Syt Proteins Research Articles

Synaptotagmins: Beyond Presynaptic Neurotransmitter Release

Synaptotagmins (Syts) are well-established primary Ca2+ sensors to initiate presynaptic neurotransmitter release. They also play critical roles in the docking, priming, and fusion steps of exocytosis, as well as the tightly coupled exo-endocytosis, in presynapses. A recent study by Awasthi and others (2019) shows that Syt3 Ca2+-dependently modulates the postsynaptic receptor endocytosis and thereby promotes the long-term depression (LTD) and the decay of long-term potentiation (LTP). This work highlights the importance of Syt3 in modulating long-term synaptic plasticity and, importantly, extends the function of Syt proteins from presynaptic neurotransmitter release to a new promising postsynaptic receptor internalization.

Synaptotagmin-7 Endows a Subpopulation of Chromaffin Granules with Distinct Calcium Sensing and Fusion Properties

Syt-7 is one of two major Ca2+-sensors for regulated release from adrenal chromaffin cells. Its unique biochemistry provides cells with a highly Ca2+ sensitive pool of dense core granules for exocytosis. Syt-7 also participates in limiting the rate at which fusion pores expand via mechanisms that are not fully clear. All previous imaging studies of Syt-7's actions during exocytosis in chromaffin cells have relied on overexpression of WT or mutant Syt protein. Here, we imaged exocytosis in mouse chromaffin cells without Syt-7 for the first time, so that properties which depend on the protein could be directly determined. Our data show that cells lacking Syt-7 exhibit at least a five-fold difference in fusion efficacy compared to WT cells in response to elevated K+ depolarization. Lumenal granule cargos are also released at faster rates from Syt-7 knockout (KO) cells, consistent with data from overexpression studies. Next, we measured the responses of WT and KO cells to endogenous secretogoguges of the sympatho-adrenal synapse, acetylcholine (ACh) and pituitary adenylate cyclase-activating peptide (PACAP). Compared to WT cells, ACh-evoked release in KO cells is significantly impaired with respect to granule fusion kinetics and fusion efficacy. PACAP-evoked release, on the hand, is only mildly suppressed. Our data provide evidence for Syt-7's role in two critical aspects of exocytosis – fusion and fusion pore expansion. Moreover, the separable responses of WT and KO cells to endogenous secretogogues suggest that Syt proteins occupy distinct roles in the physiology of the chromaffin cell system.

The Synaptotagmin-7 C2AB Domain Alters Membrane Morphology in a Calcium-Dependent Manner

The proper execution of Ca2+-triggered exocytosis requires extreme changes in bilayer shape to be precisely regulated. Because membranes alone are unlikely to undergo the required changes, proteins are necessary to mediate events. However, the identity of these proteins and the conditions under which they act are not well understood. Within this context, our studies focus on membrane-targeting C2 domains from synaptotagmin-7 (Syt-7) - an isoform of the Syt protein family that is important for secretion in neuroendocrine cells. To define how Syt-7 drives changes in membrane morphology, we have used recombinant C2AB protein fragments, supported lipid bilayers (SLBs), and total internal reflection fluorescence microscopy (TIRFM). We have developed conditions for forming SLBs under which the membrane spontaneously forms long tubule-like structures extending away from the support surface. Upon addition of purified Syt-7 C2AB in the presence of Ca2+, these long tubules disappear and are replaced by shorter tubules or vesicles. The effect is reversible upon removal of Ca2+. These studies demonstrate that Syt-7 can alter membrane morphology, ostensibly by driving changes in membrane curvature. They also demonstrate the utility of a novel experimental system in which protein-mediated changes in membrane topology can be studied in aqueous media and in real-time.

