1 Department of Neurology, Baylor College of Medicine, Houston, TX 77030,
USA
2 The Verna and Marrs McLean Department of Biochemistry and Molecular Biology,
Baylor College of Medicine, Houston, TX 77030, USA
* Present address: Max-Planck Institute for Biochemistry, D82152 Martinsried,
Germany
Author for correspondence (e-mail:
hepstein{at}bcm.tmc.edu)
Accepted 20 August 2002
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Summary |
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Key words: UNC-45, Muscle differentiation, Proliferation, Chaperone, Myosin
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Introduction |
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Myosin heads irreversibly aggregate in bacterial expression systems and do
not show motor function, suggesting that necessary factors are missing
[(McNally et al., 1988); see
(Srikakulam and Winkelmann,
1999
) for discussion]. The heads of muscle and non-muscle myosin
differ in their ability to fold properly upon expression in non-muscle
(insect) cells. Heads of cytoskeletal and smooth muscle myosins II, V and VI
expressed as subfragment 1 or heavy meromyosin regions are soluble, functional
proteins (Sweeney et al.,
1998
; Wang et al.,
2000
; Wells et al.,
1999
), however cardiac (sarcomeric) myosin heads are not (J. R.
Sellers and H. L. Sweeney, personal communication). These results suggest that
non-muscle eukaryotic cells contain factors necessary for folding the
non-muscle myosin motors (see Hutagalung
et al., 2002
). Previous research suggests that molecular
chaperones assist the folding of muscle myosin heads. The chaperonin
containing TCP-1 (CCT, where TCP-1 is t complex polypeptide 1)
(Kubota et al., 1995
)
associates with and enhances the folding of nascent skeletal muscle heavy
meromyosin in reticulocyte lysates
(Srikakulam and Winkelmann,
1999
). In addition, cultured muscle (C2C12) cells but not
epithelial cells contain factors that permit the proper folding of recombinant
muscle myosin subfragment 1 (Chow et al.,
2002
). The C. elegans protein UNC-45 has been shown by
genetic experiments to be necessary for thick filament assembly
(Epstein and Thomson, 1974
;
Venolia and Waterston, 1990
)
and by biochemical experiments to be a molecular chaperone with activity for
the myosin head (Barral et al.,
2002
).
C. elegans UNC-45 protein has an apparent molecular mass of 107
kDa. It contains three amino-terminal tetratricopeptide
repeats (TPR), a 400 residue central region and a
400
residue UNC-45/Cro1/She4p (UCS) domain
(Barral et al., 1998
;
Venolia et al., 1999
). The
UNC-45 TPR domain binds the molecular chaperone Hsp90 in a stoichiometric
manner (Barral et al., 2002
).
The remainder of the UNC-45 protein binds the myosin head and has chaperone
activity on it (Barral et al.,
2002
). A temperature-sensitive mutation (unc-45 e286) in
the UCS domain of UNC-45 reduced myosin accumulation and led to disordered
assembly of the two myosin isoforms in body-wall muscle thick filaments
(Barral et al., 1998
). The
resulting thick filaments were unstable upon isolation. UNC-45 protein was
localized to the A-bands of body-wall muscle by immunostaining, and to all
muscle types of C. elegans by a GFP-reporter
(Venolia et al., 1999
;
Ao and Pilgrim, 2000
). UNC-45
also has a role in non-muscle cells, as shown by its immunodetection in
cleavage furrows and its interaction with non-muscle myosin II and
unconventional myosin V in two hybrid studies (W. Ao and D. Pilgrim, personal
communication).
Yeast and other fungal proteins containing an UCS-domain were identified in
several distinct mutant screens and show functional linkage to the
cytoskeleton. CRO1 of Podospora anserina is necessary for multiple
processes in cell division
(Berteaux-Lecellier et al.,
1998). She4p of S. cerevisiae is required for a normal
actin-based cytoskeleton, endocytosis, and a myosin-V-based molecular
transport process (Jansen et al.,
1996
; Wendland et al.,
1996
; Beach and Bloom,
2001
). A more recently identified UCS-containing protein, S.
pombe Rng3p, is crucial for cell shape, normal actin cytoskeleton, and
contractile ring assembly (Balasubramanian
et al., 1998
). It is essential for assembly of the myosin
II-containing progenitors of the contractile ring
(Wong et al., 2002
).
Widespread defects in the cytoskeleton are found in null mutants of all three
fungal proteins.
