§
* Department of Cell Biology and Anatomy, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205-2196; Laboratório de Genética Molecular, Instituto de Biologia Molecular e Celular, 4150 Porto, Portugal; and § Departamento de
Biologia Molecular, Instituto de Ciências Biomédicas Abel Salazar, Universidade do Porto, 4050 Porto, Portugal
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Abstract |
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Assembly of the higher-order structure of mitotic chromosomes is a prerequisite for proper chromosome condensation, segregation and integrity. Understanding the details of this process has been limited because very few proteins involved in the assembly of chromosome structure have been discovered. Using a human autoimmune scleroderma serum that identifies a chromosomal protein in human cells and Drosophila embryos, we cloned the corresponding Drosophila gene that encodes the homologue of vertebrate titin based on protein size, sequence similarity, developmental expression and subcellular localization. Titin is a giant sarcomeric protein responsible for the elasticity of striated muscle that may also function as a molecular scaffold for myofibrillar assembly. Molecular analysis and immunostaining with antibodies to multiple titin epitopes indicates that the chromosomal and muscle forms of titin may vary in their NH2 termini. The identification of titin as a chromosomal component provides a molecular basis for chromosome structure and elasticity.
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Introduction |
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AUTOIMMUNE diseases are characterized by the presence of multiple autoantibodies that react with
components of nuclear, cytoplasmic, or surface origin (for review see Nakamura and Tan, 1992; Fritzler, 1997
). In clinical medicine, autoantibodies have been used
to establish diagnosis, estimate prognosis, follow the progression of a specific autoimmune disease, and, finally, increase our knowledge of the pathophysiology of autoimmunity. In cell biology, autoantibodies have been extremely
useful as probes for the identification of novel proteins
and isolation of their corresponding genes. Human autoimmune sera have been particularly useful in the study
of the eukaryotic nucleus where they have identified a
wide range of nuclear antigens, including both single- and
double-stranded DNA, RNA, histones, small nuclear
RNA-binding proteins, transcription factors, nuclear lamins,
heterochromatin-associated proteins, topoisomerase I and II,
and centromere proteins (Tan, 1989
, 1991
; Earnshaw and
Rattner, 1991
; Fritzler, 1997
).
Scleroderma (systemic sclerosis) is a multisystem connective tissue autoimmune disease of unknown etiology in
which vascular lesions and tissue fibrosis are prominent
features. Even though autoantibody production may be an
epiphenomenon of autoimmune diseases, autoantibody
targets in scleroderma are very specific (White, 1996). The
autoantigens to which scleroderma sera typically react include topoisomerase I, centromere proteins, RNA polymerases, fibrillarin, and several other nucleolar antigens
(LeRoy, 1996
). However, autoantibodies of rare occurrence have been reported that react with antigens localized to metaphase chromosomes and to the centrosome
(Jeppesen and Nicol, 1986
; Nakamura and Tan, 1992
).
Here, we report on the isolation of a Drosophila gene
using a scleroderma serum that recognized an epitope on
condensed mitotic chromosomes from both human cultured cells and early Drosophila embryos. Using this serum to screen a Drosophila expression library, we isolated
the gene that encodes the chromosomal protein that proved to be the Drosophila homologue of vertebrate titin
(D-Titin). Titin is a sarcomeric protein responsible for the
elasticity of striated muscle and may also function as a molecular scaffold for the assembly of myofibrils (for review
see Keller, 1995; Labeit and Kolmerer, 1995
; Trinick, 1996
;
Labeit et al., 1997
; Maruyama, 1997
; Squire, 1997
). We
show that D-Titin is expressed early and continuously in
striated muscle and that antibodies directed against two
different, nonoverlapping domains of Drosophila TITIN
label the Z-disks of Drosophila sarcomeres. The D-TITIN
antibodies also stain condensed human and Drosophila
mitotic chromosomes, consistent with the staining observed with the original scleroderma serum. Immunofluorescence with monoclonal and polyclonal antibodies against multiple epitopes of vertebrate titin further supports its localization to condensed mitotic human chromosomes, suggesting a role for titin not only in myofibrillar assembly
and muscle elasticity, but potentially in the architecture of
mitotic chromosomes.
As the name implies, titin is a giant protein. Individual
filamentous titin molecules, which range in molecular
mass from 2,993 to 3,700 kD, span a half-sarcomere from
the Z-disk to the M-line, a distance of ~1.2 µm in sarcomeres of relaxed skeletal muscle (Labeit and Kolmerer,
1995; Kolmerer et al., 1996
; Sorimachi et al., 1997
). Nearly
90% of titin's mass is comprised of Ig-like and fibronectin
type III (FN3)1-like repeats which are distributed throughout most of the protein (Labeit et al., 1990
; Maruyama et al.,
1993
; Labeit and Kolmerer, 1995
). The I-band region of
vertebrate titin also contains a domain rich in proline (P),
glutamic acid (E), valine (V), and lysine (K) that varies
from 163 to 2,200 residues, the so-called PEVK domain.
