Department of Cell Biology, Weill Medical College of Cornell University,
1300 York Avenue, York Avenue, New York, NY 10021, USA
* Present address: Department of Biochemistry, University of Washington,
Seattle, WA 98 195, USA
Author for correspondence (e-mail:
fisch{at}med.cornell.edu)
Accepted 14 June 2002
![]() |
Summary |
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Key words: C-protein, H-protein, Myosin, Muscle, Myofibril assembly, Immunoglobulin, Light meromyosin
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Introduction |
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In addition to myosin, each thick filament is composed of several major
proteins including MyBP-C (Offer et al.,
1973) and MyBP-H (Starr and
Offer, 1982
). MyBP-C is an asymmetric protein of
130 kDa that
accounts for approximately 2% of myofibril protein mass
(Offer et al., 1973
). Three
isoforms have been identified: cardiac, fast skeletal and slow skeletal, each
encoded by separate genes (Vaughan et al.,
1993a
; Vaughan et al.,
1993b
). MyBP-H is
55 kDa and is mainly found in fast skeletal
muscle; only one isoform and one gene have been identified. Within the
sarcomere, MyBP-C and MyBP-H are distributed in highly regular patterns that
are species- and fiber-type specific. MyBP-C is found in the C zone of the
A-band (Squire, 1990
) in
association with a set of 11 transverse stripes, 43 nm apart, in each half
A-band (Craig and Offer, 1976
;
Dennis et al., 1984
). In the
chicken pectoralis muscle, MyBP-H has a similar distribution to MyBP-C
(Bahler et al., 1985
), but
different patterns have been described in mammalian muscle
(Bennett et al., 1986
).
Nucleotide sequencing of MyBP-C and MyBP-H mRNAs has shown that both
proteins are constructed of two repeating modules: the immunoglobulin I (IgI)
and fibronectin III (FnIII) motifs (Fig.
1). Skeletal MyBP-C is composed of a linear array of seven IgI and
three FnIII domains, each containing 100 amino acids, in the order of
IgI-IgI-IgI-IgI-IgI-FnIII-FnIII-IgI-FnIII-IgI, numbered C1 to C10 from the N-
to C-terminus (Vaughan et al.,
1993a
; Weber et al.,
1993
). MyBP-H consists of a unique N-terminal leader sequence
containing two motifs of alternating alanine and proline residues; it is this
N-terminal leader domain that causes the anomalously slow mobility of chicken
MyBP-H. The C-terminus of MyBP-H is composed of four repeating modules with
the order FnIII-IgI-FnIII-IgI, an identical arrangement to the C-terminus of
MyBP-C (Vaughan et al.,
1993a
). The sequence homology between the C-termini of both
proteins is approximately 50%. Most studies of MyBP-C have focused on the
protein's biochemical properties, of which the best characterized is its
affinity for myosin (Moos et al.,
1975
; Offer et al.,
1973
). The protein binds to F-actin
(Moos et al., 1978
) and titin
(Freiburg and Gautel, 1996
).
In vitro binding studies demonstrated that the LMM-binding domain of MyBP-C
resides in the C-terminal IgI (C10) domain
(Okagaki et al., 1993
). A
comparable LMM-binding domain resides in the C-terminal IgI motif (H4) of
MyBP-H (Alyonycheva et al.,
1997
). A titin-binding site on MyBP-C has been localized within
the C7-C10 module (Freiburg and Gautel,
1996
). Using transient transfections in cultured embryonic
myocytes, it has been demonstrated that the final four domains of both MyBP-C
and MyBP-H are necessary for proper localization of these proteins in the
A-band (Gilbert et al., 1998
;
Gilbert et al., 1996
).
Deletion of the C-terminal IgI domain of either MyBP-H or MyBP-C prevents the
proteins from incorporating into the A-band. Mutants containing the C-terminal
IgI domain, but lacking the adjacent three upstream modules, do not properly
localize to the A-band, even though they include the myosin rod-binding IgI
domain (Gilbert et al., 1998
;
Gilbert et al., 1996
). In a
recent study designed to determine amino acids on the surface of C10 that may
interact with LMM, it was found that four surface regions of C10 interact with
the myosin rod. All of these contain charged residues, presumably involved in
ionic interactions with complementary residues on the myosin rod. These data
suggest that C10 behaves as a multivalent ligand, crosslinking three or four
molecules of the myosin rod (Miyamoto et
al., 1999
).
|
In the present study we have used the COS cell transfection system
(Moncman et al., 1993;
Vikstrom et al., 1993
) to
define the regions of MyBP-C and MyBP-H that may be responsible for
crosslinking myosin in thick filaments. We found that the C-terminal IgI
module in both MyBP-C and MyBP-H is both necessary and sufficient to induce
cable formation when these MyBPs are co-expressed with MyHC. In addition, we
present evidence for previously unidentified myosin-interaction sites within
domains C7-9 and H1-3 of MyBP-C and MyBP-H, respectively.
