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Vol. 14, Issue 6, 2410-2424, June 2003
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Department of Cell Biology and Human Anatomy, University of California, Davis, Davis, California 95616
Submitted July 30, 2002;
Revised December 1, 2002;
Accepted February 11, 2003
Monitoring Editor: Vivek Malhotra
 |
ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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-subunit of erythrocyte spectrin. In this study, we
show that a screen of a 
gt11 expression library resulted in the
isolation of an 
5-kb partial cDNA from a Madin-Darby bovine kidney (MDBK)
cell line, which encoded a polypeptide of 1697 amino acids with low, but
detectable, sequence homology to spectrin (37%). A blast search revealed that
this clone overlaps with the 5' end of a recently identified spectrin
family member Syne-1B/Nesprin-1, an alternately transcribed gene with
muscle-specific forms that bind acetylcholine receptor and associate with the
nuclear envelope. By comparing the sequence of the MDBK clone with sequence
data from the human genome database, we have determined that this cDNA
represents a central portion of a very large gene (500 kb), encoding an
25-kb transcript that we refer to as Syne-1. Syne-1 encodes a large
polypeptide (8406 amino acids) with multiple spectrin repeats and a region at
its amino terminus with high homology to the actin binding domains of
conventional spectrins. Golgi localization for this spectrin-like protein was
demonstrated by expression of epitope-tagged fragments in MDBK and COS cells,
identifying two distinct Golgi binding sites, and by immunofluorescence
microscopy by using several different antibody preparations. One of the Golgi
binding domains on Syne-1 acts as a dominant negative inhibitor that alters
the structure of the Golgi complex, which collapses into a condensed structure
near the centrosome in transfected epithelial cells. We conclude that the
Syne-1 gene is expressed in a variety of forms that are multifunctional and
are capable of functioning at both the Golgi and the nuclear envelope, perhaps
linking the two organelles during muscle differentiation. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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),
dystrophin (Koenig et al.,
1988), utrophin (Blake et
al., 1996), and 
-actinin
(Davison et al.,
1989). All of these proteins are functionally similar in that they
are involved in the linkage of membranes with cytoplasmic structures, thereby
providing structural support for the cell surface and, in some cases, the
formation of distinct membrane domains. The structural similarity of these
proteins lies in the conservation of two distinct structural motifs: 1) a
highly conserved amino terminal actin binding site
(Byers et al., 1989),
which facilitates cytoskeletal assembly by linking spectrin family members to
actin filaments; and 2) an 106 amino acid spectrin repeat domain, present
in multiple copies (Davison et
al., 1989; Koenig and
Kunkel, 1990; Pascual et
al., 1997), which dictates the elongated shape of these
proteins. Aside from these two conserved motifs, however, spectrin superfamily
members are otherwise structurally diverse, and this diversity likely forms
the basis for differences in function.
Functional diversity of spectrin superfamily members is exemplified by
differential tissue and subcellular distributions of these proteins.
Tissue-specific isoforms of spectrin and dystrophin are found in variety of
tissue types, with major isoforms in muscle (dystrophin/spectrin;
Bonilla et al., 1988;
Bloch and Morrow, 1989), brain
(dystrophin/spectrin; Lazarides and
Nelson, 1983; Lidov et
al., 1990), blood cells (spectrin;
Bennett, 1990), and epithelial
tissues (spectrin; Nelson and Veshnock,
1987; Nelson and Hammerton,
1989). Although differential tissue distribution of superfamily
members implies distinct tissue-specific functions for these proteins, the
fact that individual cells may possess multiple isoforms, each with their own
distinct plasma membrane localizations, indicates an additional level of
functional diversity (Lazarides and
Nelson, 1983). Moreover, additional studies have revealed that
spectrin isoforms localize to intracellular sites other than the plasma
membrane, such as the Golgi complex (Beck et al.,
1994,
1997; Devarajan et
al., 1996) and the nuclear envelope
(Apel et al., 2000;
Zang et al., 2001),
an observation that implies an even greater potential spectrum of functions
for the spectrin superfamily.
Evidence for the existence of a Golgi-localized membrane cytoskeleton has
come from the identification of Golgi-specific isoforms of the membrane
skeleton proteins spectrin and ankyrin (for review, see
Beck and Nelson, 1998). Initial
studies involved the use of antibodies raised against the erythrocyte isoform
of spectrin that were found to react with a Golgi-specific antigen in
nonerythroid cells (Beck et al.,
1994). In addition, a high molecular mass (271-kDa) variant of
erythrocyte spectrin has been reported to localize to the Golgi complex
(Stankewich et al.,
1998). Two distinct isoforms of Golgi-specific ankyrins have also
been identified. One of these, Ank195, is a 195-kDa Golgi-specific protein
that cross-reacts with erythroid ankyrin-specific antibodies
(Beck et al., 1997).
The other, AnkG119, is a truncated form of the major brain ankyrin AnkG
(Devarajan et al., 1996).