The PEVK domain and the tandemly arranged Ig domains
of the I-band region of titin confer elasticity to the titin filament (Linke et al., 1996
; Trombitas et al., 1998
). Titin has
phosphorylation sites (Sebastyén et al., 1995
), recognition
sites for muscle-specific calpain proteases (Sorimachi et
al., 1995
; Kinbara et al., 1997
) and a serine/threonine kinase domain near the COOH terminus (Labeit et al., 1992
;
Takano-Ohmuro et al., 1992
).
Titin may function as the scaffold upon which the sarcomeres are assembled into myofibrils (Keller, 1995; Trinick,
1996
). Titin mRNA is expressed in myoblasts before fusion (Colley et al., 1990
), and titin mRNA and protein are
among the earliest molecules to localize within the developing sarcomere (Fulton and Alftine, 1997
; van der Ven
and Fürst, 1997
). Titin binds to different proteins in each
region of the sarcomere. In the Z-disk, the NH2 terminus
of titin binds to the COOH-terminal region of
-actinin,
an actin-binding protein that cross-links titin to actin filaments (Ohtsuka et al., 1997a
,b; Sorimachi et al., 1997
; Turnacioglu et al., 1997
). In cardiac muscle, the NH2 terminus of titin binds to actin in the Z-disk near the Z/I-band junction (Linke et al., 1997
; Trombitas and Granzier, 1997
;
Trombitas et al., 1997
). The A-band region of titin provides a molecular template for the regular assemblies of
thick filament proteins such as myosin, MyBP-C, and
MyBP-H (C- and H-protein; Itoh et al., 1988
; Fürst et al.,
1989
; Soteriou et al., 1993
; Houmeida et al., 1995
; Freiburg
and Gautel, 1996
; Trombitas et al., 1997
). Titin may also
bind to myosin II (Eilertsen et al., 1994
). In the M-line, titin binds to M-protein and phosphorylated myomesin,
two myosin-binding proteins that cross-link titin to myosin
filaments (Eppenberger et al., 1981
; Obermann et al.,
1996
, 1997
). Functional evidence that titin acts as a myofibrillar scaffold derives from experiments where an NH2-terminal fragment of titin was fused to green fluorescent protein. This fusion protein, which localizes to the Z-disk,
causes myofibrillar disassembly when overexpressed (Turnacioglu et al., 1997
). The identification of titin, a gigantic
protein important in both the structure and elasticity of
muscle, as a chromosomal component has significant ramifications for understanding chromosome condensation
and chromosome integrity during mitosis.
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Materials and Methods |
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Staining of HEp-2 cells
Fixed HEp-2 cells (Kallestad, Chaska, MN) were blocked for 30 min at RT in PBS plus 0.1% Triton X-100 and 3% BSA (PBSTB), incubated for 1 h at room temperature in primary antibody, washed 3× for 5 min in PBSTB, incubated for 1 h with fluorescently labeled secondary antibody and washed 3× for 5 min in PBSTB. Cells were then incubated for 5 min at room temperature in 1.25 µg/ml propidium iodide (Sigma Chemical Co., St. Louis, MO) and washed twice in PBS. RNaseA (100 µg/ml; Sigma Chemical Co.) was included during antibody incubations. Images were collected on a confocal microscope (Noran Instrument, Middleton, WI). Forty scleroderma sera, provided by D. Isenberg (King's College, London, UK) were tested at a range of dilutions from 1:25 to 1:5,000 in the initial screen. The chosen scleroderma serum was used at a 1:200 dilution for subsequent experiments. Vertebrate titin polyclonal and monoclonal antibodies were used at dilutions of 1:25 and 1:100, respectively (provided by S. Labeit, EMBL, Heidelberg, Germany and J. Trinick, Bristol University, Bristol, UK). All fluorescently labeled secondary antibodies were used at a dilution of 1:200 (Vector Laboratories Inc., Burlingame, CA). Several fixative procedures (acetone/MeOH, acetone, formaldehyde based), with and without prior Triton X-100 permeabilization, were tested and produced the same staining patterns.
Antibody Production
The -LG polyclonal antiserum was made by immunizing rabbits with 450 µg of
-gal:D-TITIN fusion protein administered subcutaneously. The
-LG antiserum was affinity-purified as described (Earnshaw and Rattner, 1991
) and used at a dilution of 1:4. To produce the
-KZ antiserum,
an XhoI/EcoRI fragment of the most 5' cDNA (see Fig. 2) encoding 636 residues was ligated in-frame to the pTrcHisA expression vector (Invitrogen Corp., Carlsbad, CA) and transformed into BL21 (DE3) cells. Protein
was purified from inclusion bodies 3 h after induction with 0.1 mM IPTG
(Rio et al., 1986
). Rat polyclonal antibodies were raised (Covance Inc.,
Denver, PA) against 1 mg of renatured inclusion body protein. The
-KZ
antiserum was used at 1:5,000 dilution for embryo immunostaining in Fig. 3
and at 1:500 for all other experiments. Equivalent dilutions of preimmune
-LG and
-KZ sera were used as controls, as well as secondary antibodies alone.