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Materials and Methods |
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Cell culture
COS-1 cells were propagated in Dulbecco's modified Eagle's medium (DMEM)
containing 10% fetal bovine serum (FBS), 2 mM L-glutamine, 50 units/ml
penicillin G and 50 µg/ml streptomycin (penstrep) in 100 mm dishes with
glass coverslips. Transfections were performed with cell cultures of 40 to 60%
confluence using lipofectamine PLUS reagents (Life Technologies, Rockville,
MD) according to the manufacturer's instructions. COS cells were processed for
analysis two days after transfection. Primary cultures of myoblasts were
prepared from 11-day-old embryonic chicken pectoralis muscle as described
previously (Gilbert et al.,
1996). The myoblasts were cultured on collagen-coated, two-well
chamber slides (NUNC, Naperville, IL) in DMEM, 10% FBS, 2 mM L-glutamine and
2% chick embryo extract. One day after plating, the cells were transfected
using lipofectamine PLUS reagent. On the following day the cells were switched
to DMEM containing 10% horse serum instead of FBS. The media was changed every
two days and the cells allowed to differentiate for five days after
transfection before being fixed and processed for immunostaining.
Western blots
Transfected COS cells were prepared for western blotting by twice washing
with phosphate-buffered saline (PBS), then scraping the cells into 200 µl
of Laemmli sample loading buffer (Laemmli,
1970). Cell suspensions were briefly sonicated and boiled for
three minutes. 10 to 40 µl of each cell lysate sample, with the exception
of cells transfected with pC10, were subjected to electrophoresis on a 10% SDS
PAGE gel. pC10-transfected cells were analyzed on a 15% SDS-PAGE gel. After
electrophoresis, the proteins were transferred electrophoretically to
nitrocellulose for 1 hour at 100 V in 25 mM Tris base, 192 mM glycine, 0.1%
SDS and 20% methanol. Blots were then immersed in 5% dry milk in PBS, 0.05%
Tween 20 and 1% BSA (blocking solution) for a minimum of 30 minutes. The
membranes were then incubated with monoclonal antibodies (mAbs) against GFP
(Covance, Princeton, New Jersey) and myc (9E10) at a dilution of 1:10,000 and
undiluted, respectively. Following primary antibody incubations, the COS cells
were reacted with horseradish peroxidase (HRP)-conjugated goat
affinity-purified anti-mouse antibody (Promega, Madison, WI). COS cells
transfected with MyBP-H constructs were reacted with a 1:1000 dilution of
purified anti-MyBP-H polyclonal antibody
(Gilbert et al., 1998
)
followed by horseradish peroxidase (HRP)-conjugated affinity-purified goat
anti-rabbit antibody (Promega, Madison, WI). The antibody complexes were then
visualized by enhanced chemiluminescence (Amersham Pharmacia Biotech, Inc.,
Piscataway, NJ).
Indirect immunofluorescent labeling
COS cells cultured on coverslips and myoblasts cultured on chamber slides
were both processed at room temperature. COS cells were first washed twice
with PBS and then fixed with 2% formaldehyde in PBS. The cells were
permeabilized with 1% triton in PBS for 10 minutes and blocked with 1% BSA,
0.05% Tween 20 in PBS for 30 minutes. For both primary and secondary antibody
incubations, all dilutions were made in blocking buffer. Cells were first
probed with a 1:10 dilution of F59, a mAb specific for sarcomeric MyHC (gift
of Frank Stockdale, Stanford University) (Miller et al., 1985) for 1 hour.
After the F59 incubation, the cells were incubated for 30 minutes with
Alexa-594-conjugated goat affinity-purified anti-mouse antibody (Molecular
Probes, Eugene, OR) at a dilution of 1:200. The cells were then reacted with
biotinylated conjugated 9E10 (Covance, Princeton, NJ) at a dilution of 1:10.