The existence of two distinct isoforms of Golgi ankyrin suggests the
possibility of multiple forms of the Golgi membrane cytoskeleton, each perhaps
facilitating a unique function. Therefore, to gain a complete understanding of
Golgi membrane cytoskeletal functions, Golgi-specific isoforms of membrane
skeleton proteins must first be definitively identified and characterized at
the molecular level. To achieve this aim we have set out to clone a
Golgi-specific isoform of spectrin by screening a gt11 expression
library with an erythrocyte spectrin-specific antibody (Spec-1) that was
previously shown to cross-react with a Golgi-specific antigen in a variety of
nonerythroid cells. We chose this approach over the use of polymerase chain
reaction (PCR) amplification because we recognized that the great sequence
diversity of spectrin superfamily members would likely render the latter
approach ineffective. This screen has resulted in the isolation of a partial
5-kb cDNA clone that expresses a polypeptide that interacts with the Golgi
with the same characteristics as the Golgi-specific spectrin previously
identified with the Spec-1 antiserum. A blast search revealed that the
MDBK clone is a bovine homolog of spectrin-related protein
Syne-1B/Nesprin-1 (Apel et
al., 2000; Zang et
al., 2001), which has previously been reported to bind to the
acetylcholine receptor and localize to the nuclear envelope. We further show
that Syne-1B/Nesprin-1 represents an alternative transcript of an
25-kb parent transcript. Expression of dominant negative inhibitory
fragments of Syne-1 results in altered Golgi morphology, suggesting that one
of the functions of this novel spectrin family member is to maintain the
structural organization and cytoplasmic distribution of the Golgi complex.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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),
human embryonic kidney (HEK) cells (293), Madin-Darby bovine kidney (MDBK),
COS-1, bovine kidney epithelial cells and LB10 cells were grown in
high-glucose Dulbecco's modified Eagle's (DME) medium containing 10% fetal
bovine serum, 100 U/ml penicillin, and 100 mg/ml streptomycin (Invitrogen,
Carlsbad, CA). Cultures were maintained at 37°C with 5% CO2 in
air. For aluminum fluoride pretreatment experiments, washed cells were
pretreated with 30 mM sodium fluoride and either 50 or 250 µM aluminum
chloride for 20 min at 37°C and then incubated with 5 µM brefeldin A
(BFA) for an additional 10 min. 293 cell extracts were prepared from confluent
cultures grown on 10-cm2 dishes. Immunofluorescence staining and
detergent extractions were performed as described previously (Beck et
al., 1994,
1997).
Library Screening
Library screening was performed using the 5'-Stretch cDNA expression
system from BD Biosciences Clontech (Palo Alto, CA). This library was
constructed from cDNAs, expressed as -galactosidase fusion proteins in
gt11, derived from cultured MDBK cells. The screening procedure was
performed according to the manufacturer's instructions (BD Biosciences
Clontech). Positive plaques were excised, extracted, and the resulting phage
stocks were subjected to two successive rounds of additional screening. A
large-scale liquid culture of gt11 clone 4 was prepared and DNA was
isolated with Lambda kits (QIAGEN, Santa Clara, CA) as described by the
manufacturer. The insert was excised by digesting with EcoRI
(Promega, Madison, WI) and subcloned into Bluescript (Stratagene, La Jolla,
CA) for sequencing.
To identify additional 5' sequences of human Syne-1, we performed 5'-rapid amplification of cDNA ends (5'-RACE) with primers complementary to the 5' end of the human sequence for the partial bovine clone. Human sequences corresponding to this region were derived from the human clone KIAA1262, which overlapped with the 5' end of the bovine cDNA. 5'-RACE PCR reactions were performed using the SMART race cDNA amplification system (BD Biosciences Clonetech), according to the manufacturer's instructions. Typically, nested primers were used. The first round of PCR was performed with the 3'-most primer and the product was diluted 1:50 and used as template for a second round of amplification by using the 5'-most primer. PCR products were then subcloned using the TOPO TA cloning system (Invitrogen) and sequenced. The resulting sequences where then submitted to the Human Genome Database at the National Center for Biotechnology Information. The submitted sequences were found to be identical to predicted exons residing within contigs of genomic DNA from chromosome 11. The bovine Syne-1 cDNA was also found to be homologous with portions of these contigs. Additional 5' sequence was then identified by predicting exons within the entire genomic contig that were upstream of the exons corresponding to the RACE products. The sequences of these predicted 5' exons were then used to generate new PCR primers and the process was repeated.
Sequencing and Sequence Analysis
Automated DNA sequencing was performed using an ABI Prism 377 DNA sequencer
(Applied Biosystems, Foster City, CA). Multiple sequence alignments were made
using the CLUSTAL W program. Spectrin repeats were originally identified with
the ProfileScan Server of the Swiss Institute for Bioinformatics
(http://www.isrec.isb-sib.ch/software/PFSCAN_form.html).
This server uses "pfscan" program to search protein sequences
against database of profiles.
Antibodies
The spec-1 and Eank-2 antisera, which react with Golgi antigens in
MDCK and MDBK cells (Beck et al.,
1994,
1997), were raised against
canine erythrocyte spectrin and ankyrin as described previously
(Beck et al., 1994).
The following antibodies were purchased: mouse monoclonal anti-Golgi p58
(Sigma-Aldrich, St. Louis, MO), mouse monoclonal anti-COP
(Sigma-Aldrich), mouse monoclonal anti-HA epitope tag (Babco, Richmond, CA),
and mouse monoclonal anti-clathrin adapter 
AP-1 (Sigma-Aldrich).
Antibodies were raised against a bacterially expressed glutathione
S-transferase (GST) fusion protein composed of amino acids
1–469 of the bovine clone. To construct this fusion protein, nucleotides
368–839 of MDBK clone 4 were amplified by PCR by using forward and
reverse primers complementary to the appropriate flanking sequences. The
forward primer was modified by adding a XhoI site to the 5'
end. To the reverse primer we added a stop codon followed by a NotI
site. The amplified PCR product was purified, digested with XhoI and
NotI, and ligated into a modified pGEX-3 x-M1 (kindly provided
by Dr. Yih-Tai Chen, Stanford University, Stanford, CA) previously digested
with XhoI and NotI (Promega). The resulting construct
encoded a fusion protein containing an amino-terminal GST in frame with the
GS1.5 region of MDBK clone 4. The protein was expressed in Escherichia
coli strain XL1-blue. Peptide antibodies were prepared against the
following peptides: EAKASSPEMDISADC (SN357-1 and SN357-2) and EESGEEGTNSEISSC
(SN119 and SN120). Peptide synthesis, conjugation to KLH, and antibody
production was performed at SynPep (Dublin, CA).