The Membrane Bending Action of the Syt-1 C2AB Studied on Supported Lipid Bilayers

Calcium-triggered exocytosis is vital to the cellular and physiological homeostasis of higher organisms. The broad rules of exocytosis, including its regulation by SNARE proteins and Ca2+, have now been known for several years. However, there is a gap in our knowledge with respect to the molecular events that regulate fusion pore expansion and concomitant content dispersal. Within this context, the studies focus on the Ca2+-binding Synaptotagmin (Syt) family of proteins. Syt actions during exocytosis are often tied to its ability to bend and deform membranes. But very little real-time evidence exists that Syt bends membranes or that membrane bending is an essential part of its function. In this project, we utilized synthetic membranes and purified Syt proteins to answer a specific question: Does Syt bend membranes in a Ca2+-dependent manner? To answer this question, we used a combination of in vitro approaches, including recombinant Syt C2AB proteins, supported lipid bilayers (SLBs), and a curvature-sensitive optical technique called polarized total internal reflection fluorescence microscopy (pTIRFM). Although the results are preliminary, we find that purified Syt proteins added to supported lipid bilayers (SLBs) do create localized membrane deformations in the presence of Ca2+. Future work will investigate how the bending process is regulated, whether it differs between synaptotagmin isoforms, and ultimately, how it relates to the function of synaptotagmin in living cells undergoing exocytosis.

Probing the Structural Origins of Unusually Strong Target Membrane Affinity of Synaptotagmin 7 C2A and C2Ab Domains

Synaptotagmin (Syt) proteins serve as Ca2+ sensitive triggers in many exocytotic pathways, with the seventeen different human isoforms active in various cell types. Syt proteins contain two C2 domains, C2A and C2B, which bind membranes in response to Ca2+ to drive vesicle fusion. Much is known about the biophysical mechanism of function for Syt1, which triggers fusion for rapid neurotransmitter secretion, but less mechanistic information is available for other isoforms. Syt7 typically operates in slower pathways requiring smaller peak Ca2+ concentrations, and its C2 domains are known to bind membranes with a much higher Ca2+ sensitivity compared to Syt1. using kinetic and equilibrium fluorescence measurements of C2A domain docking to synthetic liposomes approximating the lipid composition of physiological membranes, we report that the differences between the two isoforms include kinetic and solute effects consistent with much greater hydrophobic membrane contact for Syt7 C2A. A strong hydrophobic contribution to the membrane docking mechanism of Syt7 C2A stands in contrast to the known electrostatic membrane interaction of Syt1 C2A, and is somewhat surprising given the 90% conserved amino acid polarity between the two domains. In order to test our proposed hydrophobic docking model for Syt7 C2A and probe its structural origins, we use a combination of site-directed mutagenesis, equilibrium and kinetic protein-membrane docking assays, and electron paramagnetic resonance-based depth measurements. In addition, single-molecule measurement of protein lateral diffusion on supported lipid bilayers is used to report on contributions of intra- and intermolecular protein-protein contact to the membrane-docked states of individual C2 domains and C2AB tandems. The results are interpreted to provide information on the structural origins of differences in function between these two isoforms.

Epigenetic features of human mesenchymal stem cells determine their permissiveness for induction of relevant transcriptional changes by SYT-SSX1.

BackgroundA characteristic SYT–SSX fusion gene resulting from the chromosomal translocation t(X;18)(p11;q11) is detectable in almost all synovial sarcomas, a malignant soft tissue tumor widely believed to originate from as yet unidentified pluripotent stem cells. The resulting fusion protein has no DNA binding motifs but possesses protein-protein interaction domains that are believed to mediate association with chromatin remodeling complexes. Despite recent advances in the identification of molecules that interact with SYT-SSX and with the corresponding wild type SYT and SSX proteins, the mechanisms whereby the SYT-SSX might contribute to neoplastic transformation remain unclear. Epigenetic deregulation has been suggested to be one possible mechanism.Methodology/Principal FindingsWe addressed the effect of SYT/SSX expression on the transcriptome of four independent isolates of primary human bone marrow mesenchymal stem cells (hMSC). We observed transcriptional changes similar to the gene expression signature of synovial sarcoma, principally involving genes whose regulation is linked to epigenetic factors, including imprinted genes, genes with transcription start sites within a CpG island and chromatin related genes. Single population analysis revealed hMSC isolate-specific transcriptional changes involving genes that are important for biological functions of stem cells as well as genes that are considered to be molecular markers of synovial sarcoma including IGF2, EPHRINS, and BCL2. Methylation status analysis of sequences at the H19/IGF2 imprinted locus indicated that distinct epigenetic features characterize hMSC populations and condition the transcriptional effects of SYT-SSX expression.Conclusions/SignificanceOur observations suggest that epigenetic features may define the cellular microenvironment in which SYT-SSX displays its functional effects.