The present investigation was aimed at identifying mammalian UNC-45 gene products and gaining insight into their functions. While there is a single C. elegans unc-45 gene, we found that humans and mice express two UNC-45-like gene products, one being present in all organs and the other highly expressed only in striated muscle. The expression of the general cell isoform decreased while the striated muscle isoform increased during muscle differentiation in vitro. Antisense experiments with the murine UNC-45 isoforms in the skeletal myogenic cell line C2C12 suggest that they have distinct roles.
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Materials and Methods |
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Radiation hybrid chromosomal mapping of the human general cell UNC-45
locus
PCR with total human genomic DNA and primers 5'-CCA AGG CTC ATG CAC
ACG CTA CCT ATT GTG G-3' and 5'-TGA TAG CAG GCT CAC AGC TGG TGA
GGC TGC-3' amplified only the 325 bp of the 3' UTR of human
general cell UNC-45. This primer pair was utilized at Research Genetics
(Huntsville, AL) for dual mapping on the Stanford and G3 human/hamster hybrid
panels (Cox et al., 1990).
Northern blotting
Total RNA was isolated from mouse tissues or C2C12 myogenic cells following
Chomcznski and Sacci (Chomcznski and Sacci, 1987) or with TRIzol (Life
Technologies, Rockville, MD), or was purchased (ovary, whole embryo) (Ambion,
Austin, TX). Methods for the RNA gels and northern blotting were modified from
Sambrook et al. (Sambrook et al.,
1989). RNA molecular weight standards ranging from 0.24 to 9.46 kb
(Life Technologies) were used to generate a linear plot of migration distance.
Ten µg of each sample was separated on a 1.5% agarose-formaldehyde gel.
Blots were pre-hybridized and hybridized in UltraHyb solution (Ambion) at
65°C. Two different general cell UNC-45 probes were used, one was 955 bp
of cDNA from the UCS region, the product of primer set 5'-CTC GGC ATT
GGT CAA TTG CAC CAA CAG C-3' and 5'-GGA TCT CCA GGA CCT CAC TCT
CCA TCA GGG-3', while the other was 550 bp, half of which is 3'
UTR and was the product of primer set 5'-CTC ACC TCC ATG CGG CCA
CAC-3' and 5'-GAT GCT CCC AGC ATG TGA GGA TGC-3'. The
striated muscle UNC-45 probe was 755 bp of cDNA, 350 of which was 3'
UTR, and was the product of primers 5'-TAC GGC AGG CAG CCA CCG AAT GCA
TGT G-3' and 5'-CAG TCT ACA GCC CGT TAT CTG GCC TGC-3'.
Twenty-five ng of each probe was labeled with [32P]-dCTP (Amersham,
Piscataway, NJ) by the random nonomer method to specific activities of
5x108-1x109 cpm/mg, and used at
1x106 cpm/ml. Blots were washed at room temperature with
2x sodium citrate/sodium chloride [SSC (see
Sambrook et al., 1989
)], 0.1%
sodium dodecyl sulfate (SDS) for 2 minutes, and then at 63°C with three 15
minutes washes with 0.1x SSC, 0.1% SDS and 1 wash with 0.1x SSC,
0.5% SDS. Internal standards were obtained by re-labeling with a probe
specific for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Ambion) or, in
the case of multiple tissues constitutively expressing varying levels of GAPDH
mRNA, with an 18 S RNA probe. Blots were exposed to Kodak BioMax MS film with
BioMax intensifying screens for 3 days (general cell UNC-45), 11 hours
(striated muscle UNC-45) or 2 hours (18S RNA and GAPDH) at -80°C. Films
were digitized by scanning, and Photoshop (Abobe Systems, Mountain View, CA)
was used for sizing and labeling. Absolute and relative amounts of general
cell and striated muscle UNC-45 mRNA were obtained from films with UN-SCAN-IT
software (version 5.1, Silk Scientific, Orem, UT) providing total pixels per
band, and resulting data were analyzed using the Excel program (Microsoft,
Redmond, WA).
In situ hybridization
The mouse general cell and striated muscle UNC-45 cDNA templates were
identical to those used for northern blotting. Single-stranded sense and
anti-sense digoxygenin-labeled RNA probes were generated as directed by the
manufacturer (Roche Molecular Biochemicals, Indianapolis, IN). Whole-mount in
situ hybridization was performed on mouse embryos of different ages as
described in Conlon and Rossant (Conlon
and Rossant, 1992). Labeled embryos were placed on a layer of
agarose and photographed through a dissecting microscope with Kodak Ektachrome
160 T slide film. Slides were digitized via a Sprint Scan scanner (Polaroid,
Wayland, MA) and images were adjusted for scaling and size with the Photoshop
program.