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Drosophila Immunostaining and In Situ Hybridization
Embryo fixation and antibody staining were performed as described (Reuter et al., 1990). In situ hybridizations to whole-mount embryos were carried out as described (Tautz and Pfeifle, 1989
), except formaldehyde was
used in place of paraformaldehyde and levamisole was omitted from the
staining reaction. Images were collected on an Axiophot microscope (Carl
Zeiss, Inc., Thornwood, NY). Adult thoracic muscle and larval gut muscle
were prepared for immunostaining and stained as described (Saide et al.,
1989
; Lakey et al., 1993
). Texas red-phalloidin was used at a concentration of 0.1 U/ml (Molecular Probes, Inc., Eugene, OR). Images were collected on a Noran confocal microscope.
Immunoblot Preparation
Samples were homogenized in 2× Laemmli sample buffer and electrophoresed on 2.5-7.5% SDS-PAGE gradient gels (Laemmli, 1970); the
stacking gel was 0.6% agarose in 0.1% SDS, 0.125 M Tris-glycine, pH 6.8. Proteins were transferred to nitrocellulose filters (Schleicher & Schuell,
Inc., Keene, NH) for 2 h at 900 mA as described (Wang et al., 1989
). Samples from each extract were run on the same gel, transferred to nitrocellulose and cut into strips for antibody incubations. Immunoblots were incubated for 1 h at room temperature with blocking solution (5% nonfat
dried milk in 0.1% Triton X-100 in PBS [PBT]), incubated with primary
antibody for 2.5 h at room temperature (1:200 dilution of
-LG and 1:500
dilution of
-KZ), and washed 3× for 10 min in blocking solution. Filters
were incubated in either a 1:4,000 dilution of HRP-conjugated anti-rabbit
IgG (
-LG and LG preimmune; Amersham Pharmacia Biotechnology
Inc., Piscataway, NJ) or a 1:300 dilution of HRP-conjugated anti-rat F(ab)2 IgG (
-KZ and KZ preimmune; Amersham Pharmacia Biotechnology Inc.), washed 3× for 10 min in blocking solution and 2× for 5 min in
PBS. Immunoreactive bands were visualized by chemiluminescence (Pierce Chemical Co., Rockford, IL).
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Results |
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Human Scleroderma Serum Stains Mitotic Chromosomes
To identify novel nuclear components and to isolate and characterize the corresponding genes in Drosophila, we screened for human autoimmune sera that recognized nuclear components with cell cycle-dependent distribution in both human cells and early Drosophila embryos. Sera from 40 patients diagnosed with the autoimmune disease scleroderma were studied and only one serum was identified that gave chromosomal staining on both human epithelial HEp-2 cells and Drosophila 0-2 h embryos (Fig. 1 A). During interphase, when chromosomes are decondensed, low level staining was visible throughout the nucleus, with the exception of the nucleoli. During prophase, staining with this serum colocalized with the condensing chromosomes. From metaphase through telophase, chromosomes were stained uniformly.
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Cloning of Drosophila Titin
To isolate the corresponding gene in Drosophila, the human autoimmune scleroderma serum was used to screen a
Drosophila genomic expression library (Goldstein et al.,
1986). Out of 5 × 106 plaque-forming units screened, five
independent, overlapping genomic clones were isolated,
each encoding several copies of a 71-amino acid repeat
rich in proline, valine, glutamic acid, and lysine residues
(Fig. 2 C). The largest clone (designated LG) was expressed in Escherichia coli, and the corresponding fusion protein was purified and used to immunize rabbits.
-LG
affinity-purified antibodies gave the same chromosomal
staining pattern on both human HEp-2 cells and Drosophila 0-2 h embryos in all stages of the cell cycle as was
initially observed with the human serum (Fig. 1 B). We
subsequently isolated additional exons from this Drosophila gene (see below) and used a different domain of
the protein to raise a second polyclonal antiserum in rat
(designated
-KZ). The
-KZ antiserum reproduced the
staining pattern observed with the human autoimmune serum and the
-LG antibody, that is, nuclear staining during interphase (not shown) and staining of condensed
chromosomes during mitosis (Fig. 1 C).