Characterization of the role of the Synaptotagmin family as calcium sensors in facilitation and asynchronous neurotransmitter release

Ca(2+) influx into presynaptic nerve terminals activates synaptic vesicle exocytosis by triggering fast synchronous fusion and a slower asynchronous release pathway. In addition, a brief rise in Ca(2+) after consecutive action potentials has been correlated with a form of short-term synaptic plasticity with enhanced vesicle fusion termed facilitation. Although the synaptic vesicle protein Synaptotagmin 1 (Syt1) has been implicated as the Ca(2+) sensor for synchronous fusion, the molecular identity of the Ca(2+) sensors that mediate facilitation and asynchronous release is unknown. To test whether the synchronous Ca(2+) sensor, Syt1, or the asynchronous Ca(2+) sensor is involved in facilitation, we analyzed whether genetic elimination of Syt1 in Drosophila results in a concomitant impairment in facilitation. Our results indicate that Syt1 acts as a redundant Ca(2+) sensor for facilitation, with the asynchronous Ca(2+) sensor contributing significantly to this form of short-term plasticity. We next examined whether other members of the Drosophila Syt family functioned in Ca(2+)-dependent asynchronous release or facilitation in vivo. Genetic elimination of other panneuronally expressed Syt proteins did not alter these forms of exocytosis, indicating a non-Syt Ca(2+) sensor functions for both facilitation and asynchronous release. In light of these findings, the presence of two presynaptic Ca(2+) sensors can be placed in a biological context, a Syt1-based Ca(2+) sensor devoted primarily to baseline synaptic transmission and a second non-Syt Ca(2+) sensor for short-term synaptic plasticity and asynchronous release.

Immunostaining for SYT protein discriminates synovial sarcoma from other soft tissue tumors: analysis of 146 cases

Synovial sarcoma in its classic biphasic form can be distinguished readily from other soft tissue lesions; however, monophasic and poorly differentiated forms are diagnostically more problematic. For this reason, we assessed the efficacy of immunostaining for SYT and SSX1 proteins, the gene products resulting from unique synovial sarcoma translocation, to distinguish synovial sarcoma from other soft tissue lesions. A total number of 146 cases were analyzed, including 47 synovial sarcoma cases (all of which were verified by FISH to have t(X; 18) translocation and SYT–SSX fusion gene) and 99 soft tissue tumors of various types. A polyclonal IgG antibody against SYT was used to stain formalin-fixed paraffin embedded tissues. Forty-one out of 47 (87%) synovial sarcoma displayed strong positive nuclear staining (ranging from 80 to 90% of the tumor cells) for SYT antibody. Nineteen of 99 (19%) non-synovial sarcoma cases showed variable nuclear and cytoplasmic staining with SYT, which ranged from 20 to 60% of tumor nuclei, and included malignant peripheral nerve sheath tumor (5/25), solitary fibrous tumor (2/14), Ewing sarcoma (2/6), low grade fibromyxoid tumor (2/4), extraskeletal mesenchymal chondrosarcoma (2/6), gastrointestinal tumor (4/17), epithelioid sarcoma (2/2). The remaining non-synovial sarcomas were negative. This is the first study demonstrating SYT protein expression in tissue sections of synovial sarcoma. This method could provide an easy, rapid and widely applicable means of assisting in the diagnosis of synovial sarcoma, particularly when material and/or resources are unavailable for PCR or FISH-based testing. However, as variable weak staining for SYT may be encountered in a small percentage of non-synovial sarcoma sarcomas, a positive interpretation should be made only when the staining is strong, nuclear and present in the majority of cells.

