Tmod
(D) for 4 h. After washes and incubation with alkaline
phosphatase-conjugated streptavidin, followed by incubation with pNPP
substrate, binding of Tmod was determined by a colorimetric reaction at
A405. The saturation absorbance was used along
with the obtained c and F values, and the
association equilibrium constant was calculated as
Ka = 0.227 × 109 liters/mol for
Sk-Tmod binding to M1-M2-M3 and 0.067 × 109 liter/mol
for E-Tmod binding to M1-M2-M3. The inverse of the
Ka values yields the Kd values of
4.4 nM for Sk-Tmod and 15.0 nM for E-Tmod
binding to M1-M2-M3.
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Fig. 8.
Tropomyosin does not affect binding of
biotinylated E-Tmod or Sk-Tmod to the nebulin fragment, M1-M2-M3.
100 nM recombinant M1-M2-M3 nebulin fragment was adsorbed
onto microtiter plates and incubated with 2.5 nM
biotinylated E-Tmod (circles) or biotinylated Sk-Tmod
(squares) in the presence of increasing concentrations of
purified rabbit skeletal tropomyosin. After washes and incubation with
alkaline phosphatase-conjugated streptavidin, followed by pNPP
substrate, bound biotinylated Tmod was determined by a colorimetric
reaction at A405. The graphs are representative
of two experiments. Values are the mean of duplicates ± S.D.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
APPENDIX
REFERENCES
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Fig. 9.
Molecular model for the association of
sarcomeric components at the pointed end of the thin filaments.
Tmod (blue) binds the extreme N-terminal region of
nebulin (green), as well as the terminal tropomyosin
(red) and actin (gray) molecules at the pointed
ends of the thin filaments. Each tropomyosin molecule of the two
polymers per thin filament binds one troponin complex (composed of
troponins T, I, and C) (yellow). Whether the pointed ends of
the actin filaments are capped by one or two Tmods per actin filament
awaits further clarification. The stoichiometry as determined
biochemically in skeletal myofibrils is between 1.2 and 1.6 Tmods per
thin filament (30). The stoichiometry determined by measuring capping
activity predicts one Tmod per filament end (33). Additionally, whether
there are one, two, or four nebulin molecules per thin filament awaits
further clarification (6, 9, 50).
We found that the N-terminal region of nebulin specifically binds to both Sk-Tmod and E-Tmod. However, Sk-Tmod binds to nebulin with a higher affinity compared with the binding of E-Tmod to nebulin. These two Tmod isoforms are products of different genes but are 62% identical and 75% similar to each other at the amino acid level in chicken (32, 58). Previous in vitro studies have not detected differences in their ability to cap pure actin filaments or tropomyosin-coated actin filaments (24, 32). However, our studies now reveal that the two Tmod isoforms differ in their interactions with the N-terminal region of nebulin. Although it is not yet known if this ~4-fold difference in affinity is physiologically significant, it is tempting to speculate that it may explain the previously reported differences in the cellular properties of Sk-Tmod and E-Tmod.
First, the distinct interactions of Sk-Tmod and E-Tmod with nebulin may
explain the previously reported differences in the subcellular
localization patterns of these isoforms in skeletal tissue. In chicken
slow muscle fibers, the predominant Tmod isoform expressed is E-Tmod,
which is associated with the thin filament pointed ends (32). In
chicken fast skeletal muscle fibers, which co-express both Tmod
isoforms, there is distinct targeting of E- and Sk-Tmods to different
actin filament-containing structures (32). In these fibers, E-Tmod
predominantly colocalizes with -spectrin in costameric
subsarcolemmal domains, while Sk-Tmod is present at the pointed ends of
the thin filaments in sarcomeres of those same cells (32). Therefore,
the higher affinity interaction of nebulin with Sk-Tmod compared with
E-Tmod may cause Sk-Tmod to outcompete E-Tmod for the nebulin-binding
site at the pointed ends of the thin filaments in fast muscle.