Myc-tagged MyBPs were then detected using Alexa 488 strepavidin (Molecular
Probes, Eugene, OR) at a dilution of 1:200. Coverslips were mounted in Airvol
(Air Products, Allentown PA) with 100 mg/ml 1,4-diazobicyclo (2.2.2)-octane
(Sigma, St. Louis, MO) to reduce photobleaching. Cultured myoblasts were
processed in a similar manner but probed only with F59 and
Alexa-594-conjugated goat, affinity-purified anti-mouse antibody. The cells
were examined with a Nikon epifluorescence microscope using a 100x
objective. Images were digitally captured using the Sony DKC-5000 digital
camera system (Sony Corporation, Japan) using P2 system Software (P2 System,
Budapest, Hungary).
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Results |
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|
In an earlier publication, we showed that myc-tagged MyBP-C, MyBP-H and
C7-10 proteins encoded by expression plasmids could incorporate into the
A-bands of myofibrils in cultured, differentiating myotubes
(Gilbert et al., 1998). To
test whether the GFP-tagged versions of these recombinant proteins were
competent to interact properly with thick filaments, transient transfections
of 11-day-old embryonic chick myoblasts were performed. Expression of GFP/C,
GFP/C7C10 or GFP/H resulted in the clear localization of the expressed protein
to the A-band (Fig. 3B,E,F,
respectively). When muscle cells transfected with GFP/C were probed with F59,
a mAb specific for sarcomeric myosin, we observed that distribution of the
GFP/C was similar to the distribution of myosin
(Fig. 3C). This co-distribution
is obvious when the overlapping staining patterns of both proteins were
examined (Fig. 3D). The
colocalization of GFP/C7-10 and GFP/H and myosin also was observed when these
transfected muscle cells were stained with mAb F59 (data not shown). These
data demonstrate that the GFP moiety did not interfere with the ability of
these recombinant proteins to localize properly to the A-band.
|
Intracellular distribution of expressed sarcomeric proteins
Sarcomeric MyHCs, when expressed in COS cells, form clusters of
spindle-shaped filament aggregates throughout the cytoplasm of the transfected
cells (Moncman et al., 1993;
Rindt et al., 1993
;
Straceski et al., 1994
;
Seiler et al., 1996
;
Vikstrom et al., 1992
). By
contrast, GFP/C, GFP/C7-10 and GFP/H, when expressed in COS cells, were
distributed diffusely throughout the cytoplasm
(Fig. 4A-C). We occasionally
observed an apparent nuclear localization of the transiently expressed MyBPs,
(Fig. 4A-C), but we did not
test whether this signal was coming from within or around the nuclei. When
co-expressed with MyHC, MyBP-C and MyBP-H had a striking effect on the
organization of MyHC (Seiler et al.,
1996
). In the presence of the MyBPs, the MyHC formed long
peri-nuclear cables rather than the spindle-like filament aggregates observed
with MyHC alone. Double immunostaining revealed that MyBP-C or MyBP-H
colocalized with the MyHC in these cables. Transfection of GFP/C with MyHC
also resulted in co-polymer cable formation of GFP/C
(Fig. 4D) and MyHC
(Fig. 4G). The overlap of the
GFP/C and MyHC was not complete (Fig.
4J); although the MyBP-C co-distributed with the MyHC, some MyHC
remained in spindle form, and these spindle aggregates did not contain MyBP-C.
We interpret this as reflecting a sub-stochiometric concentration of GFP/C
relative to myosin. Since co-distribution of GFP/C and MyHC was observed only
in the cables, we conclude that MyBP-C is required for cable formation.
|
The same phenomenon was observed when GFP/H was co-expressed with MyHC. Both GFP/H (Fig. 4F) and MyHC (Fig. 4I) organized into cables. The overlapping distribution of GFP/H and MyHC indicates that the both proteins can effectively colocalize. Although, the majority of GFP/H was found in the peri-nuclear cables (Fig. 4L) some myosin was detected in spindle-like aggregates lacking GFP/H. GFP/C7-10 was also an efficient promoter of cable formation (Fig. 4H). Often cells were found in which GFP/C7-10 colocalized with MyHC into co-polymer cables (Fig. 3E). However, we often observed self-aggregation of the GFP/C7-10 peptide in COS cells (Fig. 4E). This phenomenon was not observed in differentiating muscle (data not shown). In view of the fact that there were such high levels of GFP/C7C10 expression in the COS cells, some co-distribution of the two proteins may have been obscured by the intense GFP fluorescence in the transfected cells. These experiments demonstrate that GFP-tagged MyBPs are competent to induce co-polymer cable formation with MyHC. They also show that cable-forming activity is within the C-terminal four domains of MyBP-C.