SYT, a partner of SYT-SSX oncoprotein in synovial sarcomas, interacts with mSin3A, a component of histone deacetylase complex

Synovial sarcomas are soft-tissue tumors predominantly affecting children and young adults. They are molecular-genetically characterized by the SYT-SSX fusion gene generated from chromosomal translocation t(X; 18) (p11.2; q11.2). When we screened new gene products that interact with SYT or SSX proteins by yeast two-hybrid assay, we found that mSin3A, a component of the histone deacetylase complex, interacts with SYT but not with SSX. These results were confirmed by mammalian two-hybrid and pull-down assays. Analyses with sequential truncated proteins revealed a main mSin3A-interaction region on the SYT amino-terminal 93 amino acids, and another one on the region between 187th amino acid and break point. In luciferase assay, mSin3A repressed the transcriptional activity of reporter promoter mediated by SYT and hBRM/BRG1. Our results suggest that the histone deacetylase complex containing mSin3A may regulate the transcriptional activation mediated by SYT.

Transcriptional co-activator activity of SYT is negatively regulated by BRM and Brg1.

The t(X;18)(p11.2;q11.2) translocation found in synovial sarcomas results in the fusion of the SYT gene on chromosome 18 to the SSX gene on chromosome X. Although the SYT-SSX fusion proteins may trigger synovial sarcoma development, the biological functions of SYT, SSX and SYT-SSX genes are unclear. Transfections of Gal4 DNA binding domain fusion protein constructs demonstrate that SYT protein acts as a transcriptional co-activator at the C-terminal domain and that the activity is repressed through the N-terminus. The N-terminal 70 amino acids of SYT bind not only to BRM, but also to Brg1, both of which are subunits of SWI/SNF chromatin remodelling complexes. Here, we have investigated the functions of BRM and Brg1 on the repression of SYT activity. The negative regulation of SYT transcriptional co-activator activity is dependent on the ATP-hydrolysis of BRM and Brg1 in the protein complexes. This indicates that the SWI/SNF protein complexes regulate SYT activity using the chromatin remodelling activity.

SYT Associates with Human SNF/SWI Complexes and the C-terminal Region of Its Fusion Partner SSX1 Targets Histones

A global transcriptional co-activator, the SNF/SWI complex, has been characterized as a chromatin remodeling factor that enhances accessibility of the transcriptional machinery to DNA within a repressive chromatin structure. On the other hand, mutations in some human SNF/SWI complex components have been linked to tumor formation. We show here that SYT, a partner protein generating the synovial sarcoma fusion protein SYT-SSX, associates with native human SNF/SWI complexes. The SYT protein has a unique QPGY domain, which is also present in the largest subunits, p250 and the newly identified homolog p250R, of the corresponding SNF/SWI complexes. The C-terminal region (amino acids 310-387) of SSX1, comprising the SSX1 portion of the SYT-SSX1 fusion protein, binds strongly to core histones and oligonucleosomes in vitro and directs nuclear localization of a green fluorescence protein fusion protein. Experiments with serial C-terminal deletion mutants of SSX1 indicate that these properties map to a common region and also correlate with the previously demonstrated anchorage-independent colony formation activity of SYT-SSX in Rat 3Y1 cells. These data suggest that SYT-SSX interferes with the function of either the SNF/SWI complexes or another SYT-interacting co-activator, p300, by changing their targeted localization or by directly inhibiting their chromatin remodeling activities.