C2C12 mouse skeletal myogenic cell culture
Mouse C2C12 skeletal muscle cells were obtained from the American Type
Culture Collection (ATCC CRL-1772) (Yaffe
and Saxel, 1977; Blau et al.,
1983
; Silberstein et al.,
1986
). Cells were expanded and passaged 3 times, and frozen
aliquots were used for antisense experiments. Cells were propagated in a
humidified incubator at 10% CO2 in growth medium consisting of
Dulbecco's Minimal Essential Medium containing 4.5 mg glucose/L, 110 mg sodium
pyruvate/L and supplemented with 10% fetal bovine serum (FBS), 0.05 mg/ml
gentamicin and a 1:400 dilution of Fungizone (all components from Invitrogen,
Carlsbad, CA). Differentiation was induced by medium containing 2% horse serum
instead of 10% FBS. Phase-contrast images were taken with a 10x lens on
Kodak TriX Pan film at ASA 400. Negative film images were digitized by
scanning and imported into Photoshop for contrast adjustment and cropping.
Antisense oligonucleotide experiments
Phosphorothioate 21-mer oligonucleotides (Sigma-Genosys, Woodlands, TX)
designed to anneal at the start codon were used for the suppression of the
mouse general cell UNC-45 (5'-GCC ACT CAC AGT CAT CAC GAA-3') and
striated muscle UNC-45 (5'-TTC AGC CTC TGC CAT AGT CTT-3'). The
control oligonucleotide had a base composition similar to the UNC-45
oligonucleotides but randomized (avoiding tandem repeats) (5'-TAA GCA
CTA GGA CAC CTC CAC-3'). Second-generation chimeric 18-mer
oligonucleotides (trademark of Oligos Etc. Inc, Wilsonville, OR) were also
used: general cell UNC-45 antisense: 5'-CGC ATT TGA ACA GCT
CGT-3', a control oligonucleotide that reversed the order of the
previous bases (5'-TGC TCG ACA AGT TTA CGC-3'), and striated
muscle UNC-45 antisense: 5'CCA TGA GGC TGC AGA TTC-3'.
Oligonucleotides were resuspended in 10 mM Tris, pH 8.5 and lyophilized in
aliquots. They were added daily, starting 1 hour after plating, to between
1.25 and 5 µM. Proliferating cells were treated for up to 5 days, while
myotube-forming cells were treated for up to 9 days. Cell number was assessed
by three methods: counting adherent cells in defined areas, counting
trypsinized cells resuspended in trypan blue stain, and by the CyQUANT
proliferation assay (Molecular Probes, Eugene, OR). For the latter, 96 well
culture plates were seeded with 2,000 cells/well. Eight wells or triplicates
of eight wells were used for each 3-day treatment. Frozen cells were lysed
with the kit's lysis buffer supplemented with NaCl to 180 mM and EDTA to 1 mM,
and 2 Kunitz units of RNase A/ml. After 1 hour at room temperature, one volume
of GR dye solution was added to a final concentration of 2x dye, and the
fluorescence resulting from GR dye binding to DNA
(Jones et al., 2001) was read
on a CytoFluor plate reader at 485 nm excitation and 530 nm emission, with 3
reads per scan and a gain of 50. A standard curve showing a linear
relationship was obtained using 2, 4, 8, 12, and 16x103 C2C12
cells. Data was tabulated and averages±standard deviations were
calculated for the general cell or striated muscle antisense data separately
paired with the control data using the Excel program. The Student's
t-test was applied using the two-tailed, equal variance
parameters.
Immunofluorescence microscopy
Cells were grown on Aclar coverslips (SPI Supplies, West Chester, PA)
coated with 25 µg/ml of mouse basement membrane laminin (Sigma, St Louis,
MO). Coverslips were washed twice briefly with DMEM minus serum, and fixed
with 100% methanol at -20°C for 30 minutes. Coverslips were air-dried and
stored under desiccating conditions at -80°C. Cells were hydrated and
further permeabilized in phosphate-buffered saline (0.14 M NaCl, 2.5 mM KCl, 8
mM Na2HPO4, 1.8 mM KH2PO4, pH 7.4)
with 0.05% Tween 20 (Sigma) (PBST), blocked with 5% normal goat serum in PBST,
and stained with antibodies diluted into PBST for 1 hour at room temperature.
Primary antibodies were mouse monoclonal IgG clone EA53 (Sigma) against
sarcomeric -actinin, diluted 1:400 and supernatant of mouse monoclonal
IgG2b MF20 directed against fast skeletal muscle, diluted 1:8 [Developmental
Studies Hybridoma Bank (Bader et al.,
1982
)]. Alexa 488- or Alexa 594-labeled secondary antibodies
(Molecular Probes, Eugene, OR) were used at 1:500 dilutions. DNA was labeled
with 0.1 µg/ml 4'-6-diamidino-2-phenylindole (DAPI; Sigma).