Ectopic Expression of Syne-1 Fragments
For ectopic expression of various regions of Syne-1, epitope-tagged
constructs were prepared using the identical strategy described above for
construction of GST fusion proteins. PCR products digested with XhoI
and NotI where ligated with a modified pCDM8 (Invitrogen) previously
digested with NotI and XhoI. The modified pCDM8 (kindly
provided by Dr. Yih-Tai Chen, Stanford University) encodes a 5' epitope
tag derived from the influenza hemagglutinin protein
(Chen et al., 1993).
The resulting constructs encoded fusion proteins containing an amino-terminal
HA epitope tag in frame with various bSyne-1 segments. For transient
transfections with constructs subcloned into pCDM8, 0.5 µg of DNA was mixed
with 100 µl of serum-free DME (Invitrogen) containing 5 µl of
LipofectAMINE reagent (Roche Diagnostics, Indianapolis, IN). After incubation
for 45 min at 22°C, 800 µl of serum-free DME was added to bring the
total volume to 1 ml. This mixture was added to MDBK cells plated on
collagen-coated cover slips or 293 cells grown on plastic culture dishes.
After a 6-h incubation at 37°C, the cells were washed in serum-free DME
and incubated at 37°C for 36–48 h in DME containing 5% fetal bovine
serum.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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spec-1 antiserum resulted in the isolation of four
positive clones. Their sizes were determined by PCR amplification of lambda
DNA isolates (Figure 1A). Only
one of these clones, clone 4 (Figure
1A), was large enough (5 kb) to be considered a candidate spectrin
homolog. Sequencing of clone 4 identified a single uninterrupted open reading
frame of 1594 amino acids. Because the open reading frame started immediately
at the 5' end of the cDNA and extended throughout the length of the
clone we concluded that the 4.5-kb lambda clone 4 represented a partial cDNA
missing additional 5' and 3' sequences.
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A blast search of the sequence database revealed significant homology of
the MDBK cDNA with two known cDNAs: KIAA1262, a partial cDNA of a human gene
with unknown function that overlapped with the 5' end of MDBK clone 4;
and Syne-1B/Nesprin-1, a novel spectrin family member previously shown
to associate with the nuclear envelope
(Apel et al., 2000;
Zang et al., 2001).
Sequence comparison of the bovine clone with both of these cDNAs revealed a
high level of homology at both the nucleotide (85%,
Figure 1B) and amino acid level
(Table 1), indicating that the
MDBK clone represents the bovine homolog of KIAA1262 and
Syne-1/Nesprin-1. As was reported for Syne-1/Nesprin-1
(Apel et al., 2000;
Zang et al., 2001),
the MDBK cDNA (referred to herein as bSyne-1, GenBank accession number
AF525163
[GenBank]
) showed a low but detectable homology with conventional spectrin as
well as other spectrin family members
(Table 1). Sequence comparisons
revealed that bSyne-1 is as distantly related to erythroid -spectrin as
it is to dystrophin, the spectrin family member having the lowest overall
homology to -spectrin (17% identity and 39% homology). However, bSyne-1
is no more closely related to dystrophin than it is to any other spectrin
family member (17% identity and 38% homology;
Table 1), suggesting that it is
not a dystrophin isoform. It also possesses very low overall sequence homology
with a previously identified Golgi-specific -spectrin
(Stankewich et al.,
1998). In contrast, the Golgi -spectrin is highly homologous
to erythroid -spectrin (62% identity and 77% homology), whereas bSyne-1
shows the same low level of homology with erythroid -spectrin as is does
with -spectrin (Table
1). Analysis of the primary amino acid sequence of bSyne-1
therefore indicates that although the clone was isolated by cross-reactivity
with a spectrin-specific antibody, it is only distantly related to known
spectrin family members and likely represents a novel spectrin superfamily
member.
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Syne-1/Nesprin-1 has been show to be expressed in multiple forms
(Apel et al., 2000;
Zang et al., 2001).
Syne-1B/Nesprin-1 is expressed as an 10-kb transcript with broad
tissue distribution (Apel et al.,
2000; Zang et al.,
2001), whereas Syne-1A/Nesprin-1 is a muscle specific 4.5-kb
transcript that corresponds to a C-terminal portion of Syne-1B
(Apel et al., 2000;
Figure 1D). As mentioned above,
the MDBK clone 4 cDNA encodes a single open reading frame that overlaps with
the 5' end of Nesprin-1, implying that either the proposed
transcription start site for Nesprin-1 is inaccurate or that
Nesprin-1 actually represents a truncated alternate transcript of a
larger parent molecule. To examine the expression of bSyne-1 in kidney
epithelial cells, we performed a Northern blot of MDBK cDNA with a probe
derived from the MDBK clone 4 sequence
(Figure 1D). As was previously
reported for Syne-1B/Nesprin-1 (Apel
et al., 2000; Zang
et al., 2001), a prominent 10-kb transcript was
detected (Figure 1C). We also
detected a less abundant 25-kb species, indicating the existence of an
additional, previously unidentified transcript of the Syne-1 gene.
The identification of a 25-kb Syne-1 transcript, together with the fact that the original bovine cDNA was a continuous open reading frame lacking a 5' start site, necessitated a search for upstream sequences. To accomplish this, we used an approach that used a combination of 5'-RACE and comparison of the resulting new 5' sequences to the human genome database (see MATERIALS AND METHODS). This allowed us to "walk" along the sequence in the 5' direction. Using this approach we detected an additional 15 kb of potential mRNA sequence upstream of the original MDBK clone (Figure 1D). The final 5'-RACE experiment gave rise to a sequence encoding a putative 5' untranslated region with no significant open reading frame. The coding region initiates with a Kozak consensus site. Importantly, just downstream from this putative start site we identified a region with high homology to the actin binding domain found in many spectrin-like proteins (Figure 2). Because actin binding domains are located at the extreme amino-termini of all known spectrin family members, this result confirmed that we had identified the 5' end of Syne-1. Using human genome sequence data, we constructed a hypothetical cDNA that was 26,089 nucleotides in length and encoded a polypeptide of 8406 amino acids (Figure 1D). To verify that the predicted transcript for human Syne-1 (hSyne-1, GenBank accession number BK000543 [GenBank] ) is expressed in intact cells, we performed reverse transcription-polymerase chain reaction (RT-PCR) with HEK-293 cell cDNA by using primers designed to detect overlapping fragments of hSyne-1. Additionally, each of these PCR products was subjected to one round of DNA sequencing to demonstrate that the appropriate sequence was amplified (data not shown). In all cases, the primer pairs generated fragments of the appropriate size (Figure 1D).