Description of myc-tagged MyBP-C and MyBP-H truncation mutants
The cloning of myc-tagged MyBP-C and MyBP-H mutants has been described in
an earlier publication (Gilbert et al.,
1998; Gilbert et al.,
1996
). Four of those plasmids expressing myctagged MyBP-C were
reused in the present study (Fig.
1B): full-length MyBP-C; C7-10, a C-terminal fragment containing
the A-band localization region; C10 (previously termed
1-9), encoding
the C-terminal IgI-domain-containing the major myosin rod-binding domain; and
C1-9 (previously termed
10), a MyBP-C fragment lacking the C-terminal
IgI domain. Four myc-tagged MyBP-H constructs were also tested for myosin
binding and cable formation in COS cells
(Fig. 1C): full-length MyBP-H;
H
U, a MyBP-H peptide lacking the unique N-terminal region; H
U1,
which lacks the both the unique region and the first FnIII domain; and
H
4, a recombinant molecule missing the C-terminal myosin rodbinding IgI
domain. Our earlier studies showed that truncation mutants of the MyBPs that
lack the C-terminal IgI domain, for example, C1-9 and H
4, lose the
ability to localize in the A-bands of transfected embryonic myotubes. Before
studying the cellular distribution of myc-tagged MyBPs mutants in COS cells,
we tested the stability of these proteins using western blots
(Fig. 5). Whole cell lysates
from COS cells transfected with MyBP-C, C7-10, C10 and C1-9 were prepared two
days after transfection, western blotted and probed with an anti-myc mAb. All
constructs generated proteins that reacted with the myc antibody and
eletrophoresed at the expected relative mobilities
(Fig. 5A). Likewise,
transfection of the four MyBP-H constructs produced immunoreactive products
with the myc mAb. Of note, only H
U and H
U1 ran with motilities
consistent with their molecular mass of 47 and 35 kDa. MyBP-H and H
4,
with predicted molecular masses of 60 and 50 kDa, contain a unique proline and
alanine leader region, and these ran slower than their expected masses. In
general, MyBP-C constructs produced less myc-tagged protein in transfected COS
cells than did the MyBP-H constructs. These studies demonstrated that
myc-tagged full-length MyBPs and truncation mutants can be stably expressed in
COS cells.
|
We next tested the antigenicity of the MyBP-H mutants with a previously
described MyBP-H-specific pAb (Gilbert et
al., 1998) (Fig.
5C). This pAb reacted with a single band in lysates from COS cells
transfected with MyBP-H. This band had an identical relative mobility to
full-length MyBP-H (Fig. 5C)
and proved that recombinant myc-tagged MyBP-H, expressed in transient
transfections of COS cells, was immunoreactive with a pAB specific for chicken
skeletal MyBP-H. Also showing immunoreactivity were H
U and H
4
lysates. COS cells transfected with H
U1, although immunoreactive with
the anti-myc pAb (Fig. 5B), did
not react with the MyBP-H pAb (Fig.
5C). The positive reactivity of H
U and the absence of
reactivity with H
U1 indicate that the epitope for this polyclonal
antibody resides in domain H1.
Domain C10 contains the cable formation activity of MyBP-C
Of the MyBP-C recombinant fragments tested, only full-length MyBP-C and
C7-10 targeted to the A-bands of transfected, differentiating myotubes
(Gilbert et al., 1996). C10
and C1-9 did not localize at all to the A-bands. Biochemical studies, however,
have shown that the C-terminal 14 kDa fragment of MyBP-C (C10) contains a
LMM-binding domain (Okagaki et al.,
1993
). Furthermore, a second myosin-binding domain within the
C1-C2 region of cardiac MyBP-C has been identified
(Gruen and Gautel, 1999
). To
delineate the functional domain responsible for cable formation, we
transfected COS cells with full-length MyHC in combination with MyBP-C, C7-10,
C10 or C1-9. The cells were then fixed and doubly immunostained with mAbs
against sarcomeric myosin and myc (Fig.