Synaptotagmin IX Regulates Ca2+-dependent Secretion in PC12 Cells

Synaptotagmin (Syt) I-deficient phaeochromocytoma (PC12) cell lines show normal Ca(2+)-dependent norepinephrine (NE) release (Shoji-Kasai, Y., Yoshida, A., Sato, K., Hoshino, T., Ogura, A., Kondo, S., Fujimoto, Y., Kuwahara, R., Kato, R., and Takahashi, M. (1992) Science 256, 1821-1823). To identify an alternative Ca(2+) sensor, we searched for other Syt isoforms in Syt I-deficient PC12 cells and identified Syt IX, an isoform closely related to Syt I, as an abundantly expressed dense-core vesicle protein. Here we show that Syt IX is required for the Ca(2+)-dependent release of NE from PC12 cells. Antibodies directed against the C2A domain of either Syt IX or Syt I inhibited Ca(2+)-dependent NE release in permeable PC12 cells indicating that both Syt proteins function in dense-core vesicle exocytosis. Our results support the idea that Syt family proteins that co-reside on secretory vesicles may function cooperatively and redundantly as potential Ca(2+) sensors for exocytosis.

The synovial sarcoma associated protein SYT interacts with the acute leukemia associated protein AF10.

As a result of the synovial sarcoma associated t(X;18) translocation, the human SYT gene on chromosome 18 is fused to either the SSX1 or the SSX2 gene on the X chromosome. Although preliminary evidence indicates that the (fusion) proteins encoded by these genes may play a role in transcriptional regulation, little is known about their exact function. We set out to isolate interacting proteins through yeast two hybrid screening of a human cDNA library using SYT as a bait. Of the positive clones isolated, two were found to correspond to the acute leukemia t(10;11) associated AF10 gene, a fusion partner of MLL. Confirmation of these results was obtained via co-immunoprecipitation of endogenous and exogenous, epitope-tagged, SYT and AF10 proteins from cell line extracts and colocalization of epitope-tagged SYT and AF10 proteins in transfected cells. Subsequent sequential mutation analysis revealed a highly specific interaction of N-terminal SYT fragments with C-terminal AF10 fragments. The N-terminal interaction domain of the SYT protein was also found to be present in several SYT orthologs and homologs. The C-terminal interaction domain of AF10 is located outside known functional domains. Based on these results, a model is proposed in which the SYT and AF10 proteins act in concert as bipartite transcription factors. This model has implications for the molecular mechanisms underlying the development of both human synovial sarcomas and acute leukemias.

Erratum to: Drosophila AD3 mutation of synaptotagmin impairs calcium-dependent self-oligomerization activity (FEBS 24173) : [ FEBS Letters 482 (2000) 269–272

The legend for ¢gure 4 was omitted. It should have read: `Diagram describing Ca2‡-independent and -dependent Syt II (or I) self-oligomerization of wild-type and AD3 (Y312N) mutant proteins. Schematic representations of Ca2‡-independent and -dependent oligomerization of wild-type (A), heterozygous (B), and homozygous AD3 mutant (C) Syt proteins. Transmembrane domain (TM) and C2 domains are represented by open boxes and hatched boxes, respectively. The shaded region around the transmembrane domain indicates Ca2‡-independent self-oligomerization, which was observed in every case (A^C). In A, wild-type proteins show e¤cient Ca2‡-dependent oligomerization (large double arrow) that is inhibited by IP4 [38,51]. In B, AD3 mutant proteins interact with wild-type proteins in a Ca2‡-dependent manner (small double arrow), whereas in C, AD3 mutant proteins fail to interact with each other in the presence of Ca2‡.'

Delineation of the Protein Domains Responsible for SYT, SSX, and SYT-SSX Nuclear Localization