Coverslips were rinsed with glass-distilled water and mounted with Fluoromount
G (Southern Biotechnology Associates, Birmingham, AL). Microscopy was done
using an Olympus BX 60 (Olympus America, Melville, NY) or a Zeiss Axioplan 2
(Carl Zeiss, Thornwood, NY) epifluorescence microscope. Fluorochrome emission
was examined individually, photographed on Fujichrome Provia 1600 slide film
and developed with E-6 push processing prior to digitization and importation
into Photoshop. Digital images as jpeg or Zeiss vision files were taken from
the Axioplan microscope using Zeiss Axiovision software.
Immunoblotting
C2C12 myotubes cultures treated for 8 days plus or minus 2.5 µM
antisense oligonucleotides were rinsed several times with Hank's balanced salt
solution. Cells within one well of a six-cell plate were lysed with 0.2 ml of
150 mM NaCl, 20 mM Tris pH 7.4, 2 mM EDTA, 2 mM adenosine triphosphate, 5 mM
dithiothreitol, 1% Triton X-100, and Complete protease inhibitor cocktail
(Boehringer Mannheim, Indianapolis, IN) to 5x concentration. SDS was
then added to 1%. Proteins separated on 7.5% polyacrylamide gels were
transferred to Immobilon-NC filters in 20% methanol in Laemmli running buffer,
at 80 volts for 15 hours in the cold. Use of Kaleidoscope molecular weight
markers (BioRad, Richmond, CA) and subsequent Commassie Blue-staining of the
gels confirmed transfer. Blots were blocked with 1% nonfat dry milk in 150 mM
NaCl, 50 mM Tris pH 7.6, 0.05% Tween-20, reacted with 2 µg/ml MF20
monoclonal anti-skeletal muscle myosin antibody
(Bader et al., 1982), and
counter-stained with 1:1000 dilution of horse radish peroxidase-labeled
anti-mouse IgG. SuperSignal enhanced chemiluminescence (Pierce, Rockford, IL)
and exposure to X-ray film detected labeling.
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Results |
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Like C. elegans UNC-45, the predicted sequences for the mammalian proteins consist of three distinct regions: an amino-terminal triple TPR motif, a unique central region, and a C-terminal UCS domain. Although the two mammalian UNC-45 isoform proteins predicted from full-length cDNA sequences are each 31-32% identical and 53-54% similar to C. elegans UNC-45, they are quite different from one another. Overall, the mammalian GC and SM UNC-45 isoforms are only 55-56% identical and 74% similar in amino acid sequence. The most similar isoforms are 94-95% identical and 96-98% similar between mouse and human. Blocks of up to 10 consecutive identical and up to 25 conserved amino acids are present throughout the isoform sequences.
As indicated in Fig. 1,
residues associated with dysfunctional mutations in C. elegans unc-45
(red asterisks: in order, with residue number referring to that protein,
unc-45 b131 (G427E), su2002 (L559S), m94/r450
(E781K) and e286 (L822F) or S. pombe rng3 (blue asterisks:
rng3-A3 (L483P) and rng3-65 (G688E) are identical or
conserved in the mammalian UNC-45 proteins
(Barral et al., 1998;
Wong et al., 2000
). The
phenylalanyl and lysyl residues identified at the chymotrypsin and the trypsin
cleavage sites, respectively, in C. elegans UNC-45 (J.M.B. and
H.F.E., unpublished) are also closely conserved in the predicted mammalian
proteins (indicated by blue and red arrows in
Fig. 1). Five specific residues
at the equivalent positions for interacting with and forming the C-terminal
aspartyl di-carboxylate clamp with Hsp90
(Russell et al., 1999
;
Scheufler et al., 2000
) are
identical or conserved (arginine replacing lysine) in the mammalian UNC-45 TPR
domains.
The GC and UNC-45 isoforms are predicted to have molecular weights of
103.450 and 103.41x103. Structural prediction programs
indicate that the pI of the GC UNC-45 protein would be 6.0, close to that of
the C. elegans protein. The SM UNC-45 protein is predicted to have a
more basic pI of 8.2. The GC and SM UNC-45 proteins are each predicted to
consist of about 3:1 -helical: random coil content with negligible
amounts of beta sheet [(Rost,
1996
)
www.embl-heidelberg.de/predictprotein].