It is also possible that the isoform-specific interaction of Tmod with nebulin may provide the molecular basis for the observed variations in thin filament lengths between slow and fast skeletal muscle fibers as well as during muscle differentiation (e.g. see Refs. 59 and 60). For instance, E-Tmod is located at the pointed ends of thin filaments in embryonic chicken pectoralis muscle. This embryonic tissue contains longer thin filaments with a wider length distribution (~0.95-1.1 µm) compared with adult pectoralis muscle, which has Sk-Tmod at the pointed ends and shorter, more uniform thin filaments (all of the thin filaments are ~0.9 µm long) (60). We speculate that this phenomenon is a result of the differential nebulin-Tmod isoform interaction that functions to regulate thin filament lengths. Specifically, the tighter association of Sk-Tmod with nebulin, compared with the association of E-Tmod with nebulin, may cause tighter capping at the pointed ends, resulting in a reduced actin monomer exchange at the pointed ends and, hence, more precise thin filament lengths in adult fast skeletal muscle fibers.
Finally, it is striking that cardiac muscle, which expresses E-Tmod (32) but does not appear to contain nebulin (11) has a significantly broader range of thin filament lengths (e.g. 0.6-1.1 µm in rat) as compared with skeletal muscle (e.g. 1.1 ± 0.03 µm in rabbit) (3, 4). Small proteins with homology to nebulin have recently been identified in heart, but these proteins do not localize to the pointed ends of the thin filaments. These include nebulette, which associates with the Z-disc and is likely to extend only about 25% along the length of the thin filament, and N-RAP, which is found at intercalated discs (14, 45, 46, 61). Thus, in cardiac muscle, E-Tmod may work with proteins other than nebulin to regulate thin filament lengths at the pointed ends, although the particular regulatory mechanism appears to be less precise than that for skeletal muscle, which contains full-length nebulin.
Investigations into the physiological significance of the Tmod/nebulin
interaction are needed and are expected to be pivotal for deciphering
the mechanisms of thin filament regulation in skeletal muscle. Since
the precise organization of the sarcomere is crucial for the
contractile activity of striated muscle, understanding the function of
the Tmod/nebulin interaction also may provide clues for understanding
specific muscular diseases. For example, investigating nebulin is of
clinical relevance, since mutations in this molecule are associated
with autosomal recessive nemaline myopathy (62, 63), and the integrity
of the nebulin filament is severely reduced in Duchenne muscular
dystrophy (64). Finally, given that actin is the most abundant
cytoskeletal component of most, if not all, eukaryotic cells and that
nebulin-related proteins have recently been identified (14, 45, 46,
61), as well as the recent finding that a Tmod isoform (TMOD3; Ref. 58)
is ubiquitously expressed, investigations into the function of the Tmod/nebulin interaction will aid in understanding actin filament dynamics in a variety of cell types.
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ACKNOWLEDGEMENTS |
---|
We gratefully acknowledge Ryan Mudry, Adam Geach, Jeannette Moyer, Thomas Centner, and Dietmar Labeit for assistance with recombinant protein purification. We thank Adam Geach and Ryan Mudry for help with the figures, Paul St. John for insightful discussions on binding studies, Edwin Marengo for assistance with the dcELISA data analysis, and members of the Gregorio laboratory for critical reading of the manuscript.
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FOOTNOTES |
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* This work was supported by the National Institutes of Health Grants HL57461 and HL03985 (to C. C. G.), HL07249 (to A. S. M.), and GM3425 (to V. M. F.), Deutsche Forschungsgemeinschaft Grant La 668/3-3 (to S. L.), and an award from the Human Frontier Science Program (to C. C. G., V. M. F., and S. L.).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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) X83957.
** To whom correspondence should be addressed: Dept. of Cell Biology and Anatomy, LSN455, The University of Arizona, 1501 N. Campbell Ave., Tucson, AZ 85724. Tel.: 520-626-8113; Fax: 520-626-2097; E-mail: gregorio@u.arizona.edu.
Published, JBC Papers in Press, October 2, 2000, DOI 10.1074/jbc.M005693200
2 R. Littlefield, A. Almenar-Queralt, and V. M. Fowler, submitted for publication.
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ABBREVIATIONS |
---|
The abbreviations used are: Tmod, tropomodulin; Sk-Tmod, skeletal Tmod; E-Tmod, erythrocyte Tmod; pNPP, p-nitrophenyl phosphate; ELISA, enzyme-linked immunosorbent assay; dcELISA, direct calibration ELISA.