In the vast majority of synovial sarcomas the N-terminal part of the SYT protein is fused to the C-terminal part of an SSX protein, either SSX1 or SSX2. The wild-type proteins, as well as the resultant SYT-SSX1 and SYT-SSX2 fusion proteins, are localized in the nucleus. Recent studies in experimental systems indicated that the SYT protein may function as a transcriptional activator whereas the SSX proteins may act as transcriptional repressors. In the present work we created a series of deletion mutants and found that SYT and SSX depend on N-terminal and highly conserved C-terminal domains for nuclear localization, respectively. Our results also show that the SYT-SSX proteins colocalize with SSX2, a feature that depends on the presence of the C-terminal SSX sequences in the chimeric proteins. Absence of these sequences led to an altered subcellular localization, coinciding with that of SYT. Besides, we found that endogenously expressed SSX proteins colocalize with polycomb-group proteins and condensed chromosomes during mitosis, features that are also conferred by the C-terminus of SSX. Taken together, these results led us to conclude that the SSX moiety, especially the most C-terminal 34 amino acids, of the SYT-SSX fusion proteins is crucial for aberrant spatial targeting and transcriptional control within the nucleus.

Molecular mechanisms underlying human synovial sarcoma development.

Synovial sarcomas are rather common among soft-tissue tumors, occurring at any age but affecting mainly young adults. The vast majority of synovial sarcomas carries a t(X;18)(p11.2;q11.2) chromosomal translocation, in about one-third of the cases as the sole cytogenetic anomaly. Several studies have indicated that the t(X;18) translocation arises exclusively in synovial sarcomas, therefore being an excellent tool to diagnose this malignancy. The breakpoint-associated genes were recently isolated: SYT, from chromosome 18, and SSX1 and SSX2, both from the X chromosome. This discovery enabled the detection of SYT-SSX fusion transcripts by specific reverse transcriptase-polymerase chain reactions. This molecular genetics methodology has now been applied to numerous tumor samples and has led to the finding that, in contrast to tumors carrying SYT-SSX2 fusions, SYT-SSX1-positive tumors more often exhibit a biphasic histology, show a higher proliferation rate, and are associated with a poorer clinical outcome. It has also been shown that the SYT and SSX proteins are localized in the nucleus, where they appear to play a role in transcriptional regulation, SYT as an activator of transcription and the SSX proteins as transcriptional repressors. It was also found that SYT interacts and colocalizes in the nucleus with the BRM protein, a transcriptional coactivator, and that the SSX proteins colocalize in the nucleus with polycomb group proteins, which are transcriptional corepressors. Together, these studies have provided mechanistic clues about how the SYT-SSX fusion proteins may trigger synovial sarcoma development.

SSX and the synovial-sarcoma-specific chimaeric protein SYT-SSX co-localize with the human Polycomb group complex.

Chromosome translocation t(X;18)(p11.2;q11.2) is unique to synovial sarcomas and results in an 'in frame' fusion of the SYT gene with the SSX1 or closely-related SSX2 gene. Wild-type SYT and SSX proteins, and the SYT-SSX chimaeric proteins, can modulate transcription in gene reporter assays. To help elucidate the role of these proteins in cell function and neoplasia we have performed immunolabelling experiments to determine their subcellular localization in three cell types. Transient expression of epitope-tagged proteins produced distinctive nuclear staining patterns. The punctate staining of SYT and SYT-SSX proteins showed some similarities. We immunolabelled a series of endogenous nuclear antigens and excluded the SYT and SYT-SSX focal staining from association with these domains (e.g. sites of active transcription, snRNPs). In further experiments we immunolabelled the Polycomb group (PcG) proteins RING1 or BMI-1 and showed that SSX and SYT-SSX proteins, but not SYT, co-localized with these markers. Consistent with this we show that SSX and SYT-SSX associate with chromatin, and also associate with condensed chromatin at metaphase. Noteably, SSX produced a dense signal over the surface of metaphase chromosomes whereas SYT-SSX produced discrete focal staining. Our data indicate that SSX and SYT-SSX proteins are recruited to nuclear domains occupied by PcG complexes, and this provides us with a new insight into the possible function of wild-type SSX and the mechanism by which the aberrant SYT-SSX protein might disrupt fundamental mechanisms controlling cell division and cell fate.

Functional domains of the SYT and SYT-SSX synovial sarcoma translocation proteins and co-localization with the SNF protein BRM in the nucleus.