The homologous human and mouse cDNAs for the ubiquitously expressed general cell UNC-45 (see below) that we cloned are represented in the GenBank database as human SMAP1 (direct submission, acc. no. BAB20273.1) and a human colon adenocarcinoma cDNA (direct submission, acc. no. AAH06214) and as a mouse cDNA from an induced mammary tumor cDNA (direct submission, acc. no. AAH04717). There is no full-length cDNA in the GenBank database for either the homologous human or mouse striated muscle UNC-45 isoform; (acc. nos. requested). The human SM UNC-45 gene product predicted in the NCBI database (acc. no. XP_091530; gene LOC146862, encoded by acc. no. XM_091530.1) is incorrect. It contains three extra exons encoding 122 additional residues when compared to the SM UNC-45 protein deduced from multiple PCR products.
Radiation hybrid mapping placed the human general cell gene locus on chromosome 15q25-26, correlating well with the human genome project placement of the gene at that location between 96,331,340-96,352,778 bp. The predicted mRNA XM_038413 and protein XP_038413 are in agreement with our cloned cDNA and the appropriate BAC sequences. No human disease loci have yet been mapped near this region.
The mouse GC UNC-45 gene mapped to chromosome 7q14-q21.3 at locus 39 (954788 in Mouse Genome Database, chromosome 7D1 in Map Viewer), as determined by the site of an EST (acc. no. AW538196) that encodes the carboxyl 96 residues plus 330 bases of 3' UTR. The loci of the human and mouse GC unc-45 are syntenic. The mouse coding sequence is within acc. no. XM_124930, on NCBI contig NW_000327.
The human SM UNC-45 gene is entirely contained in a bacterial artificial chromosome (acc. no. AC022916) and mapped to chromosome 17q11, between 33,872 and 33,834 Kb. The SM UNC-45 gene spans 38 kb, and consists of 19 exons. By synteny, the mouse SM UNC-45 gene would be on mouse chromosome 11 at locus 47.5 cM, and is in fact included in a mouse chromosome 11 BAC (acc. no. AL603745). The mouse gene is similar in size to the human gene, 34-kb. No human disease loci have yet been mapped to the SM UNC-45 locus.
Both the human and mouse GC UNC-45 genes are over 10 kb smaller than the SM UNC-45 genes, due to longer introns since total exonic sequences are nearly identical in length. Both GC UNC-45 genes contain one exon more than the SM UNC-45 genes, which encodes the extreme amino terminus. The intron/exon boundaries of the respective mouse and human genes for an isoform are identical, although the introns are of different sizes. The exons of the GC and SM UNC-45 genes are also identical within a species (Fig. 1).
We assembled and aligned predicted UCS domains from the NCBI database (www.ncbi.nlm.nih.gov) (Fig. 2). The 17 full length UCS domains are from the species already mentioned and in addition from Neurospora crassa, Aspergillus fumigatus, a second genus of nematode, the mosquito Anopheles gambiae, Drosophila, the Danio rerio zebrafish, the pufferfish Fugu rubripies, and cow. Nearly complete UCS domains were assembled for the frogs Xenopus tropicalis and Xenopus laevis. Blocks of high identity clearly show the divergence of vertebrates from invertebrates. Only 15 residues of the approximately 400 UCS residues are identical in all species studied. The most conserved block includes the sequence LTNL. The fungal proteins have extra residues in five locations, causing gaps in the alignment. The invertebrate UCS domains have C-terminal extensions. The vertebrate UCS domains contain four extra residues corresponding to residues 640-643 and 657-660 in the SM and GC UNC-45 proteins (top right in Fig. 2). These additions and deletions may represent specializations in function.
|
The presence of two UNC-45 isoforms in the pufferfish gives rise to the
notion that the second UNC-45 gene arose sometime during the chordate
radiation [see accompanying Commentary
(Hutagalung et al., 2002)].
Pair-wise comparisons of identity showed that one of the Fugu UNC-45
isoforms is 72% identical and 84% similar to the human and mouse SM UNC-45.
The other Fugu UNC-45 isoform is 64% identical and 78% similar to the
human and mouse GC UNC-45 isoform.
