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Cytosolic SYT/SS18 isoforms are actin-associated proteins that function in matrix-specific adhesion.

Kim J, Swee M, Parks WC - PLoS ONE (2009)

Bottom Line: Disruption of the actin cytoskeleton also led to a breakdown of the filamentous organization of SYT isoforms in the cytosol.RNAi ablation of SYT/L alone or both isoforms markedly impaired formation of stress fibers and focal adhesions but did not affect formation of cortical actin bundles.These findings indicate that cytoplasmic SYT isoforms interact with actin filaments and function in the ability cells to bind and react to specific extracellular matrices.

View Article: PubMed Central - PubMed

Affiliation: Center for Lung Biology, University of Washington, Seattle, Washington, United States of America.

ABSTRACT
SYT (SYnovial sarcoma Translocated gene or SS18) is widely produced as two isoforms, SYT/L and SYT/S, that are thought to function in the nucleus as transcriptional coactivators. Using isoform-specific antibodies, we detected a sizable pool of SYT isoforms in the cytosol where the proteins were organized into filamentous arrays. Actin and actin-associated proteins co-immunoprecipitated with SYT isoforms, which also co-sedimented and co-localized with the actin cytoskeleton in cultured cells and tissues. The association of SYT with actin bundles was extensive yet stopped short of the distal ends at focal adhesions. Disruption of the actin cytoskeleton also led to a breakdown of the filamentous organization of SYT isoforms in the cytosol. RNAi ablation of SYT/L alone or both isoforms markedly impaired formation of stress fibers and focal adhesions but did not affect formation of cortical actin bundles. Furthermore, ablation of SYT led to markedly impaired adhesion and spreading on fibronectin and laminin-111 but not on collagen types I or IV. These findings indicate that cytoplasmic SYT isoforms interact with actin filaments and function in the ability cells to bind and react to specific extracellular matrices.

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SYT antibodies.(A) Total lysate (40 µg protein) from U2OS cells was immunoblotted (IB) with affinity-purified panSYT antibody (pSYT) or antibody specific for the SYT/L isoform. The pSYT antibody reacted with both SYT/S (S; 50.5 kD) and SYT/L (L; 56 kD) isoforms, whereas the SYT/L antibody reacted only with the long isoform. (B) Total cell lysates were immunoprecipitated with pSYT or SYT/L-specific antibodies or purified IgG. (C) Cos-1 cells were processed for immunofluorescence staining with SYT/L antibody in the presence of increasing molar ratios (relative to antibody) of antigenic peptide. Bar = 20 µm. (D) Total U2OS lysate was immunoprecipitated with SYT/L-specific antibody (1° IP), and the supernatant was re-immunoprecipitated with pSYT (2° IP). Both immunoprecipitates, as well as the starting lysate (Pre IP) and the supernatant after the second immunoprecipitation (Post IP), were immunoblotted with pSYT antibody. (E) Lysates from control U2OS cells or from cells 3 days post transfection with RNAi duplex 894, which targets only SYT/L transcripts (see Figure S1), were immunoblotted with pSYT and SYT/L-specific antibodies.
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pone-0006455-g001: SYT antibodies.(A) Total lysate (40 µg protein) from U2OS cells was immunoblotted (IB) with affinity-purified panSYT antibody (pSYT) or antibody specific for the SYT/L isoform. The pSYT antibody reacted with both SYT/S (S; 50.5 kD) and SYT/L (L; 56 kD) isoforms, whereas the SYT/L antibody reacted only with the long isoform. (B) Total cell lysates were immunoprecipitated with pSYT or SYT/L-specific antibodies or purified IgG. (C) Cos-1 cells were processed for immunofluorescence staining with SYT/L antibody in the presence of increasing molar ratios (relative to antibody) of antigenic peptide. Bar = 20 µm. (D) Total U2OS lysate was immunoprecipitated with SYT/L-specific antibody (1° IP), and the supernatant was re-immunoprecipitated with pSYT (2° IP). Both immunoprecipitates, as well as the starting lysate (Pre IP) and the supernatant after the second immunoprecipitation (Post IP), were immunoblotted with pSYT antibody. (E) Lysates from control U2OS cells or from cells 3 days post transfection with RNAi duplex 894, which targets only SYT/L transcripts (see Figure S1), were immunoblotted with pSYT and SYT/L-specific antibodies.

Mentions: We generated two polyclonal antibodies: one against the N-terminal half of SYT, which is common to all isoforms, and the other against the peptide sequence coded by exon 8, which is present only in SYT/L. Both antibodies were affinity purified using the recombinant or peptide antigens, and we confirmed the specificity of these antibodies by several approaches. Immunoblotting and immunoprecipitation of human osteosarcoma U2OS cell lysates demonstrated that the panSYT (pSYT) antibody reacted with both SYT/S (50.5 kD) and SYT/L (56 kD) and that the SYT/L-specific antibody detected only SYT/L (Figure 1A, B). Immunofluorescence revealed signal in both the nucleus and cytosol, which was ablated with excess antigen (Figure 1C). Immunoprecipitation with SYT/L-specific antibody cleared lysates of the larger isoform yet retained signal for SYT/S, which was brought down by subsequent immunoprecipitation with pSYT (Figure 1D). After immunodepletion with both antibodies, lysates were cleared of all SYT immunoreactivity.