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All spectrin family members are related structurally by the presence of a
conserved amino-terminal actin binding domain
(Byers et al., 1989),
as well as multiple, sequential copies of a 106 amino acid spectrin repeat
domain (Koenig et al.,
1988; Pascual et al.,
1997). Therefore, to find additional evidence that the sequences
predicted from human genome data were contiguous with our MDBK clone, we
examined the amino acid sequence of these upstream segments to identify
spectrin-like domains. As mentioned above, at the extreme amino terminus of
hSyne-1 we detected an 250 amino acid region with extensive homology to a
spectrin-actin binding domain (Figure
2). Throughout this region we found good correspondence between
the amino acid sequence of hSyne-1 with that of other spectrins. If fact, the
homology within this region was much greater than the overall homology between
Syne-1 and other family members. However, the putative actin binding domain of
hSyne-1 differed from that of the other spectrins in that it contained a
serine- and proline-rich insert (Figure
2). Importantly, this insert lies within one of the three helical
segments of the utrophin actin binding domain that have been shown to
associate directly with actin. This suggests that Syne-1 may have altered
actin binding activity. When the hSyne-1 amino acid sequence was submitted as
a query to the ProfileScan Server of the Swiss Institute for Experimental
Cancer Research (see MATERIALS AND METHODS), numerous spectrin repeat domains
were detected along the entire length of the molecule (our unpublished data).
Importantly, spectrin repeats were found not only in the regions corresponding
to Syne-1A and B, as has been reported previously
(Apel et al., 2000;
Zang et al., 2001),
but they were also present within the MDBK clone 4 sequence as well as the
5' upstream sequences predicted from human genome data (our unpublished
data). This confirms that all of these sequences are derived from the same
gene. Because all of these features of the hSyne-1 sequence upstream of our
original bovine clone are consistent with a spectrin-like protein, and because
RT-PCR results show overlapping identity with the original sequence, we
conclude that the upstream sequence identified by our 5'-RACE approach
is indeed contiguous with MDBK clone 4.
Functional Analysis of Syne-1
Because the initial screen of the gt11 library was performed with
an antibody raised against erythrocyte -spectrin, we were surprised by
the low level of sequence homology of the isolated cDNA with spectrin. To
verify that the antibody used in the initial screening does indeed react with
the bSyne-1 protein, we expressed three epitope-tagged fragments of bSyne-1
(HAf3, HAf4, and HAf5; Figure
3A) in 293 cells and analyzed extracts of these cells by PAGE and
immunoblotting (Figure 3B). The
electrophoretic mobilities of these three fragments were determined by
immunoblotting with an epitope-tag specific antibody
(Figure 3B, a, c, and e). On
immunoblotting with the spec-1 antibody, we found that two of these
fragments, HAf5 and the N-terminal portion of HAf5, HAf3
(Figure 3B, d and e),
cross-reacted with the spectrin-specific antiserum. In contrast, HAf4, the
c-terminal half of HAf5, did not react with spec-1
(Figure 3B, f). It should be
noted that because of relatively low transformation frequency, neither of the
ectopically expressed fragments could be detected in these extracts by general
protein stain (Coomassie Blue and Ponceau S; our unpublished data), indicating
that the reactivity with the spec-1 antibody was not due to nonspecific
binding to an exceptionally abundant protein. Furthermore, the specificity of
the spec-1 antibody is also demonstrated by the absence of reactivity
with HAf4, which was expressed at levels roughly equivalent to the other two
fragments. These results indicate that despite the low-level homology with
spectrin family members the MDBK protein does indeed react with the
spectrin-specific antibody.
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To establish whether bSyne-1 is a Golgi resident protein, we first expressed recombinant fragments of bSyne-1 in intact MDCK cells and human LB10 epithelial cells and determined their subcellular distributions by indirect immunofluorescence microscopy (Figure 4). Recombinant fragments (HAf1-5, Figure 3A) were epitope tagged with eight amino acids derived from the influenza HA protein. Transient transfections of MDCK cells were performed and the localization of recombinant proteins was determined by staining with HA-specific antibodies (Figure 4). Golgi localization was observed with HAf2 (Figure 4a), HAf3 (Figure 4, c, g, and i), and HAf5 (Figure 4e). Note that HAf5 contains the sequence of HAf3 within it (Figure 3). With all of these fragments, we observed distinct localization to perinuclear Golgi structures (Figure 4, arrows) that costained with a variety of Golgi markers, including a Golgi-specific ankyrin antibody EAnk-2 (Figure 4, b and f), KDEL receptor (Figure 4h), and the cis Golgi/intermediate compartment marker p58 (Figure 4j). In addition to the Golgi localized signal, we also observed diffuse cytoplasmic localization with these fragments, consistent with a cytoplasmic pool of protein in equilibrium with a Golgi bound fraction. Somewhat less obvious Golgi localization was observed with the HAf2 fragment (Figure 4a) than with HAf3. Typically, Golgi-specific staining was more difficult to detect with HAf2 fragment due to higher cytoplasmic staining compared with that observed with HAf3, suggesting that the HAf2 fragment binds Golgi with a lower affinity. Golgi localization was not observed with HAf4, which gave a diffuse cytoplasmic signal, or HAf1, which localized to discrete cytoplasmic puncta that did not correspond to any known organelle (our unpublished data). These results demonstrate a capacity for Syne-1 to bind and localize to the Golgi complex. Furthermore, they also identify at least two distinct Golgi binding sites on Syne-1, a low-affinity site in the vicinity of amino acids 4606–4945, and a high-affinity site within amino acids 5015–5410. It should be noted that we did not observe nuclear envelope localization with any of the fragments tested.