The two murine UNC-45 isoform genes are differentially expressed in
adult tissues
The two murine UNC-45 mRNAs are differentially expressed in the adult. GC
UNC-45 mRNA was detected in uterus, large intestine, kidney, spleen, lung,
brain, liver and ovary using gene-specific labeling of duplicate northern
blots containing total RNA from various adult organs
(Fig. 3A). The mRNA was
relatively less abundant in cardiac and skeletal muscle than in the
non-striated muscle tissues. The GC UNC-45 mRNA was also found in whole 12 day
mouse embryos. The demonstration of GC UNC-45 mRNA in every tissue examined
here confirms the multi-organ expression pattern demonstrated by EST database
searches which showed other GC UNC-45 expressing cells or organs include skin,
bone marrow, T- cells, urinary bladder, mammary gland, optic nerve, various
parts of the eye, germ cells, testis, prostate, pancreas, parathyroid gland
and placenta. In addition, over a dozen tumors of various cell-types express
GC UNC-45, some to a higher than normal level as suggested by SAGE
(serial analysis of gene
expression) analysis [see UniGene Cluster Hs.26110 Homo sapiens in
the NCBI database
(www.ncbi.nlm.nih.gov)].
|
The SM UNC-45 mRNA is abundant in skeletal muscle and the heart, both of
which consist predominantly of striated muscle fibers
(Fig. 3B). The same size mRNA
was also present in whole embryo samples. However, SM UNC-45 mRNA was not
detected in uterus and large intestine, which are rich in smooth muscle cells
nor in non-muscle organs such as kidney, liver and ovary. The source of the
minor SM reaction in the lung sample is unknown. A TBLASTN query
(Altschul et al., 1997) of the
EST database reveals that the partial SM UNC-45 sequences are present in cDNA
libraries derived mostly from heart and segments of embryo containing the
developing heart, such as embryonic body between the diaphragm region and the
neck, as well as tissues containing skeletal muscle such as limbs and total
head tissue.
The two unc-45 genes are differentially expressed during
embryogenesis
To determine the location of early expression of the GC and SM UNC-45 RNA
species, in situ hybridization was performed on whole mouse embryos using
sense controls and anti-sense RNA probes. The SM UNC-45 gene was strongly
expressed in the heart at 8.75 days when it is already beating
(Fig. 4A), and was not
expressed in other organs above the background level seen in a sense control
embryo (Fig. 4B). The GC UNC-45
gene was expressed at high levels in all tissues of an 8-day embryo, the
earliest stage embryo obtainable, whereas by 9.75 days the most robust
expression was in regions of intense development such as the branchial arches
and the forelimb bud (data not shown).
|
GC UNC-45 and SM UNC-45 mRNAs are differentially expressed during
muscle differentiation in vitro
Having determined that the mRNAs for the two isoforms of mammalian UNC-45
are differentially expressed during development and in adult tissues, we
addressed when during muscle differentiation the striated muscle isoform is
expressed. To assess the relative expression of the two UNC-45 isoform mRNAs,
total RNA was isolated from C2C12 cells proliferating in growth medium, and
from three stages of muscle differentiation induced by changing confluent
cultures to differentiation medium, i.e. fusing myoblasts present at 2 days,
young myotubes present at 3.5 days, and older myotubes present after 6 days of
differentiation, some of which twitched
(Fig. 5A). Total RNA from mouse
skeletal muscle and uterus were used as respective positive controls for SM
UNC-45 and GC UNC-45 mRNA.
|
As demonstrated in Fig. 5B, only GC UNC-45 mRNA was expressed in the proliferating C2C12 myoblasts. GC UNC-45 mRNA expression was at the highest level in these cells relative to the differentiating cultures at any stage, as shown by the total pixel number/band normalized to the internal standard of GAPDH (Fig. 5C). SM UNC-45 mRNA was first expressed when aligned myogenic cells were actively fusing (Fig. 5B). This population of fusing myoblasts and the earliest, narrow diameter myotubes expressed GC UNC-45 mRNA at 73% of the level in proliferating cells. Young myotubes in the process of assembling and remodeling myofibrils had the highest relative expression of SM UNC-45, 1.5 times greater than that in fusing myoblasts. The level of GC UNC-45 expression continued to decline during myotube maturation, from 25% in young myotubes to 14% in older myotubes, relative to that in proliferating cells. SM UNC-45 mRNA expression decreased in older myotubes to about half of the maximum found in younger myotubes.
GC UNC-45 functions in cell proliferation
Antisense experiments were performed to test whether the GC and SM UNC-45
isoforms exhibited different functions. The differential tissue expression of
the two isoforms suggested that the GC UNC-45 may have a role in cytoskeletal
functions and the SM UNC-45 may have a more specialized role in sarcomere
assembly and function. Therefore the antisense treatments of C2C12 cells
focused on cell proliferation and muscle differentiation. For studies of the
effects of suppression of UNC-45 isoform mRNA in proliferation, C2C12 cells
were plated at low density and antisense oligonucleotides were added daily to
2.5 µM for three days. This concentration permitted only partial mRNA
suppression. However, higher concentrations of oligonucleotides had obvious
toxic effects, evidenced by cell death in control cultures.