Attempts to clone the entire genomic region corresponding to the chromosome-associated protein gene were unsuccessful most likely because of its repetitive structure. The isolation of cDNAs was similarly difficult due in part to the repetitive structure of the gene but also because of the predicted large size of the corresponding mRNA (see below). Nonetheless, cDNAs mapping to several discrete regions of the gene, encoding a total of 1,608 amino acids, were isolated and characterized (Fig. 2). The partial cDNAs, designated KZ, NB, and JT, were named according to the libraries in which they were found. See Fig. 2 legend for the details of cloning. All of the cDNA clones and genomic phage clones 1-5 map to cytological position 62C1-2 in a region known to contain only a single gene.
Notably, every open reading frame (ORF) identified
from the chromosome-associated protein gene shows significant similarity to vertebrate titins (Fig. 2 B). Using the
conceptual translation of the KZ cDNA to do a BLAST
search, the two proteins with greatest similarity are
chicken skeletal titin (P = 2.7e99) and human cardiac titin
(P = 1.2e
80). The ORF within the unprocessed NB
cDNA also shows significant similarity to vertebrate titins.
An alignment between the ORFs derived from the KZ
and NB cDNAs and the chicken skeletal and human cardiac titins is shown in Fig. 2 B. In the region of overlap, the
ORF encoded by the KZ cDNA shows 28.6% identity/
58.3% similarity to chicken skeletal titin, and 27.4% identity/56.8% similarity to human cardiac titin. The ORF encoded by the NB cDNA shows 18.4% identity/48.9% similarity to chicken skeletal titin, and 17.2% identity/47.7%
similarity to human cardiac titin in the region of overlap.
The sequence conservation among the ORFs from the LG
and JT clones and vertebrate titins is not as great; however, in these clones, the frequency of P, E, V, and K residues (63% for LG, 56.4% for JT) strongly suggests that
these ORFs correspond to the elastic PEVK domain of
vertebrate titin, which is 70% P, E, V, K (Fig. 2 C). Thus,
starting with a human autoimmune scleroderma serum, we
have cloned a Drosophila gene encoding a nuclear protein that localizes to chromosomes and is homologous to vertebrate titins.
D-Titin in Striated Muscles and Their Precursors
To determine whether the gene that encodes the nuclear,
chromosome-associated form of titin also encodes the
muscle form of titin, we examined both transcript and protein accumulation in embryos, and determined the subcellular localization of the protein in muscle. Analysis of
RNA expression by in situ hybridization to whole-mount
Drosophila embryos revealed RNA accumulation as early
as the germ band extended stage in both somatic and visceral muscle precursors (Fig. 3 A). Protein was initially detected during late stage 11 in the precursors of both somatic and visceral muscles (Fig. 3 A'), before myoblast
fusion (stage 13; Hartenstein, 1993). This early accumulation of protein in Drosophila muscle precursors parallels
vertebrate titin accumulation in early myoblasts (Colley et al., 1990
). Expression of both RNA and protein in all
visceral and somatic muscles persisted throughout embryogenesis (Fig. 3, B-H'). These muscles include the somatic or body wall muscles, the pharyngeal muscles, and
the visceral musculature which surrounds the digestive
system. We did not detect RNA or protein in embryonic cardiac muscle or cardiac muscle precursors.
To determine if the protein localizes to specific regions
in the sarcomere, we immunostained adult thoracic muscle
with antibodies directed against two different domains of
the protein (-KZ and
-LG; Fig. 2) and with the original
human autoimmune scleroderma serum. The
-KZ antiserum stained the Z-disks of each sarcomere, which can be
identified as the phase-dark bands on myofibrils (Fig. 4 A).
A double-stained image of a myofibril stained with
-KZ
and Texas red-phalloidin, which stains the filamentous actin of the I-band, supports this localization (Fig. 4 B). Double staining with the
-KZ antiserum and either the human autoimmune scleroderma serum (Fig. 4 C) or the
-LG affinity-purified antibodies (Fig. 4 D) also revealed
Z-disk staining. Both the
-LG antibodies and the scleroderma serum also stained the M-line suggesting potential cross-reactivity to other antigens. The scleroderma serum,
but not the
-LG antibodies, also stained along the length
of the myofibril (Fig. 4 C), suggesting the presence of additional, nontitin antibodies in the serum. Two antibodies
against vertebrate titin recognized epitopes on Drosophila
myofibrils: serum from a patient with myasthenia gravis,
which recognizes the major immunogenic region (MIR)
epitope in the I-band near the I/A-band junction (Fig. 4 E;
Gautel et al., 1993
), and anti-Zr5/Zr6, a polyclonal antiserum that was raised to the expressed
-actinin binding
Z-repeat motifs Zr5/Zr6 (Fig. 4 F; Sorimachi et al., 1997
).