|
As an alternative approach to demonstrate Golgi localization for Syne-1, we
prepared a polyclonal antiserum, GS1.5, to a recombinant bSyne-1 fragment that
corresponds to the HAf2 region (Figure
3A). We also prepared antibodies to two different peptides derived
from the Syne-1 sequence (Figure
3A, SN 357, SN119, and SN120). When these antibodies were used to
stain MDBK and LB10 cells by indirect immunofluorescence
(Figure 5), we found that the
predominant feature of the resulting staining patterns were reticular,
perinuclear Golgi structures. These immunoreactive structures costained with
antibodies to two markers of the Golgi complex, p58
(Figure 5, b and n) and the
Golgi-specific coat protein -COP
(Figure 5d). To test the
specificity of the GS1.5 antiserum, we affinity purified the antibody by using
two different affinity columns. The first was composed of recombinant HAf2,
the antigen used to prepare the antiserum. Material bound to this column was
eluted and tested for Golgi staining activity
(Figure 5e). As expected, Golgi
staining was observed with antibody affinity purified against the original
antigen. In contrast, Golgi-specific immunoreactivity was not observed with
the eluate from an affinity column composed of purified erythrocyte spectrin
(Figure 5g), indicating that
the GS1.5 antiserum is specific for bSyne-1 and does not cross-react with
erythroid spectrin. Thus, the ability of the GS1.5 antiserum to stain the
Golgi complex correlates only with its ability to bind bSyne-1. Golgi staining
observed with peptide antibodies SN120 and SN119 was also specific because it
could be blocked by preabsorption with peptide
(Figure 5, j and l).
Furthermore, it is worth noting that the SN120 antibody was raised against a
peptide that resides within the HAf3 fragment
(Figure 3A) and that when we
transfected MDBK cells with HAf3 and stained them with SN120 we observed
increased intensity of both cytoplasmic and Golgi-specific staining in
transfected cells (Figure 4d,
arrow) verses nontransfected cells (Figure
4d, arrowhead). This indicates that the SN120 does indeed react
with the antigen to which it was raised, even when that antigen was presented
in fixed cells prepared for immunofluorescence microscopy. Finally, specific
Golgi staining was not only observed with antibodies raised against epitopes
that resided within the original MDBK clone 4. We also observed specific Golgi
staining with two separate antibodies (SN357-1, our unpublished data; and
SN357-2, Figure 5m) raised
against a peptide derived from a site far upstream from the MDBK clone 4
homology region (Figure
3A).
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The antibody used to identify bSyne-1, spec-1, had been previously
shown to react with a Golgi-specific spectrin isoform
(Beck et al., 1994).
Having established that Syne-1 is a Golgi-localized protein structurally
similar to spectrin at the level of primary sequence data, we next set out to
determine whether Syne-1 shares functional properties with the Golgi-specific
spectrin previously identified with the spec-1 antiserum. First, it had
previously been shown that Golgi spectrin, identified with spec-1,
associates with the Golgi complex in the form of a detergent insoluble
structure that was referred to as a Golgi ghost
(Beck et al., 1997).
To localize Syne-1 to a detergent-insoluble Golgi ghost, we extracted cells in
Triton X-100 before fixation and examined the distribution of Syne-1 by
immunofluorescence microscopy (Figure
6). We have previously shown that although the extraction
conditions used in these experiments are sufficient to fully extract proteins
and lipids from the Golgi, both Golgi spectrin and ankyrin remain associated
with a cytoplasmic structure that is morphologically identical to the Golgi
complex (Beck et al.,
1997). When we examined the distribution of Syne-1 in cells
extracted under identical conditions, we found that the protein remained
localized to a cytoplasmic structure
(Figure 6c) that costained with
the Golgi marker mannosidase II (Figure
6d) and was morphologically equivalent to the Golgi of
nonextracted cells (Figure 6, a and
b).
|
Previous studies have also shown that the Golgi-specific spectrin isoform
identified with spec-1 (which we refer to here as Golgi spectrin)
associates with Golgi membranes in a BFA-sensitive manner (Beck et
al., 1994,
1997). Like Golgi spectrin,
bSyne-1 rapidly dissociates from Golgi membranes after 10-min treatment with
BFA (Figure 7, e and q), as
revealed by a diffuse cytoplasmic staining
(Figure 7e) and a lack of
colocalization with the Golgi marker mannosidase II
(Figure 7, q and r).
BFA-induced dissociation of both Golgi spectrin and bSyne-1 is fully
reversible upon washout of BFA (data not shown). Furthermore, the effects of
BFA on the distributions of Golgi spectrin and the bSyne-1 are blocked by
aluminum fluoride (Figure 7, o and
m). This effect, which was also observed with other BFA-sensitive
coat proteins such as AP-1 (Figure
7j; Wong and Brodsky,
1992), suggests a role for GTPases in the regulation of Golgi
membrane skeleton assembly. Interestingly, the concentrations of aluminum
fluoride required to affect the assembly of both bSyne-1 and Golgi spectrin
(Figure 7, m and o) differed
from those affecting the activity of AP-1
(Figure 7, j and l). A fivefold
higher concentration of aluminum fluoride was required to protect Golgi
spectrin and bSyne-1, suggesting that although Golgi spectrin and other coat
proteins (i.e., clathrin and coatamer coats) may be regulated in a similar
manner, the actual agents that facilitate this regulation are different.