The extent of suppression of UNC-45 mRNA expression by treatment with
antisense oligonucleotides was determined by northern blotting. Three days of
treatment of proliferating C2C12 cultures with 2.5 µm GC UNC-45 antisense
oligonucleotides reduced GC UNC-45 mRNA expression to half the amount in the
control or SM UNC-45 antisense treated cultures
(Fig. 6A). Cell proliferation
was judged from total DNA as determined by fluorescence
(Jones et al., 2001). The GC
UNC-45 antisense oligonucleotides suppressed cell proliferation to 68-75% of
values obtained by treatment with the control reverse GC oligonucleotide
within 3 days of treatment (Fig.
6B,C). This reduction is significant (P=0.00008).
Treatment with the SM UNC-45 antisense oligonucleotides did not significantly
effect the proliferation rate (P=0.36). Cell viability was not
affected by 2.5 µM oligonucleotide, as assessed by all cultures having less
than 1% of trypan blue-stained cells.
|
SM UNC-45 and GC UNC-45 have distinct roles in muscle cell
differentiation
To gain insight into the role of each UNC-45 isoform in muscle
differentiation, C2C12 cells were treated with 2.5 µM antisense
oligonucleotides throughout the proliferative phase and for up to a week of
differentiation. At this point, twitching myotubes with robust striations were
present in cultures receiving none or negative control oligonucleotides. The
SM UNC-45 antisense treatment specifically reduced SM UNC-45 mRNA expression
to half the control levels (Fig.
7A). SM UNC-45 mRNA is expressed at control levels in the GC
antisense-treated cultures, reflecting their limited differentiation
(Fig. 7A).
|
For quantitative analysis of differences in myotube and sarcomere formation
in C2C12 cultures treated with antisense oligonucleotides, over 600 cells from
each treatment were scored for the number of nuclei per cell, expression of
muscle-specific sarcomeric -actinin as viewed in low magnification, and
the pattern of staining of sarcomeric
-actinin viewed in high
magnification. GC UNC-45 antisense treatment severely reduced myoblast fusion
so that the positively stained cells contained predominantly one to four
nuclei (Fig. 7B,D). Treatment
with the SM UNC-45 antisense oligonucleotide reduced fusion but to a lesser
extent. These cells were able to fuse to form multinuclear myotubes containing
over a dozen nuclei (Fig.
7B,D). Reduction of SM UNC-45 mRNA affected sarcomere formation
more directly, so that about half the myotubes had only small submembraneous
structures with striated
-actinin while the interior of the myotubes
was largely unstriated (Fig.
7C).
Considering the paucity and instability of thick filaments in C.
elegans unc-45 temperature-sensitive mutants
(Barral et al., 1998), we
addressed whether reduction of SM UNC-45 had an effect on the total amount of
skeletal myosin heavy chain in the C2C12 myotube cultures. Skeletal muscle
myosin heavy chain (MHC) was specifically detected by immunoblotting using MF
20 antibody against equivalent amounts of total protein from myotube cultures
treated 8 days plus or minus negative control or antisense oligonucleotides
directed against GC or SM UNC-45. Reduction of SM UNC-45 mRNA had no
significant effect on the amount of skeletal MHC in the myotube cultures
compared to untreated and negative control antisense-treated cultures
(Fig. 8). GC UNC-45
antisense-treated cultures had less than half as much skeletal MHC as the
other samples, consistent with the suppression of myotube formation
(Fig. 8). In these cultures
skeletal MHC was derived from the small myotubes containing two to four nuclei
and the rare larger myotubes, which were positively stained by the MF20
antibody, in contrast to the larger multi-nucleated myotubes in controls (see
Fig. 7B). The effects of
antisense suppression upon sarcomere formation therefore do not appear to be
related to changes in the amounts of MHC or other major proteins.
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Discussion |
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The C2C12 skeletal myogenic cell line allows one to examine general
cytoskeletal functions in the proliferative stage as well as muscle-specific
functions during differentiation. Consistent with its widespread expression in
organs, GC UNC-45 mRNA is expressed in proliferating, non-differentiated
myoblasts. This expression level is greater than any stage of muscle
differentiation. By the time more robust myotubes have developed, the levels
of GC UNC-45 mRNA are only 14% of those in proliferating cells. Reducing the
GC UNC-45 mRNA to about a half-normal level by antisense treatment decreased
C2C12 cell proliferation to 68-75% of control values. The GC UNC-45 antisense
treatment also inhibited myoblast fusion. In contrast to the GC isoform, SM
UNC-45 mRNA was not expressed until myogenic cells started fusing, with the
highest levels of expression in young myotube cultures. Assembly and
remodeling of myofibrils is highest during young myotube formation, consistent
with SM UNC-45 being involved in the process of thick filament assembly and
sarcomere formation. Unlike the GC UNC-45 antisense-treated cells, cells
treated with antisense SM UNC-45 oligonucleotides were able to fuse and form
myotubes. However, half of these myotubes lacked striated myofibrils, as
identified by staining for sarcomeric -actinin.