The nearly complete overlap of the signals with
-KZ and
the MIR serum suggests that the resolution of confocal microscopy was insufficient to allow visual separation of
Z-disk staining from I/A-band staining in nonstretched
Drosophila myofibrils. We also found that the
-KZ and
-LG antibodies stained the Z-disks of visceral muscles from third instar larvae (Fig. 4 G). Drosophila visceral
muscle, unlike vertebrate smooth muscle, is striated.
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We have named the gene isolated with the human autoimmune scleroderma serum D-Titin for Drosophila Titin. This name is based on the high level of similarity to vertebrate titins, the expression pattern of this gene during embryogenesis (Fig. 3), the localization of two different domains of the protein to the Z-disks in sarcomeres by immunofluorescence (Fig. 4) and the size of the protein on immunoblots (Fig. 5; see below).
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D-TITIN Migrates in the Megadalton Size Range
Vertebrate muscle titin isoforms range in molecular mass
from 2,993 to 3,700 kD (Labeit and Kolmerer, 1995; Kolmerer et al., 1996
; Sorimachi et al., 1997
). Because the
mini-titins identified in Drosophila are smaller (500-1,200
kD; Ayme-Southgate et al., 1991
, 1995
; Fyrberg et al.,
1992
; Lakey et al., 1993
), they are unlikely to span a half-sarcomere from the Z-disk to the M-line. Mutational analysis further suggests that these proteins do not provide the
elasticity and proposed scaffolding functions of vertebrate titin. Our data shows that NH2-terminal regions of D-TITIN
localize to the Z-disk, that D-TITIN has significant homology to vertebrate titins, and that D-TITIN is expressed
early and continuously in striated muscles in Drosophila.
These characteristics make D-TITIN a good candidate for
the Drosophila homologue of vertebrate muscle titin. If
it is indeed the homologue, D-TITIN should be in the 2-4-MD size range. To test this prediction, total protein
extracts from 8-24 h embryos were prepared (in muscle
precursors, D-TITIN is first detected at ~7 h by immunostaining of embryos), proteins were separated on denaturing
polyacrylamide gradient gels (2.5-7.5%), and were transferred to nitrocellulose filters. Immunoblots incubated with both
-LG and
-KZ detected a discrete band in the
megadalton size range, consistent with vertebrate titin
(Fig. 5 a, lanes 2 and 4).
-LG and
-KZ preimmune sera
revealed no cross-reacting polypeptides (Fig. 5 a, lanes 3 and 5). Thus, D-TITIN is likely to be the Drosophila homologue of vertebrate sarcomeric titin based on size, sequence similarity, developmental expression and subcellular localization.
To determine the size of chromosome-associated D-TITIN
in nonmuscle cells, total protein extracts were prepared
from both 0-2 h Drosophila embryos (myogenesis does
not begin until several hours later) and from HeLa cells
(epithelial cells). In the 0-2 h embryonic extracts, we detected a discrete high molecular mass polypeptide of identical size on immunoblots with both -LG (Fig. 5 b, lane 2)
and
-KZ (Fig. 5 b, lane 4). No cross-reacting polypeptides were detected with either
-LG or
-KZ preimmune sera
(Fig. 5 b, lanes 3 and 5). Using total cell extracts from
HeLa cells, we also detected a megadalton polypeptide
with both
-LG and
-KZ antisera (Fig. 5 c, lanes 2 and 4),
with no staining with the preimmune sera (Fig. 5 c, lanes 3 and 5). Thus, antibodies to D-TITIN detected a very high
molecular mass polypeptide in nonmuscle cells from
Drosophila and in human epithelial cells. Since, by immunofluorescence, the only detectable staining with these antibodies on Drosophila 0-2 h embryos and HEp-2 cells is
chromosomal, we concluded that the chromosomal form
of D-TITIN migrates in the megadalton size range and is
approximately as large as the muscle form.
Antibodies to Vertebrate Muscle Titin Stain Human Chromosomes
Given that Drosophila TITIN localized to condensed
Drosophila mitotic chromosomes and that antibodies directed against this protein also stained human chromosomes, we were curious whether antibodies to vertebrate
muscle titin also stained condensed chromosomes. We
used a panel of eight antibodies directed against different epitopes of vertebrate titin to immunostain HEp-2 cells.
Six of the eight antibodies directed against vertebrate titin
stained the condensed chromosomes in a pattern indistinguishable from that observed with the original scleroderma serum and the antibodies to the D-TITIN protein
(Fig. 6, A and B). The antibodies that gave chromosomal
localization include three mouse monoclonals, two of
which recognize distinct epitopes in the A-band (BD6 and CE12; Whiting et al., 1989) and one of which recognizes
the PEVK domain in the I-band (9D10; data not shown;
Wang et al., 1991
). We also observed chromosomal staining with two rabbit polyclonal antibodies to vertebrate
titin: N2A, which recognizes an I-band epitope in skeletal
titin, and A168, which recognizes an M-line epitope (Linke
et al., 1996
). Finally, the MIR human autoimmune serum,
which recognizes an I/A-band epitope (Gautel et al., 1993
), also stained condensed mitotic chromosomes of HEp-2
cells although additional staining of the mitotic apparatus
was visible with this serum (Fig. 6 A). The two vertebrate
titin antibodies that did not recognize titin on HEp-2 chromosomes, anti-Zr5/Zr6 and T12, are directed against NH2-terminal regions of titin that either map to the Z-disk and
bind to
-actinin (anti-Zr5/Zr6; Sorimachi et al., 1997
) or
map to the Z-disk/I-band junction (T12; Fürst et al., 1988
).