Importantly, in all cases the bSyne-1 behaved identically to the
Golgi-localized spectrin originally identified with the spec-1
antiserum, indicating that these two molecules are functionally
equivalent.
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A Role for Syne-1 in Maintaining Golgi Structure
As mentioned above, the recombinant Syne-1 fragment HAf3 localized to the
Golgi (Figure 4). While
performing these experiments, we noticed that the structure of the Golgi
complex was often altered in cells expressing this fragment. This was evident
only in MDBK cells, which possess a Golgi complex that is highly reticular in
appearance and tends to form a crescent shape that follows the contour of the
nucleus (Figure 5 a, c, and m). MDBK cells expressing HAf3 and HAf5 tended to have a more compacted Golgi
(Figure 4, d and f). This
altered Golgi morphology was more evident in cells expressing high levels of
Golgi binding fragments of Syne-1 (Figure
8). Figure 8 shows
several MDBK cells expressing the Syne-1 fragments HAf3, the Golgi binding
fragment; HAf5, a larger fragment that contains the sequence encoding HAf3;
and HAf 4, the C-terminal half of HAf4 that does not bind Golgi. We judged the
expression levels of these fragments to be high in these experiments because
the accumulation of cytoplasmic fragment obscured the Golgi-localized
material. Significant compaction of the Golgi complex was observed in cells
expressing Golgi binding Syne-1 fragments
(Figure 8, d and f, arrows),
whereas untransfected cells (Figure 7, b
and f, arrowheads) and cells expressing HAf4
(Figure 8h), which does not
bind Golgi, had Golgi complexes with normal crescent-shaped reticular
morphologies. Compaction of the Golgi apparatus upon expression of HAF3 was
also observed in C2C12 myoblasts but was not observed in COS-7 cells, which
normally possess a more compacted Golgi complex (data not shown). We interpret
these results as an indication that the Golgi binding fragments of Syme-1 act
as dominant negative inhibitors of Syne-1 function at the Golgi, perhaps by
competing with endogenous Syne-1 for Golgi binding sites. Furthermore, we
conclude that the altered Golgi morphology observed upon expression of these
fragments indicates that at least one function of Golgi-associated Syne-1 is
to maintain Golgi structure and distribution. This effect is apparently cell
type specific. Finally, the ability of Syne-1 fragments to alter Golgi
structure also confirms our general conclusion that Syne-1 can reside and
function at the Golgi.
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|
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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spec-1,
was raised against canine erythrocyte -spectin, and despite the low
overall sequence homology of the isolated clone with other spectrin family
members (Table 1), this
antibody reacted with at least two distinct domains encoded by the MDBK clone
(Figure 3B). Previous reports
have shown that the spec-1 antiserum reacts with a Golgi-localized
protein in nonerythroid cells, including a variety of epithelial cell lines
such as MDBK, the source of the cDNA used in our screen. Functionally, the
MDBK clone isolated in this study behaves identically to the proposed
Golgi-spectrin isoform originally identified with the spec-1 antiserum.
Like Golgi spectrin, MDBK clone 4 localizes to the Golgi complex, as indicated
by two different approaches used in this study: ectopic expression of MDBK
clone 4 cDNA fragments (Figure
4) and indirect immunofluorescence microscopy with four different
antibody preparations (Figure
5). Syne-1 associates with Golgi membranes as a
detergent-insoluble complex (Figure
6) and, like spec-1–reactive epitope, its association
with the Golgi is sensitive to BFA and aluminum fluoride treatment
(Figure 7). Thus, in all of the
functional tests performed herein, Syne-1 consistently behaved identically to
the Golgi spectrin isoform identified with the spec-1 antiserum, the
same antiserum used to isolate the clone.
Additional evidence that Syne-1 can reside and function at the level of the
Golgi complex comes from our observation that ectopic expression of Syne-1
fragments alters the morphology of the Golgi
(Figure 8), suggesting that
Syne-1 serves to maintain Golgi structural organization. This function is
consistent with Syne-1 being a spectrin family member because it is well
established that one of the functions of spectrin and spectrin-like proteins
is to maintain the structural integrity of the plasma membrane. As for the
mechanism whereby Golgi structure is perturbed by the Syne-1 fragments, it is
interesting to note that the condensed Golgi complex that we have observed in
transfected cells is remarkably similar to Golgi structure changes observed
after perturbation of the actin cytoskeleton by treatment with cytochalasin D
(Valderrama et al.,
1998) or by expression of the transforming N-Ras mutant K61
(Babiá et al.,
1999), suggesting that Syne-1 may exert its effects on Golgi
structure by interacting with the actin cytoskeleton,. This is also consistent
with Syne-1 being a spectrin family member with a conserved actin binding
domain (Figure 2). Furthermore,
it is well established that Golgi structural organization is dependent on
microtubules and microtubule motor proteins such as kinesin and dynein
(Thyberg and Moskalewski,
1985). We have shown previously that Golgi-specific ankyrin and
spectrin are constituents of a detergent-insoluble Golgi ghost that is
anchored in the cytoplasm to a drug and temperature stable population of
microtubules (Beck et al.,
1997). In this study, we show that Syne-1 also localizes to this
structure (Figure 6),
suggesting that Syne-1 could serve to affect Golgi structure by linking Golgi
membranes to microtubules. In support of this, previous studies using the same
-spectrin antibody used herein to clone Syne-1, spec-1, have shown
that Golgi spectrin associates with the dynactin complex, a regulator of
dynein function (Holleran et al.,
1996). In addition, recent studies in our laboratory have
identified a binding site on Syne-1 for the trimeric kinesin KIF3B (Fan and
Beck, unpublished data), suggesting that Syne-1 could effect Golgi structure
through both plus and minus end directed microtubule motors. Finally, it
should be pointed out that the Syne-1–dependent Golgi structure changes
that we have observed occurred in MDBK cells but not in COS cells, and that
the condensed Golgi observed in transfected MDBK cells resembles the normal
Golgi morphology observed in COS cells. This suggests that Syne-1 is involved
in aspects of Golgi structure that may give rise to cell type-specific Golgi
morphology.
