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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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Ablation of SYTs impairs formation of stress fibers and focal adhesions.(A) At 3 d post transfection with control, panSYT229, or SYT/L894-specific RNAi duplexes, U2OS cells were stained with phalloidin (actin; red) or co-stained with phalloidin and FAK, pan-phosphotyrosine, or paxillin antibodies (green). The inset in the middle column highlights the exaggerated filopodia, presence of cortical F-actin, and absence of stress fibers in cells transfected with panSYT229 RNAi duplexes. (B) Similar experiment with different (panSYT97 and panSYT471) panSYT RNAI duplexes. Bars = 20 µm or 5 µm (inset in A).
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pone-0006455-g007: Ablation of SYTs impairs formation of stress fibers and focal adhesions.(A) At 3 d post transfection with control, panSYT229, or SYT/L894-specific RNAi duplexes, U2OS cells were stained with phalloidin (actin; red) or co-stained with phalloidin and FAK, pan-phosphotyrosine, or paxillin antibodies (green). The inset in the middle column highlights the exaggerated filopodia, presence of cortical F-actin, and absence of stress fibers in cells transfected with panSYT229 RNAi duplexes. (B) Similar experiment with different (panSYT97 and panSYT471) panSYT RNAI duplexes. Bars = 20 µm or 5 µm (inset in A).

Mentions: We transfected U2OS cells with control (similar GC content), panSYT97, panSYT229, panSYT471, or SYT/L-specific RNAi duplex 894 and assessed the formation of stress fibers 3 days later (Figure 7). Whereas well-formed cytoskeleton was seen in cells transfected with control or SYT/L-specific RNAi's, stress fibers were not detected in cells transfected with panSYT RNAi's (Figure 7). Total actin levels were not affected in RNAi-ablated cells (Figure 3A). Furthermore, in the absence of total SYT, cortical actin was still detected and we noticed robust membrane ruffling and exaggerated filopodia formation (Figure 7, inset), indicating that SYT is not needed for actin polymerization within certain compartments. Importantly, a similar phenotype (lack of stress fibers but retention of cortical actin bundles) was recently reported for fibroblasts isolated from Syt- mouse embryos [22]. In addition, focal adhesions, identified by staining for FAK, paxillin, and phosphotyrosine, were disrupted in panSYT RNAi-transfected cells (Figure 7A). We saw no overt change in stress fiber formation or focal adhesion markers in cells with specific knock-down of SYT/L, suggesting that SYT/S compensates for loss of the larger isoform in adherent cells.


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

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

Ablation of SYTs impairs formation of stress fibers and focal adhesions.(A) At 3 d post transfection with control, panSYT229, or SYT/L894-specific RNAi duplexes, U2OS cells were stained with phalloidin (actin; red) or co-stained with phalloidin and FAK, pan-phosphotyrosine, or paxillin antibodies (green). The inset in the middle column highlights the exaggerated filopodia, presence of cortical F-actin, and absence of stress fibers in cells transfected with panSYT229 RNAi duplexes. (B) Similar experiment with different (panSYT97 and panSYT471) panSYT RNAI duplexes. Bars = 20 µm or 5 µm (inset in A).
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Related In: Results  -  Collection

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pone-0006455-g007: Ablation of SYTs impairs formation of stress fibers and focal adhesions.(A) At 3 d post transfection with control, panSYT229, or SYT/L894-specific RNAi duplexes, U2OS cells were stained with phalloidin (actin; red) or co-stained with phalloidin and FAK, pan-phosphotyrosine, or paxillin antibodies (green). The inset in the middle column highlights the exaggerated filopodia, presence of cortical F-actin, and absence of stress fibers in cells transfected with panSYT229 RNAi duplexes. (B) Similar experiment with different (panSYT97 and panSYT471) panSYT RNAI duplexes. Bars = 20 µm or 5 µm (inset in A).
Mentions: We transfected U2OS cells with control (similar GC content), panSYT97, panSYT229, panSYT471, or SYT/L-specific RNAi duplex 894 and assessed the formation of stress fibers 3 days later (Figure 7). Whereas well-formed cytoskeleton was seen in cells transfected with control or SYT/L-specific RNAi's, stress fibers were not detected in cells transfected with panSYT RNAi's (Figure 7). Total actin levels were not affected in RNAi-ablated cells (Figure 3A). Furthermore, in the absence of total SYT, cortical actin was still detected and we noticed robust membrane ruffling and exaggerated filopodia formation (Figure 7, inset), indicating that SYT is not needed for actin polymerization within certain compartments. Importantly, a similar phenotype (lack of stress fibers but retention of cortical actin bundles) was recently reported for fibroblasts isolated from Syt- mouse embryos [22]. In addition, focal adhesions, identified by staining for FAK, paxillin, and phosphotyrosine, were disrupted in panSYT RNAi-transfected cells (Figure 7A). We saw no overt change in stress fiber formation or focal adhesion markers in cells with specific knock-down of SYT/L, suggesting that SYT/S compensates for loss of the larger isoform in adherent cells.

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