The NH2-terminal region of the D-Titin isoform encoded
by the KZ cDNA does not contain regions homologous to the
-actinin-binding regions of vertebrate titin. It is likely that cDNAs encoding the NH2 terminus of the muscle
D-TITIN isoform will reveal homologies to the
-actinin-
binding regions since (a) the COOH-terminal sequence of
-actinin from Drosophila is highly homologous to the
COOH terminus of human
-actinin (Fyrberg et al., 1990
)
and (b) the anti-Zr5/Zr6 antiserum stains Drosophila myofibrils but not chromosomes. Thus, titin localizes to chromosomes in both Drosophila embryos and human cells although the chromosomal and muscle forms of titin may
vary in their NH2 termini.
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Discussion |
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Proposed Function for Titin in Chromosome Structure
The uniform distribution of titin along condensed chromosomes suggests a structural role for titin in chromosome
condensation, a role similar to the one titin plays as a scaffolding element in the sarcomeres (Trinick, 1994, 1996
).
Chromosome condensation during mitosis is essential for
proper segregation and for compacting chromosomes so
that they are no longer at the cleavage furrow during cytokinesis (for review see Hirano, 1995
; Koshland and Strunnikov, 1996
). Chromosome condensation is thought
to occur by a deterministic process based on the fixed
length and banding patterns of individual chromosomes in
a given cell-type, the invariant position of specific sequences within a chromosome, and the fixed axial diameter of mitotic chromosomes (Koshland and Strunnikov, 1996
). The invariant axial diameter of condensed mitotic
chromosomes suggests the involvement of a protein that
functions in part as a "molecular ruler", a function that has
already been ascribed to titin in muscles (Trinick, 1994
,
1996
). Thus, we can envision the chromosomal form of
titin functioning in the assembly of the higher-order structure observed in condensed mitotic chromosomes, perhaps
determining the length and/or axial diameter of condensed chromosomes.
Titin is the elastic component of sarcomeres where it
acts as a molecular spring that prevents sarcomere disruption when muscles are overstretched. Likewise, the chromosomal form of titin could provide elasticity to chromosomes and resistance to chromosome breakage during
mitosis. The elastic properties of purified titin (Kellermayer et al., 1997; Rief et al., 1997
; Tskhovrebova et al.,
1997
) correspond well to the recently described elastic
properties of chromosomes in living cells (Houchmandzadeh et al., 1997
). Studies on vertebrate myofibrils have
shown that the PEVK domain and the Ig/FN3 repeats constitute a two-spring system acting in series to confer reversible extensibility to titin (Linke et al., 1996
; Trombitas
et al., 1998
). Under physiological stretching conditions, the
Ig/FN3 domains straighten and the PEVK domain reversibly unfolds. Under more extreme nonphysiological stretching conditions, the Ig and FN3 domains also unfold. However, refolding of the Ig and FN3 repeats is slow and
occurs only in the absence of stretch force. Similarly,
metaphase chromosomes from living cells also show two
levels of extensibility (Houchmandzadeh et al., 1997
).
Metaphase chromosomes from cultured newt lung cells can be stretched up to 10 times their normal length and return to their native shape. Further nonphysiological extensions of chromosomes from 10 to 100-fold are irreversible.
The discovery of titin on chromosomes integrates the mechanical properties of muscle titin with the elastic properties of eukaryotic chromosomes.
Does titin remain associated with chromosomes during
interphase? Although we detected titin in the nucleus during interphase with both the Drosophila titin antibodies
and the antibodies directed against vertebrate titin, the
resolution of confocal microscopy does not allow us to directly ask if titin remains bound to chromosomes. However, we have looked at the accumulation of D-TITIN on
salivary gland polytene chromosomes from Drosophila
third instar larvae using the -KZ antiserum. Based on
gene activity and chromatin ultrastructure, polytene chromosomes are functionally similar to diploid interphase
chromosomes (Tissièrres et al., 1974
; Elgin and Boyd,
1975
; Bonner and Pardue, 1976
; Woodcock et al., 1976
). Low level D-TITIN staining was observed throughout the
chromosomes with several discrete sites of higher accumulation (data not shown). These results suggest that titin remains associated with relatively decondensed interphase
chromosomes, consistent with the chromosome core structure being templated during interphase (Andreasson et al.,
1997
). Measurements made at different stages in the cell cycle indicate that chromosome flexibility increases during
the transition from interphase to metaphase (Houchmandzadeh et al., 1997
). Regulated phosphorylation of
titin may control the assembly of interphase chromosomes
into the higher-order structure of metaphase chromosomes and indirectly alter chromosome flexibility.
