Review by Sarah E. Brautigam
The protein titin has already been already identified
as a huge sarcomeric protein contributing to the elasticity of striated
muscle. This goal of this paper is to convince the reader that titin
is also a protein used by chromosomes in providing structure and elasticity.
The way in which chromosomes are assembled is necessary for later chromosome
condensation, segregation, and integrity. Most proteins involved
in this process have yet to be discovered. In this paper first, human
autoimmune scleroderma serum is used to identify a chromosomal protein
in human epothelial cells and Drosophila early embryos (so that
there are no muscles). The authors then used the protein information
to clone the Drosophila gene for this chromosomal protein.
They proceed to try to prove that the gene encodes for a protein which
is a homologous to the vertebrate protein titin by 1) sequence similarity
of the Drosophila protein and vertebrate titin, 2) expression of
this protein and RNA in embryo muscles, 3) presence of this protein on
specific areas of Drosophila muscle, and 4) protein size.
Finally the authors show that vertebrate titin localizes to condensed mitotic
chromosomes in human epithelial cells.
The authors first identified a serum produced by
1 person of 40 with autoimmune disease scleroderma that bound to proteins
on chromosomes in both human epithelial cells (HEp-2) and Drosophila
embryos 0-2 hours mature. Figure 1A uses immunofluorescence to show
chromosomal staining with the serum in the Drosophila early embryos
and HEp-2 cells (green). This figure also includes double-staining
of DNA with propidium iodide (red). The left and center panels, serum
identifying the protein (red left), and the DNA( green center) were then
overlapped so as to make sure that the serum was in fact binding to proteins
on the chromosomal DNA. When the two were superimposed (right) a
yellow color was the result. The figure shows the chromosomal staining
in interphase, prophase, metaphase, anaphase, and telophase. during
interphase when the chromosomes are not condensed there was low level staining
throughout the nucleus (except nucleoli). In prophase the staining
began to localize as the chromosomes condensed. Metaphase through
telophase the condensed chromosomes stained uniformly.
This scleroderma serum was then used to isolate
the Drosophila gene that produced the protein that bound to the
chromosomes in figure 1A. The serum was used to screen a Drosophila
genomic expression library in which five overlapping clones were identified
from all of the proteins in an expression library. These clones each
encoded several copies of an amino acid sequence rich in proline, valine,
glutamic acid, and lysine. It is important to note that the I-band
of vertebrate titan has also been shown to contain a domain rich in proline,
glutamic acid, valine, and lysine. Figure 2C shows 2 of the five
clones- the LG clone and the JT. The LG clone (largest clone) was
expressed in E. Coli and and used to immunize rabbits to makes anti-LG
affinity-purified antibodies. Immunofluoresence of the anti-LG antibodies
were shown to give the same chromosomal staining patterns as the serum
as seen in Figure 1B. Figure 1B only shows metaphase and anaphase
though. This does not seem to be a problem because it is already
shown that serum will bind to the chromosomes in all stages. Additional
exons were isolated from the Drosophila gene were isolated and another
portion of the protein was used to make another polyclonal antiserum in
a rat. This antiserum bound to the portion of the protein which was
encoded by the KZ region of the gene in Figure 2A. The anti-KZ antiserum
reproduced a similar chromosomal staining pattern through immunofluorescence
as the original scleroderma serum and the anti-LG antibody in Figure 1C.
Again this figure shows staining in the metaphase and anaphase and includes
staining of DNA so as to make sure that the proteins that the antibodies
are binding to are on the chromosomes.
The entire gene encoding the chromosome-associated
protein was never cloned. The authors are careful never to call the
protein titin until they have more figures to substantiate its homology
to titin in muscles and vertebrate titin. The authors account for
this unsuccessful attempt to clone the gene because of the repetitive structure
of the gene. Problems isolating the cDNAs was difficult for this
same reason of repetitiveness but also huge mRNAs. Figure 2A shows
the limited gene map that the authors were able to describe for the chromosome-associated
protein. This figure is useful in determining where a certain antibody
binds to the chromosome-associated protein (titin). Figure
2B is a protein sequence alignment which shows the similarity that the
ORFs encoded by the the NB and KZ cDNAs of the chromosome-associated
protein isolated in Drosophila have with chicken skeletal titin
and human cardiac titin. 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 encoded by the NB cDNA shows 18.4% identity/ 48.9%similarity to chicken
skeletal titin and17.2% identity / 47.7% similarity to human cardiac titin.