A search of the protein sequence database identified a high level of
homology (85%) between the MDBK cDNA and the 5' end of a recently
identified spectrin family member Syne-1B/Nesprin-1. Syne-1B is an
alternative transcript of a parent gene that was found to be expressed in at
least two forms: a muscle specific form, Syne-1A, of 4.7 kb expressed in
heart, skeletal muscle, and smooth muscle, and a larger 10-kb form,
Syne-1B, that has a more general tissue distribution
(Apel et al., 2000;
Zang et al., 2001).
We have performed multiple tissue Northern blots with probes derived from the
MDBK clone 4 region of Syne-1 and observed tissue distributions identical to
published distributions of the 10-kb Syne-1B/Nesprin-1 (our
unpublished data). The observation that the MDBK clone isolated in this study
encoded a continuous open reading frame that overlaps with the proposed
5' end of Syne-1B/Nesprin-1 inferred the existence of a larger
parent molecule that gives rise to both alternate transcripts. We determined
the sequence of the putative precursor to Syne-1A and B by 5'-RACE PCR
combined with comparison of the resulting sequences to the human genome
database. This approach identified a very large (>500-kb) gene that is
predicted to encode a transcript of 25 kb, with Syne-1B corresponding to
the 3' half of the transcript and the MDBK clone aligning roughly with
the center of the molecule. We refer to this gene and its predicted product as
Syne-1. Amino acid analysis identified additional spectrin repeats along the
sequence extending upstream of Syne-1B, as well as a highly conserved actin
binding domain at the amino terminus. While this paper was in review, two
groups reported full-length sequences for Syne-1/Nesprin and related proteins
in Drosophila and Caenorhabditis elegans that confirm the
sequence reported here (Starr and Han,
2002 and Zhang et
al., 2002)
An additional form of the Syne-1 molecule can be predicted by examining the
sequence of a partial cDNA reported in the database as KIAA1262. This partial
cDNA aligns with the 5' end of MDBK clone 4
(Figure 1D). Importantly,
KIAA1262 clearly represents the 3' end of a transcript. It encodes an
open reading frame that initiates at the first nucleotide but extends only to
nucleotide 4089. Beyond this point, no significant open reading frame can be
detected in the remaining 1500 nucleotides, indicating the presence of a
3' untranslated region. The homology of KIAA1262 with the MDBK clone 4
begins at nucleotide 2248 and extends only to the end of the open reading
frame. Examination of the predicted intron/exon structure of the Syne-1 gene
reveals that the homology between KIAA1262 and the MDBK clone ends precisely
at the 3' end of a coding exon and that the putative untranslated region
of KIAA1262 aligns with the adjacent downstream intron. These observations
predict a fourth major form of Syne-1 that represents a premature truncation
of the full-length Syne-1. We refer to this transcript as Syne-1C
(Figure 9). Importantly, we
have found herein that an antibody raised against a peptide derived from the
putative Syne-1C region (SN357-2) also stains the Golgi
(Figure 5m), suggesting that
Syne-1C can reside and function at the level of the Golgi complex, although it
is also possible that the Golgi staining observed with SN357-2 is due to
Golgi-localized full-length Syne-1 and not Syne-1C. Thus, it seems that Syne-1
gene can be expressed in a variety of different forms, including a large
full-length molecule (Syne-1), the N-terminal half alone (Syne-1C), the
C-terminal half alone (Syne-1B), and a truncated muscle-specific C-terminal
segment (Syne-1A). It is interesting to note that of the four forms of Syne-1,
only the full-length Syne-1 and the predicted Syne-1C are expected to encode
an N-terminal actin binding domain. Actin binding domains are found in all
spectrin family members with the notable exception of -spectrin,
suggesting that isoforms of Syne-1 lacking actin binding sites could represent
homologs of -spectrin and serve to form heterodimers with actin binding
isoforms. It is tempting to speculate that the C-terminal isoforms (Syne-1A
and B) could interact with N-terminal isoforms in an anti-parallel manner, as
do - and -spectrin. Perhaps these associations can occur as
intermolecular dimmers of separate Syne-1A/B and Syne-1C chains or as an
intramolecular association resulting from the folding of the full-length
syne-1 in half. Such an arrangement could explain the complicated splicing of
the Syne-1 gene.
|
The most obvious discrepancy between this study and previous reports is the
subcellular localization of Syne-1. The nuclear envelope binding site for
Syne-1A and Syne-1B has been localized to the extreme C termini of both
molecules (Apel et al.,
2000; Zang et al.,
2001), whereas the two Golgi binding domains identified in this
study are widely separated from nuclear envelope binding determinants
(Figure 9), with both being
found near the center of the full-length Syne-1 molecule, one at the N
terminus of Syne-1B and the other at the C terminus of the putative Syne-1C
(Figure 9). Syne-1B and the
full-length Syne-1, having both Golgi and nuclear envelope binding sites,
would be expected to interact with both organelles. However, because Syne-1A
lacks the Golgi binding sites detected herein, we propose that it represents a
nuclear envelope-specific form. One possible explanation for the discrepancy
observed in this study versus other reports may lie in the specificity of the
antibodies used. Previous studies showing nuclear envelope localization of
Syne-1 were performed with antibodies that recognized the nuclear envelope
binding Syne-1A (Apel et al.,
2000; Zang et al.,
2001). Because Syne-1A is the predominant isoform in muscle, and
because Syne-1A contains a nuclear envelope binding site and not a Golgi
binding site, it is not surprising that nuclear envelope staining predominates
with this antibody. In contrast, our antibodies were raised to a region of
Syne-1 that contained the Golgi binding domain. Because this domain is not
present on Syne-1A, we can conclude that we are examining the distribution of
other Syne-1 forms, perhaps including isoforms that do not associate with the
nuclear envelope. Alternatively, as will be discussed below, the Golgi complex
is known to maintain a close association with the nuclear envelope in muscle
cells. In fact, at the level of the light microscope it may be difficult to
distinguish Golgi and nuclear envelope staining muscle, indicating that that
previous observations do not necessarily rule out Golgi localization in these
cells. Other possible mechanisms that would allow Golgi localization to
predominate over nuclear envelope localization in a given cell type would be
the existence of isoforms that contain Golgi binding sites but no nuclear
envelope binding sites, the masking of the nuclear envelope binding site by
protein–protein interactions or secondary modification, or the absence
of a receptor for Syne-1 on the nuclear envelope.