Chromosomal and Muscle Forms of Titin
The chromosomal and muscle forms of titin are unlikely to
be identical. Although both forms of D-TITIN appeared
to comigrate, the resolution of the gradient gels used to
detect both the muscle and chromosomal forms of D-TITIN
may be insufficient to resolve the respective molecular
mass differences. The most 5' D-Titin cDNA isolated in
this work, which encodes an NH2 terminus, does not contain the most 5' sequences found in vertebrate muscle
titin, the so-called Z-repeats that bind to -actinin in the
Z-disk (Turnacioglu et al., 1996
; Ohtsuka et al., 1997a,b;
Sorimachi et al., 1997
). Antibodies directed against the
-actinin-binding region of vertebrate titin did not recognize titin on human chromosomes, although antibodies directed to more COOH-terminal epitopes were reactive
(Fig. 6). Furthermore, the most 5' cDNA contains a unique
81-amino acid sequence at the NH2 terminus. Titin mRNA
and protein have been detected in BHK (Jäckel et al.,
1997
). Moreover, in a subline derived from the BHK cells,
the titin gene contained deletions in the Z-disk region.
Titin mRNA was still detected in the mutant cell line. Altogether, these results suggest that muscle titin and chromosomal titin vary at least in their most NH2-terminal regions. Our results with D-TITIN suggest that the muscle
and chromosomal forms of titin are encoded by splice variants of the same gene, and that we have not cloned the exons encoding the most NH2-terminal regions of muscle
titin. Polytene chromosome in situ hybridization and genomic southern analysis revealed that D-Titin is a single-copy gene (unpublished results). Determination of whether
vertebrate chromosomal and muscle titins are also splice variants of the same gene or, instead, are encoded by two
closely related genes awaits further investigation of vertebrate titin.
The previously known components of condensed chromatin include DNA, histones, topoisomerase II, the SMC
family of proteins (for review see Chuang et al., 1994;
Earnshaw and Mackay, 1994
; Hirano and Mitchison, 1994
;
Peterson, 1994
; Hirano, 1995
; Saitoh et al., 1995
; Strunnikov et al., 1995
; Holt and May, 1996
; Koshland and Strunnikov, 1996
; Warburton and Earnshaw, 1997
), the condensins, three recently identified proteins that form a
complex with SMC family members (Hirano et al., 1997
),
and the cohesins, proteins that link condensation and sister chromatid cohesion (Guacci et al., 1997
; Michaelis et al.,
1997
). Topo II and SMC were identified as the two most
abundant chromosomal "scaffold" proteins, which by definition, comprise an insoluble fraction purified from isolated mitotic chromosomes. These scaffold proteins are
proposed to determine the characteristic shape of mitotic
chromosomes. Both genetic and in vitro depletion studies
confirm that topo II and SMC proteins are indeed required for chromosome condensation and subsequent chromosome segregation; whether their roles in chromosome condensation are structural or entirely enzymatic,
however, remains to be determined (see previously cited
reviews and Kimura and Hirano, 1997
; Sutani and Yanagida,
1997
). If titin is part of the chromosomal scaffold, why was
titin not identified in the initial biochemical analyses of
scaffold proteins? The simplest explanation is based on
the size of titin. Almost all of the protein gels used to identify topo II, SMC and other scaffold proteins were 12.5%
polyacrylamide gels and did not resolve proteins of molecular mass >200 kD. Indeed, in almost every published
photograph of a protein gel of purified scaffold components, there is a high molecular mass component that fails
to enter the gel (Adolph et al., 1977
; Paulson and Laemmli, 1977
; Laemmli et al., 1978
; Lewis and Laemmli, 1982
; Earnshaw and Laemmli, 1983
). We demonstrated that
chromosomally associated titin from HEp-2 cells and early
Drosophila embryos migrates in the megadalton size
range. Although a protein of this size can be resolved in
2.5-7.5% gradient gels, titin would not enter the 12.5%
gels typically used in chromosome scaffold studies.