The SM to GC UNC-45 mRNA expression ratio in older myotube cultures was 2.8. This ratio contrasts with the 18 to 1 ratio of SM to GC UNC-45 mRNA in adult mouse skeletal muscle. This difference is most likely a consequence of only partial differentiation in cell cultures versus in vivo. The basis for less SM UNC-45 mRNA in older as compared to younger myotube cultures is unknown. However, protein half-life may vary and in addition the relationship between protein and mRNA expression need not be proportionate. This relationship can be determined for the UNC-45 isoforms when specific antibodies become available. Further work is needed to determine whether both GC and SM UNC-45 are present in striated muscle fibers. The antisense results correlate with the mRNA expression patterns indicating that GC UNC-45 has a role in processes involving the cytoskeleton and SM UNC-45 has a more muscle-specific function in sarcomere assembly. We cannot rule out the possibility of functional overlap between the two isoforms because suppression of either isoform had an affect on cell fusion and sarcomere formation. To date, it is not known what myosins are involved in the process of myocyte fusion. Based on their mRNA expression and the different roles of the two UNC-45 isoforms in C2C12 myogenesis, these two isoforms most likely have separate and distinct activities, possibly mediating functions that involve different classes of myosin. However, either isoform may function in myosin folding, assembly, and/or contractile activity.
Previous studies have shown that proteins in the UCS domain family are
required for a variety of myosin- and actin-based processes utilizing both
conventional and unconventional myosins. She4p is required for mRNA transport
involving an unconventional myosin type V in S. cerevisiae
(Beach and Bloom, 2001;
Jansen et al., 1996
).
Temperature-sensitive mutations in the essential gene rng3, a member
of the UCS family, block the assembly of the actomyosin ring during
cytokinesis, and are synthetically lethal with mutations in the cytoskeletal
myosin II gene (Balasubramanian et al.,
1998
). C. elegans UNC-45 has been shown to directly bind
the head of muscle myosin II (Barral et
al., 2002
), and to interact with cytoskeletal type II and
unconventional type V myosins through two-hybrid analysis (W. Ao and D.
Pilgrim, personal communication). In addition, C. elegans UNC-45 was
recently shown to be a myosin-targeted chaperone since it prevents the
thermally induced aggregation of the myosin head
(Barral et al., 2002
). This
finding, in conjunction with UNC-45 binding the chaperone Hsp90, suggests that
UNC-45 might influence thick filament assembly through a role in myosin
folding. The other UCS domain proteins may function likewise for different
myosin substrates. In this regard, it is significant that wild-type S.
pombe Rng3p is sequestered only by myo2-E1 myosin II, which has
a mutation in the myosin head and leads to defective contractile ring assembly
(Wong et al., 2000
).
Based on their mRNA expression and the different effects of suppression of
the two UNC-45 isoforms in C2C12 muscle differentiation, we propose that they
are involved in distinct functions. The GC UNC-45 isoform appears to have more
of a general function, possibly being involved in cell division, whereas the
SM isoform is related to striated muscle differentiation including myofibril
formation. The functions of the two isoforms are not necessarily independent
of one another, because the cytoskeleton is needed for formation and
maintenance of myofibrils. Lack of cytoskeletal proteins such as vinculin
(Barstead and Waterston, 1991),
talin (Moulder et al., 1996
),
desmin (Li et al., 1997
;
Milner et al., 1996
), certain
isoforms of integrin (Gettner et al.,
1995
; Moorthy et al.,
2000
; Volk et al.,
1990
) and spectrin
(Hammarlunda et al., 2000
;
Norman and Moerman, 2002
) as
well as the extracellular matrix protein perlecan
(Rogalski et al., 1995
) leads
to defects in myofibril formation and maintenance. Therefore both UNC-45
isoforms may be necessary for functional sarcomeres.
Our results are consistent with a model (Fig. 9) in which the two UNC-45 isoforms have separate, but possibly overlapping functions in striated muscle differentiation. In this model, GC UNC-45 would be involved in cell proliferation and cytoskeletal maintenance of myofibrils once they have formed. SM UNC-45 would function in the development of sarcomeres. These two proteins may interact with different myosins in their respective functions.
|
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Acknowledgments |
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