The t(X;18)(p11.2;q11.2) chromosomal translocation commonly found in synovial sarcomas fuses the SYT gene on chromosome 18 to either of two similar genes, SSX1 or SSX2, on the X chromosome. The SYT protein appears to act as a transcriptional co-activator and the SSX proteins as co-repressors. Here we have investigated the functional domains of the proteins. The SYT protein has a novel conserved 54 amino acid domain at the N-terminus of the protein (the SNH domain) which is found in proteins from a wide variety of species, and a C-terminal domain, rich in glutamine, proline, glycine and tyrosine (the QPGY domain), which contains the transcriptional activator sequences. Deletion of the SNH domain results in a more active transcriptional activator, suggesting that this domain acts as an inhibitor of the activation domain. The C-terminal SSX domain present in SYT-SSX translocation protein contributes a transcriptional repressor domain to the protein. Thus, the fusion protein has transcriptional activating and repressing domains. We demonstrate that the human homologue of the SNF2/Brahama protein BRM co-localizes with SYT and SYT-SSX in nuclear speckles, and also interacts with SYT and SYT-SSX proteins in vitro. This interaction may provide an explanation of how the SYT protein activates gene transcription.

Nuclear localization of SYT, SSX and the synovial sarcoma-associated SYT-SSX fusion proteins.

Synovial sarcoma is characterized by a prevalent chromosomal translocation, t(X;18)(p11;q11). As a result of this translocation the SYT gene on chromosome 18 fuses to either the SSX1 or the SSX2 gene on the X chromosome. In this study, we generated polyclonal antibodies against the SYT and SSX2 proteins. These antibodies specifically detected both these proteins and the SYT-SSX fusion proteins in transfected COS-1 cell extracts. Indirect immunofluorescence analysis of COS-1 cells expressing tagged or untagged SYT, SSX2, SYT-SSX1 or SYT-SSX2 indicated that all these proteins are localized in the nucleus, excluding the nucleoli. The SSX2 protein exhibited a diffuse staining pattern whereas both the SYT and SYT-SSX proteins appeared in several nuclear dots. Similar nuclear dots were also detected in primary synovial sarcoma cells growing in a short-term in vitro culture. Double immunofluorescence in conjunction with confocal laser-scanning microscopy revealed that the SYT and SYT-SSX nuclear dots do not co-localize with known nuclear structures as e.g. coiled bodies, SC35 interchromatin granules or PML bodies. The similar nuclear localization patterns of SYT and SYT-SSX suggest that the SYT-SSX fusion proteins are directed to SYT-associated nuclear domains where an abnormal function may be exerted.

The SYT protein involved in the t(X;18) synovial sarcoma translocation is a transcriptional activator localised in nuclear bodies.

The t(X;18)(p11.2;q11.2) translocation found in synovial sarcomas results in the fusion of the SYT gene on chromosome 18 to either of two closely related genes SSX1 and SSX2 on chromosome X. The resulting chimaeric genes express SYT-SSX1 or SYT-SSX2 fusion proteins in which the C-terminal amino acids of SYT are replaced by amino acids from the C-terminus of the SSX proteins. Using green fluorescent protein fusions we demonstrate that the SYT, SSX and the SYT-SSX proteins are nuclear proteins. We demonstrate that whilst the SSX1 protein has a uniform nuclear distribution the SYT protein has a speckled distribution in the cell nucleus, and this distribution is retained with the SYT-SSX2 fusion protein. Since the SYT speckles do not co-localise with PML-containing bodies (PODs) or spliceosomes it is possible that they represent a novel nuclear structure. Transfection of constructs expressing GAL4 fusion proteins demonstrate that the SYT domains present in the SYT-SSX fusion proteins can activate transcription of a luciferase reporter. It is proposed that the t(X;18) translocation results in the generation of an SYT-SSX transcriptional co-activator in which the addition of the C-terminal SSX domain to SYT provides a new interacting domain that redirects the SYT activation domain to different target promoters.