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APPENDIX |
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Expression of Recombinant Nebulin Fragments-- The expression constructs encoding fragments from the N-terminal region of nebulin, as well as the expression construct encoding a fragment from the N-terminal region of the cardiac protein, nebulette, were expressed in Escherichia coli using the pET system (65) and are as follows (to simplify protein purification, histidine hexamer tags were included either as N-terminal or C-terminal fusions): 1) Neb N-terminal unique acidic domain, in pET9d + C-terminal His6 (11.3 kDa); 2) Neb N-Term + M1, in pET8c + N-terminal His6 (13.6 kDa); 3) Neb N-Term + M1-M2-M3, in pET9d + C-terminal His6 (21.8 kDa); 4) M1-M2-M3, in pET9d C-terminal His6 and in pET-M11 (13.5 kDa); 5) M1-M8, in pET9d C-terminal His6 (33.7 kDa); 6) M9-M15, in pETd C-terminal His6 (32.8 kDa); 7) nebulette N-term + R1-R2-R3 in pET9d C-terminal His6 (24.5 kDa).
Nebulin cDNA fragments were amplified by polymerase chain reaction from total human skeletal muscle cDNA (66) using combinations of the primers listed below. Skeletal muscle cDNA was used as a template (CLONTECH, Matchmaker HL4047AH). For polymerase chain reaction, about 50 ng of the amplified cDNA was used as template, and 25 cycles were performed with the following profile: 10 s, 95 °C; 2 min, 68 °C; and 5 min, 74 °C final incubation. After polymerase chain reaction amplification, the products were agarose gel-purified and subcloned into pET vectors essentially as described (67).
Primer Sequences-- All primer sequences were derived from EMBL data library accession number X83957 (20.9-kilobase pair full-length human skeletal nebulin cDNA). The following primer pairs were used for amplification of the nebulin N-terminal unique acidic domain (beginning with the amino acid sequence MADD) to the M15 module of nebulin. Small capital letters denote primer mismatch portions harboring cloning sites, whereas capital letters correspond to nebulin codons/reverse codons: neb N-term sense (MADD-sense), ttt ctcgagc GCA GAT GAC GAA GAC TATG; X83957 nebulin 444S; neb 731R (MADD-reverse), ttt acgcgt-ta-GCT AAA AAG ATC CTG CATTT; neb 663S (M1-sense), tttccatggcc-AAA GTG GAT CCT TCA AAG TTC ATG ACC CCC TAC; neb 797R (M1-reverse), ttt acgcgt-ta- AGT ATC TGT TGT GCT GGC; neb 774S (M2-sense), ttt ctcgagc-CCA TAC GCC AGC ACA ACA GA; neb 890R (M2-reverse), ttggtacc-ta-TAC GTG ACA TAT AGT CTT AGC AAC ATC ACC; neb 996S (M4-sense), ttt ctcgagct-CCT CCT GAT GCC CCT GAA CTT GTC CAG; neb 1,533S (M9-sense), tttccatg ggc-CCT GCT TCA GAG AAC CCA CAG CTT AGG CAG; neb 1,007R (M3 reverse), ttt ggtacc-ta-GGC ATC AGG AGG AAG CAG GTA CTT ATC CTT; neb 1,532R (M8-reverse), ttt acgcgt ta AAG CAC ATT ATA ATC TGCTT; neb 2,385R (M15-reverse), ttt gtcgac-ta-ATT CAT ACT CTT TGC CTT TGT CTT CTC ATA G.
As discussed above, to compare the properties of the expressed nebulin
N-terminal + M1-M2-M3 with those of a corresponding nebulette fragment,
nebulette N-terminal + R1-R2-R3 (14) was amplified with the following
primer pair: ttttccatg-ggg AGG GTC CCT GTA TTT GAG GAT (human nebulette
398S) and tttggtacc-cta-GGG CTC CTT CAT GTG GGC ATA (human nebulette 830R).
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