It is also possible that some cell types may require the activity of a
molecule that can bind to both the Golgi and the nuclear envelope. For
instance, there is evidence that the Golgi complex has special relationship
with the nuclear envelope in muscle cells. In both cardiac and skeletal
muscle, the Golgi has been found to form a uniform ring around the nucleus, a
configuration that is distinct from the perinuclear, reticular Golgi found in
nonmuscle cells (Tassin et al.,
1985a; Kronebusch and Singer,
1987). At the ultrastructural level, the muscle cell Golgi
apparatus is held at a uniform distance of 150–200 nm from the
nuclear envelope (Tassin et al.,
1985a), suggesting a specific physical connection between the two
organelles. In addition, functional coupling of Golgi and nuclear envelope in
muscle cells is indicated by the observation that the Golgi complex of muscle
cells maintains a close association with ER exit sites located in discrete
regions of the nuclear envelope (Lu et
al., 2001). Finally, the observation that Golgi morphology in
muscle cells is insensitive to nocodazole
(Tassin et al.,
1985b) suggests that mechanisms responsible for the unique
cytoplasmic positioning of the muscle Golgi differ from those of other cell
types. We propose that forms of Syne-1 that possess Golgi and nuclear envelope
binding sites (Syne-1 and Syne-1B) would be excellent candidates for
maintaining Golgi organization in muscle cells because they could serve to
tether the Golgi to the nuclear envelope. Interestingly, Syne-1B is expected
to be 1.5–2 times the size of conventional spectrin
(Figure 9), provided it folds
similarly. Because erythrocyte spectrin forms herterodimers of 100 nm,
Syne-1B could form a similar rod-like structure of 150–200 nm,
precisely the dimensions of the spacing observed between the Golgi and the
nuclear envelope in muscle cells (Tassin et al., 1985). Support for
such a role for Syne-1 in facilitating Golgi–nuclear envelope
interactions comes from the observations that factors influencing Golgi
distribution in muscle cells also effect the distribution of Syne-1. For
instance, Golgi distribution in muscle is developmentally regulated. In
myoblasts, the Golgi complex has a morphology and cytoplasmic distribution
that is typical of fibroblasts and other epithelial cells. However, upon
fusion to form myotubes, both the Golgi and centrosomes rearrange and acquire
a close association with the nuclear envelope
(Tassin et al.,
1985a; Lu et al.,
2001). Apel et al.
(2000) found that Syne-1
localizes to cytoplasmic structures in undifferentiated myoblasts, whereas
upon myoblast fusion, Syne-1 redistributes to the nuclear envelope. In muscle
fibers, close association of the Golgi with the nuclear envelope is affected
by muscle impulse activity (Ralston et
al., 2001), and in some muscle fiber types the density of
nuclear envelope associated Golgi elements is greater on nuclei associated
with neuromuscular junctions (Ralston
et al., 2001). Remarkably, studies examining localization
of Syne-1 in intact muscle fibers have found that Syne-1 is more abundant in
nuclei associated with neuromuscular synapses than in extrasynaptic nuclei
(Apel et al.,
2000).
Aside from serving to maintain Golgi structural organization and
distribution in muscle cells, Syne-1 could also serve additional
Golgi-specific functions in muscle and nonmuscle cell types. At the plasma
membrane, spectrin family members are known to function in facilitating
membrane association with the cytoskeleton, the promotion of general membrane
stability, and the formation of discrete membrane domains. These same
functions could significantly impact a variety of Golgi-specific functions,
including regulation of membrane protein compositions of discrete Golgi
compartments, the sorting and segregation of newly synthesized plasma membrane
proteins within the trans-Golgi network
(Beck and Nelson, 1998), and
various membrane-cytoskeleton interactions such as microtubule based vesicular
transport. These latter two proposals are particularly relevant to the fact
that Syne-1 was initially identified in a two-hybrid screen for acetylcholine
receptor binding proteins (Apel et
al., 2000). Although it is yet unclear why a nuclear envelope
protein would interact with a plasma membrane receptor, an interaction with a
Golgi resident protein would have more obvious implications for our
understanding of acetylcholine receptor function. Finally, with the view that
Syne-1 could function in muscle cells to physically couple Golgi to the
nuclear envelope, it is worth reconsidering our observation that expression of
Golgi binding fragments of Syne-1 in MDBK kidney epithelial cells alters Golgi
structure (Figure 7). In these
studies, we found that the Golgi complex, which is normally a crescent-shaped
reticular structure that follows the contour of the nucleus, adopts a
compacted morphology in cells expressing dominant-negative Syne-1 fragments,
as if the Golgi had been pealed away from the nuclear envelope and collapsed
around the centrosome. This observation suggests that even in kidney cells
Syne-1 may serve to tether some Golgi elements to the surface of the nucleus
and that Golgi–nuclear envelope interactions may be a widespread
phenomena occurring in various cells types.
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ACKNOWLEDGMENTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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* Corresponding author. E-mail address: kabeck{at}ucdavis.edu.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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