These numbers seem to indicate a highly conserved sequence among the previously
identified titin in humans and chickens. In this figure the authors
seem to be suggesting that because it looks like titin in other species,
it must be titin in Drosophila. The authors acknowledge the
fact that the conservation among the ORFs from the LG and JT clones and
vertebrate clones is not as great. They explain that these clones
have a high frequency of P,E,V, and K residues which the ORFs correspond
to elastic PEVK domain of vertebrate titin which contains lots of
P,E,V, and K. It would have been more convincing if the authors had
indeed done a protein alignment sequence for these ORFs and the corresponding
vertebrate regions rich in the P,E,V, and K residues
Next the authors set out to determine whether the
gene that encodes the nuclear chromosome-associated form of titin also
encodes the muscle form of titin. They examined the protein expression
and presence of RNA in Drosophila embryos. The authors
seem to be at this point be comfortable calling the chromosome-associated
protein titin. The authors state that Figure 3A to 3H shows the actual
muscles that Drosophila TITIN is expressed in all the somatic and
visceral musculature in stages of 10/11 to 16 embryogenesis. The
titin (brown) is localized to different muscles as the embryo develops.
Additionally the RNA (blue) seems to be present in all stages in the muscles.
Muscles include body wall muscles, pharyngeal muscles and others.
The authors suggest that the early RNA accumulation and protein presence
in both somatic and visceral muscle precursors parallels vertebrate titin
accumulation in early myoblasts. The presence of the RNA in 3A is
not as clear as the other figures. It seems that in the other figures
the intensity of the RNA is the same as the protein but it is not the case
with A. The RNA is hardly detectable. The authors fail to state
what they examine the presence of the proteins and mRNA with. In other
words what antibody did they use. It is unclear whether it is a new
antibody for muscle titin in Drosophila or whether it is the autoimmune
serum or or the anti-LG or KZ . This is important because the the
antibodies bind different epitopes on titin. Certain antibodies may
bind and others may not depending on the similarity of the muscle titin
and the chromosome-associated titin. It is also seems peculiar that no
RNA or protein was detected in the embryonic cardiac muscle precursors.
Determination of protein location on specific parts
of the sarcomere was examined in Figure 4. The authors hope that
this specific location of titin on the muscles will further convince the
reader that it is in fact , homologue of vertebrate muscle titin.
One of the characteristics of titin is that it localizes to muscles.
Adult thoracic muscles were immunostained with antibodies directed against
two different domains of the protein using the anti- LG and anti-KZ and
the original autoimmune serum. Both the autoimmune serum and the
anti-LG bind to the PEVK rich region of the protein and the KZ binds to
the amino end (Figure 2). It is shown in figure 4A and 4B that
anti-KZ antiserum stained the Z-disks. The authors use this evidence
to then determine whether the anti-LG and autoimmune serum bind to the
Z-disks. Figures 4C and 4D present a few problems. There is
a lot of background staining in 4C along the myofibril. The authors suggest
that this is a result of other antibodies present in the serum. This
may present a problem in the paper if in other experiments for other figures
there were other antibodies binding to other antigens besides titin.
In addition in Figure 4D and 4C show staining along the M-line suggesting
cross-reactivity with other antigens which also a potential problem.
In both cases of cross-reactivity with other antigens and the presence
of other antibodies may cause misleading data. Both 4C ad 4D suggest
some recognition of Z-disks by both serums though. Figures 4E and
4F show two other antibodies against vertebrate titin recognize epitopes
on Drosophila myofibrils. The authors state these figures
are poor in resolution so as to not allow staining on two separate regions
of the myofibrils, the Z-disk and I/A-band, to be confused.
Figure 4G shows shows that anti-KZ and anti-LG stained the Z-disks of viscertar
larvae. In the figure legend it is not clear which antibody is staining
green. There are several problems with this figure, but the authors
do address most of them. Good negative controls were performed in
A and B so that no staining was apparent with preimmune anti-KZ serum or
secondary antibodies.
At this point in the paper the authors feel comfortable
enough to officially call the gene isolated with the human autoimmine scleroderma
serum D-TITIN based on Figures 3,4, and 5. Figure 5 deals with the
molecular weight of titin. The authors are trying to really convince
the reader once and for all that D-TITIN is the homologue to vertebrate
muscle titin. The authors suggest that the D-TITIN must be 2-4 MD
in size if it is the homologue. Total protein extracts were extracted
for 8-24 h Drosophila embryos were separated on a polyacrylamide
gel and transfered to nitrocellulose. Immunoblots or western blots
were prepared with anti-LG and anti-KZ. Figure 5a is an immunoblot
with protein from 8-24 h embryos from Drosophila, 5b is an immunoblot
with protein from 0-2 h embryos from Drosophila, and 5c is protein
from HeLa cells (epithelial cells). The authors find that there
is a band of a megadalton size present in all three cases for both anti
serums 5a, 5b, and 5c lanes 2 and 4. Figure b and c were used to
show the size of chromosome-associated D-TITIN in non muscle cells because
the 0-2 h embryos have not developed muscles and there is no muscle in
epithelial cells. There appears to be good negative controls in a,
b, and c with the preimmune serums for both antibodies in which no cross-reacting
polypeptides are detected. The authors conclude that since by immunoflorescence
staining of the 0-2 h embryos and the HEp-2 cells is chromosomal (Figure
1), the chromosomal form of D-TITIN is migrates to the megdalton
size and is about the same size as the muscle form. There are a few
problems with figure. First even the authors admit there is only
a discrete band and it shows up in some lanes better than others.