6). When expressed individually, all of the MyBP-C recombinant
proteins distributed diffusely throughout the cytoplasm, with occasional
nuclear staining (Fig. 6A-D).
When co-transfected with MyHC, myc-tagged MyBP-C and C7-10 induced co-polymer
cable formation in a manner identical to their GFP-tagged counterparts
(Fig. 6E,I,M and 6F,J,N). Transfection with only the last domain of MyBP-C (C10) with MyHC also promoted
cable formation (Fig. 6G,K,O).
These results demonstrate that although the C10 domain could not localize to
the A-band of cultured myotubes, it could bundle sarcomeric myosin in this
non-muscle cell system.
|
To determine whether C10 was necessary for cable formation, we performed a complementary experiment co-transfecting a construct lacking the C-terminal IgI domain, C1-9, with MyHC. This truncation mutant did not induce cable formation (Fig. 6H,L) but did colocalize with the spindleshaped clusters of MyHC (Fig. 6P). These data suggest the presence of other myosin-binding sites within the skeletal MyBP-C molecule besides those in C10. Our MyBP-C transfection data are presented graphically in Fig. 8A. The percentage of doubly transfected cells, demonstrating a colocalization phenotype of recombinant MyBP-C protein with MyHC exceeded 90% for each of the expression constructs. MyBP-C and C7-10 were the most effective promoters of cable formation, with an efficiency of 78 and 86%. Cables also formed in 57% of the transfected cells expressing C10 and MyHC. By contrast, only 5% of the cells co-transfected with C1-9 and MyHC formed cable-like structures. These data demonstrate that the C-terminal IgI domain of MyBP-C is both necessary and sufficient for inducing MyHC cable formation in COS cells.
|
Domain 4 of MyBP-H is required for co-polymer cable formation with
MyHC
The amino-acid sequences of the four C-terminal domains of MyBP-C and
MyBP-H have 49% identity, with additional 23% conservative substitutions. Both
proteins showed similar cable formation when co-transfected into COS cells
(Fig. 7). Single transfections
of MyBP-H, HU, H
U1 and H
4 resulted in diffuse expression
of the expressed proteins (Fig.
7A-D). When myc-tagged MyBP-H was co-expressed with MyHC, it
behaved identically to GFP/H, forming large cables
(Fig. 7E,I,M). H
U, a
MyBP-H analogous to fragment C7-10, promoted cable formation
(Fig. 7F,J,N) with 80%
efficiency, the same as full-length MyBP-H
(Fig. 8B). These results
demonstrate that the unique N-terminal region of MyBP-H does not contribute to
the cable-forming properties of MyBP-H when co-expressed with MyHC. When the
unique region and first FnIII domain were deleted (H
U1), the MyBP-H
truncation mutant still formed cables with MyHC
(Fig. 7G,K,O) but with a 22%
lower efficiency than full-length MyBP-H or H
U
(Fig. 8B). Finally, we tested
the ability of construct H
4 to form cables. Similar to C1-9, H
4
lacks the C-terminal myosin rod-binding region of MyBP-H. When co-transfected
with MyHC, the expressed protein colocalized with spindle structures
(Fig. 7H,L,P) in 91% of the
doubly transfected cells (Fig.
8B). Cable-like structures, however, were rarely observed; only 6%
of the cells weakly manifested this phenotype. This demonstrated that domain
H4 of MyBP-H is necessary for copolymer formation. Since construct H
U1
co-distributed with MyHC in spindle-like structures, domains H1-3 must contain
some myosin recognition sequences that are incapable of crosslinking MyHC into
bundles.