Cytosolic SYT/SS18 isoforms are actin-associated proteins that function in matrix-specific adhesion.

Kim J, Swee M, Parks WC - PLoS ONE (2009)

SYT antibodies.(A) Total lysate (40 µg protein) from U2OS cells was immunoblotted (IB) with affinity-purified panSYT antibody (pSYT) or antibody specific for the SYT/L isoform. The pSYT antibody reacted with both SYT/S (S; 50.5 kD) and SYT/L (L; 56 kD) isoforms, whereas the SYT/L antibody reacted only with the long isoform. (B) Total cell lysates were immunoprecipitated with pSYT or SYT/L-specific antibodies or purified IgG. (C) Cos-1 cells were processed for immunofluorescence staining with SYT/L antibody in the presence of increasing molar ratios (relative to antibody) of antigenic peptide. Bar = 20 µm. (D) Total U2OS lysate was immunoprecipitated with SYT/L-specific antibody (1° IP), and the supernatant was re-immunoprecipitated with pSYT (2° IP). Both immunoprecipitates, as well as the starting lysate (Pre IP) and the supernatant after the second immunoprecipitation (Post IP), were immunoblotted with pSYT antibody. (E) Lysates from control U2OS cells or from cells 3 days post transfection with RNAi duplex 894, which targets only SYT/L transcripts (see Figure S1), were immunoblotted with pSYT and SYT/L-specific antibodies.
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Related In: Results  -  Collection

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pone-0006455-g001: SYT antibodies.(A) Total lysate (40 µg protein) from U2OS cells was immunoblotted (IB) with affinity-purified panSYT antibody (pSYT) or antibody specific for the SYT/L isoform. The pSYT antibody reacted with both SYT/S (S; 50.5 kD) and SYT/L (L; 56 kD) isoforms, whereas the SYT/L antibody reacted only with the long isoform. (B) Total cell lysates were immunoprecipitated with pSYT or SYT/L-specific antibodies or purified IgG. (C) Cos-1 cells were processed for immunofluorescence staining with SYT/L antibody in the presence of increasing molar ratios (relative to antibody) of antigenic peptide. Bar = 20 µm. (D) Total U2OS lysate was immunoprecipitated with SYT/L-specific antibody (1° IP), and the supernatant was re-immunoprecipitated with pSYT (2° IP). Both immunoprecipitates, as well as the starting lysate (Pre IP) and the supernatant after the second immunoprecipitation (Post IP), were immunoblotted with pSYT antibody. (E) Lysates from control U2OS cells or from cells 3 days post transfection with RNAi duplex 894, which targets only SYT/L transcripts (see Figure S1), were immunoblotted with pSYT and SYT/L-specific antibodies.
Mentions: We generated two polyclonal antibodies: one against the N-terminal half of SYT, which is common to all isoforms, and the other against the peptide sequence coded by exon 8, which is present only in SYT/L. Both antibodies were affinity purified using the recombinant or peptide antigens, and we confirmed the specificity of these antibodies by several approaches. Immunoblotting and immunoprecipitation of human osteosarcoma U2OS cell lysates demonstrated that the panSYT (pSYT) antibody reacted with both SYT/S (50.5 kD) and SYT/L (56 kD) and that the SYT/L-specific antibody detected only SYT/L (Figure 1A, B). Immunofluorescence revealed signal in both the nucleus and cytosol, which was ablated with excess antigen (Figure 1C). Immunoprecipitation with SYT/L-specific antibody cleared lysates of the larger isoform yet retained signal for SYT/S, which was brought down by subsequent immunoprecipitation with pSYT (Figure 1D). After immunodepletion with both antibodies, lysates were cleared of all SYT immunoreactivity.

Bottom Line: Disruption of the actin cytoskeleton also led to a breakdown of the filamentous organization of SYT isoforms in the cytosol.RNAi ablation of SYT/L alone or both isoforms markedly impaired formation of stress fibers and focal adhesions but did not affect formation of cortical actin bundles.These findings indicate that cytoplasmic SYT isoforms interact with actin filaments and function in the ability cells to bind and react to specific extracellular matrices.

View Article: PubMed Central - PubMed

Affiliation: Center for Lung Biology, University of Washington, Seattle, Washington, United States of America.

ABSTRACT
SYT (SYnovial sarcoma Translocated gene or SS18) is widely produced as two isoforms, SYT/L and SYT/S, that are thought to function in the nucleus as transcriptional coactivators. Using isoform-specific antibodies, we detected a sizable pool of SYT isoforms in the cytosol where the proteins were organized into filamentous arrays. Actin and actin-associated proteins co-immunoprecipitated with SYT isoforms, which also co-sedimented and co-localized with the actin cytoskeleton in cultured cells and tissues. The association of SYT with actin bundles was extensive yet stopped short of the distal ends at focal adhesions. Disruption of the actin cytoskeleton also led to a breakdown of the filamentous organization of SYT isoforms in the cytosol. RNAi ablation of SYT/L alone or both isoforms markedly impaired formation of stress fibers and focal adhesions but did not affect formation of cortical actin bundles. Furthermore, ablation of SYT led to markedly impaired adhesion and spreading on fibronectin and laminin-111 but not on collagen types I or IV. These findings indicate that cytoplasmic SYT isoforms interact with actin filaments and function in the ability cells to bind and react to specific extracellular matrices.

Show MeSH
Related in: MedlinePlus