Second there is a lot of background that the authors do not address but
this is possibly to be expected with western blots. The biggest problem
is the molecular weight markers in lane one. The markers should correspond
better with the weight of the titin protein so that there can be a better
estimate than "a band of megadalton size." The markers stop at 584
and the protein is much bigger than that.
Figure 6a tests 8 antibodies directed against
different epitopes of vertebrate titin. The antibodies immunostained
HEp-2 in order to determine whether antibodies to vertebrate titin bound
to chromosomes. Six out of eight antibodies stained the the chromosomes
in a pattern just as the shown in Figure 1 to indicate that vertebrate
titin does seem to bind to chromosomes. The two antibodies that didn't
recognize the the titin the HEp-2 cells were those directed to the amino
terminus of titin. The authors explain this by saying that the KZ
cDNA is not homologous to the vertebrate titin but the muscle D-TITIN probably
will reveal homologies. Figure 6b shows where on titin binds to certain
portions of the muscle.
This paper seems to indicate a couple of things.
First it seems to prove that D-TITIN, the chromosomal-associated protein
in Drosophila is in fact homologous to the vertebrate titin.
This links titin as a chromosomal-associated protein as well as a protein
associated with the muscles. This then suggests a structural role
of titin in chromosomal condensation. More research must be done
to actually see what the role of titin is in condensation of chromosomes-not
just that it is present on chromosomes and resembles muscular titin.
The authors suggest titin functions in a similar way in condensing chromosomes
and in muscles in the actual mechanism-as a molecular ruler so as not to
allow chromosome breakage during mitosis by providing elasticity. In muscles
the elastic component of titin prevents sarcomeric disruption when muscles
are overstretched. I think that a possible way to see what the role
of titin is may be to possibly remove all or certain amounts of chromosomal
titin and seeing how far mitosis precedes and if it can proceed at all.
It may be possible to tell exactly what function is missing. One
could use site-directed mutagenesis where the gene that encodes titin is
deleted in the embryo of drosophila. A restriction enzyme
could also be used to wipe out the gene. Then certain expression
vectors such as plasmids with the titin gene inserted could be added to
the organism. The vector would allow control of titin expression
thus seeing the how much titin is necessary. The chromosomes could
be observed under the microscope in order to see where in mitosis the process
were stopped. It is important that the muscle titin remain in the
cell .
Site-directed mutagenesis can also be used to create
mutations in the titin gene and thus protein (instead of complete deletion)
to help understand the exact role of chromosomal titin. Homologous
recombination could be performed in the stem cells of a mother Drosophila
and her offspring could be analyzed for the mutation and how it affected
them. In addition, after deletingt he titin gen, the expression vectors
mentioned above, could be selected for mutated forms of the gene using
resistence to ampicillan and disruption of the lac Z gene. Since
titin is such a big protein certain areas of the protein could be narrowed
down to exact roles in chromosomal condensation.
The removal of chromosomal titin may also answer
the question of whether titin remains associated with the uncondensed chromosomes
of interphase. The cell may not be affected by the lack of titin if it
is not needed in interphase. The authors suggest that titin remains
bound to the chromosomes in interphase but do not know that function.
Another question that the paper does not completely
answer is the relationship between muscle and chromosomal titin.
The western blot in Figure 5 is not sufficient to say that the proteins
are of the same mass. As stated earlier, the molecular weight markers were
very poor. The authors describe possible ways that the 2 may be different
in terms of amino ends and carboxyl ends. The authors suggest that
the findings about D-TITIN forms (muscle and chromosomal) suggest an alternative
spice sites of the same gene and that the most amino terminal regions of
muscle titin have not been cloned. An interesting expreiment would
involve investigating whether vertabrate chromosomal and muscle titin are
splice variants of the same gen or whether whether they are encoded by
two separate genes. This could be tested by selecting for various
probes for different parts of the mRNA. Titin in other species could be
used to generate these probes. The probes could possibly identify
separate candidates for carboxyl termini within the gene. With respect
to which probes hybridized to RNA blots, one could determine which of potential
polyadenylation signals are used in mRNA processing.
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