|
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Discussion |
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|
In a previous study, we proposed a space-filling model for the interaction
of C10 with the myosin rod (Miyamoto et
al., 1999). A hypothetical structure of C10, assumed to be a
ß-barrel (Okagaki et al.,
1993
), was generated on the basis of the cystallographic
coordinates of telokin, an homologous sequence within the C-terminal region of
smooth muscle myosin light chain kinase
(Holden et al., 1992
). To
determine which amino acids on C10 interact with LMM, we generated charge
reversal mutations in positively or negatively charged residues on the surface
of this domain and assayed effects on binding to or polymerization of light
meromyosin (LMM). Amino-acid residues that positively or negatively affected
binding, but did not alter C10 structure as determined by circular
dichroism measurements were hypothesized to form ionic interactions
with the LMM polymer at physiological ionic strength. By examining the
distribution of surface charges along the coiled-coil of the myosin rod, we
were able to fit C10 into a surface groove of the thick filament backbone
structure put forth by Chew and Squire, in both parallel and anti-parallel
orientations (Chew and Squire,
1995
). We concluded that three quarters of the C10 surface
interacted with myosin; a single molecule of C10 could potentially crosslink
three or four molecules of the myosin rod, thus explaining the well known
reduction in critical concentration for myosin polymerization caused by MyBP-C
(Davis, 1988
). This model of
C10 interacting with multiple myosin molecules is consistent with the
transfection data presented in the present report. In order for myosin
filaments to form higher ordered structures, for example, the cables observed
in our co-transfection experiments, single molecules of C10 must be capable of
interacting with at least two or more myosin rods. In other words, the C10
domain must be capable of crosslinking parallel myosin rods, in effect
behaving as a multivalent ligand. This property seems to be unique to the
C-terminal IgI domains of MyBP-C or MyBP-H as recombinant MyBPs lacking these
domains do not form cables in COS cells co-transfected with MyHC. The precise
location of MyBP-C in the vertebrate thick filament is still uncertain, but
clearly reactive epitopes must reside in an accessible location on the
filament since antibodies to MyBP-C strongly decorate the A-band
filaments.
Although capable of interacting with myosin in the COS cell system, C10
does not insert into the A-band when overexpressed in developing muscle cells
(Gilbert et al., 1996). This
domain of MyBP-C appears to have a lower affinity for myosin than full-length
MyBP-C does: C10 is incapable of displacing full-length MyBP-C from myosin
filaments (D.A.F., unpublished). We suggest that in the absence of full-length
MyBP-C, C10 is capable of binding to myosin filaments and crosslinking them
into bundles, but in native myofibrils this domain cannot displace endogenous
MyBP-C, thus explaining its inability to target to the A-band in developing
muscle (Gilbert et al.,
1996
).
Under suitable salt conditions, full-length myosin polymerizes in vitro to
form bipolar structures that resemble native thick filaments, but even with
these impressive self-assembly characteristics, the in vitro filaments exhibit
wide variations in length, rarely matching the in vivo situation
(Barral and Epstein, 1999;
Harrington and Joseph, 1968
;
Huxley, 1963
;
Lowey et al., 1969
;
Philpott and Szent-Gyorgyi,
1954
). LMM also aggregates in vitro at physiological ionic
strength, but its polymers are neither filamentous nor typically bipolar
(Atkinson and Stewart, 1991
).
It is likely that other components of the cell (e.g., microtubules and
intermediate filaments) and of the thick filament (e.g., titin, myomesin and
the MyBPs) play important but poorly understood roles in this process. In
differentiating chick skeletal muscle, the spatial and temporal expression of
MyBP-C coincides with the emergence of myofibrillar cross-striations
(Lin et al., 1994
).
Furthermore, C-terminal truncations of MyBP-C that lack C10 cause a dominant
interference with myofibril assembly when overexpressed in cultured myotubes
(Gilbert et al., 1996
). These
observations are consistent with the notion that MyBP-C may function as a
rate-limiting molecule in the lateral registration of thick-filaments during
A-band assembly. The impact of the MyBPs on myosin organization is readily
apparent in the prominent reorganization of MyHC spindles to long peri-nuclear
cables, which result from the co-expression of MyBP-C or MyBP-H with MyHC. A
number of publications have demonstrated that MyBP-C has a significant effect
on the length of myosin polymers formed in vitro
(Davis, 1988
;
Miyahara and Noda, 1980
). In
addition to its effects on lateral thick filament alignment, MyBP-C could
function in both the organization and stability of already formed thick
filaments.
In conclusion we have defined the essential domains of MyBP-C and MyBP-H responsible for intracellular myosin crosslinking. Our findings suggest the presence of a third myosin interaction domain shared by MyBP-C and MyBP-H. It remains to be seen how this region of the MyBPs functions within developing and mature striated muscle. Although, MyBP-H shares many structural and functional similarities with MyBP-C, especially in the C-terminal one-third of the latter molecule, MyBP-H lacks all of the putative regulatory residues found upstream in MyBP-C. Further studies on MyBP-H are clearly warranted, since its function in vivo is unknown.
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Acknowledgments |
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