Titin as an Autoantigen
As an alternative to a biochemical approach, autoimmune
sera have been successfully used as probes in the isolation
of novel chromosomal proteins and for expression cloning
of the corresponding genes (for review see Earnshaw and
Rattner, 1991; Tan, 1989
, 1991
; Fritzler, 1997
). Even
though the prevalence for autoantibodies against nuclear
components appears to be higher, the spectrum of autoantibodies identified in sera from patients with autoimmune diseases is much broader, and includes numerous cytoplasmic antigens as well as several extracellular matrix proteins (Fritzler, 1997
). Many autoantigens are proteins very
well conserved throughout evolution, ranging from species
as distant as man, fish, amphibia, Drosophila, yeast, and
plants (Snyder and Davis, 1988
; Tan et al., 1987
; Mole-
Bajer et al., 1990; Brunet et al., 1993
; Shibata et al., 1993
;
Rendon et al., 1994
; Bejarano and Valdivia, 1996
). The
work presented here is the first to identify titin autoantibodies in scleroderma sera, the first to reveal titin as a
chromosomal component and, to our knowledge, the first
successful cloning of a Drosophila gene using a human autoimmune serum.
The identification of autoantibodies against titin until
now has been restricted to a subset of patients with myasthenia gravis (MG) who have also developed thymus neoplasia (Aarli et al., 1990; Gautel et al., 1993
). However, the
stimulus for the autoimmune response to titin may be due
to molecular mimicry. This immunoreactivity is directed
exclusively to a single epitope of titin, the MIR epitope.
This epitope is shared with neurofilaments that are overexpressed in these thymomas (Marx et al., 1996
). Since
titin MIR autoantibodies can be detected in 97% of sera
from MG-thymoma patients, the MIR epitope of titin is a
sensitive marker for evaluating the presence of thymoma
in MG patients (Gautel et al., 1993
). The isolation of the
D-Titin gene in Drosophila using a scleroderma autoimmune serum raises the inevitable question of whether titin
may represent a new, unidentified autoantigen in scleroderma.
D-Titin represents the third Drosophila member of the
titin gene family. The other two family members, both of
which are referred to as mini-titins (Vibert et al., 1996), include KETTIN and PROJECTIN. KETTIN is a 500-kD
family member with low homology to vertebrate titin.
KETTIN has been proposed to be one of the structural
components of Z-disks; however, mutations in the corresponding gene have not yet been described (Lakey et al.,
1993
). PROJECTIN is a highly homologous ~1,200-kD
titin family member most closely related to TWITCHIN
(Ayme-Southgate et al., 1991
, 1995
; Fyrberg et al., 1992
), a
Caenorhabditis elegans mini-titin that binds to myosin filaments in body wall muscles. TWITCHIN has the Ig and
FN3 repeats and a myosin light chain kinase domain near
the COOH terminus, but does not have an obvious PEVK
region (Benian et al., 1989
; Benian et al., 1996
). TWITCHIN
is thought to regulate myosin activity because mutations in
twitchin (unc-22) cannot develop or sustain muscle contractions (Waterston et al., 1980
; Moerman et al., 1988
). Furthermore, mutations in twitchin can be suppressed by
mutations in the myosin heavy chain gene. Lethal alleles
of Drosophila PROJECTIN exist as mutations in the bent
locus. Homozygous bent mutant animals die as late embryos but, unlike twitchin mutant worms, have apparently normal muscle contractions (Fyrberg et al., 1992
; Ayme-Southgate et al., 1995
), suggesting normal sarcomere organization. The identification of a true titin homologue in a
genetically tractable organism will greatly facilitate the
analysis of titin function both in sarcomeres and in chromosome structure and flexibility. Indeed, we have mapped
the D-Titin gene to a cytological interval known to contain
only a single gene. A thorough characterization of D-Titin
mutations is currently underway.
![]() |
Footnotes |
---|
Received for publication 11 December 1997 and in revised form 2 February 1998.
C. Machado was supported by a Ph.D. fellowship from Junta Nacional de Investigação Cientifica e Tecnológica (JNICT) and, in part, by grants from Fundação Luso-Americana para o Desenvolvimento and Fundação Calouste Gulbenkian. This work was supported by a grant from the Council for Tobacco Research to D.J. Andrew and a grant from JNICT to C.E. Sunkel.We thank D. Isenberg for providing scleroderma sera from patients at the Immunology Unit, King's College (London, UK). We thank S. Labeit and J. Trinick for vertebrate titin antibodies. We thank J. Mason and A. Spradling for fly stocks. We thank M. Delannoy and K.D. Henderson for assistance with the NORAN confocal microscope. We thank P. Tuma for advice with the protein gradient gels. We thank D. Barrick, J.D. Castle, K.D. Henderson, A. Hubbard, C. Machamer, and K. Wilson for their critical comments on the manuscript. We also thank F. Domingues for assistance during the expression screen and all the members of the Andrew laboratory for their help and patience during the final stages of this study.
![]() |
Abbreviations used in this paper |
---|
E, glutamic acid; FN3, fibronectin type III; K, lysine; MG, myasthenia gravis; MIR, major immunogenic region; ORF, open reading frame; P, proline; V, valine.
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References |
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