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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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Cytosolic SYT.(A) Rat lung fibroblasts (RFL) and 3T3 fibroblasts were stained with pSYT antibody, and U2OS cells were stained with pSYT or SYT/L-specific antibodies. Bar = 10 µm. (B) Mouse retina was stained for SYT/L. Nuclear signal was seen in ganglion cells (GC) and cells of the inner (INL) and outer (ONL) nuclear layers and within the choroid (C). Prominent cytoplasmic signal was seen within the inner (IPL) and outer (OPL) plexiform layers, the photoreceptors (PR), and the choroid. (C) Total lysate (T) of U2OS cells was separated by centrifugation into cytoplasmic supernatant (S1) and nuclear pellet (P1). The S1 cytosolic fraction was separated further by ultracentrifugation, yielding supernatant (S2) and pellet (P2). Samples were resolved by electrophoresis and immunoblotted with antibodies against total SYT (pSYT), lamin-A/C, EEA-1, RhoGDI, and the β1 integrin subunit.
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pone-0006455-g002: Cytosolic SYT.(A) Rat lung fibroblasts (RFL) and 3T3 fibroblasts were stained with pSYT antibody, and U2OS cells were stained with pSYT or SYT/L-specific antibodies. Bar = 10 µm. (B) Mouse retina was stained for SYT/L. Nuclear signal was seen in ganglion cells (GC) and cells of the inner (INL) and outer (ONL) nuclear layers and within the choroid (C). Prominent cytoplasmic signal was seen within the inner (IPL) and outer (OPL) plexiform layers, the photoreceptors (PR), and the choroid. (C) Total lysate (T) of U2OS cells was separated by centrifugation into cytoplasmic supernatant (S1) and nuclear pellet (P1). The S1 cytosolic fraction was separated further by ultracentrifugation, yielding supernatant (S2) and pellet (P2). Samples were resolved by electrophoresis and immunoblotted with antibodies against total SYT (pSYT), lamin-A/C, EEA-1, RhoGDI, and the β1 integrin subunit.

Mentions: Immunofluorescence with either SYT antibody verified nuclear signal in both cells and tissue (Figure 2A,B); however, signal for SYT protein was also seen in the cytosol of all cells examined, typically in a filamentous pattern (Figure 2A). In addition to U2OS, RLF, and 3T3 cells, we detected filamentous cytosolic SYT in human primary keratinocytes, human HaCaT keratinocytes, human diploid fibroblasts (HDF), human primary fibroblasts (IMR90), human cervical carcinoma cells (HeLa), and monkey kidney fibroblasts (CV-1 and Cos-1) (not shown). Furthermore, prominent signal for cytosolic SYT was detected in all adult mouse tissues examined, including all cell layers of the retina (Figure 2B), intestine, lung, skin, cardiac muscle, spleen, brain, uterus, and testis, among others (not shown). Thus, the cytoplasmic distribution of SYT was seen across tissues, cell types, and species.


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

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

Cytosolic SYT.(A) Rat lung fibroblasts (RFL) and 3T3 fibroblasts were stained with pSYT antibody, and U2OS cells were stained with pSYT or SYT/L-specific antibodies. Bar = 10 µm. (B) Mouse retina was stained for SYT/L. Nuclear signal was seen in ganglion cells (GC) and cells of the inner (INL) and outer (ONL) nuclear layers and within the choroid (C). Prominent cytoplasmic signal was seen within the inner (IPL) and outer (OPL) plexiform layers, the photoreceptors (PR), and the choroid. (C) Total lysate (T) of U2OS cells was separated by centrifugation into cytoplasmic supernatant (S1) and nuclear pellet (P1). The S1 cytosolic fraction was separated further by ultracentrifugation, yielding supernatant (S2) and pellet (P2). Samples were resolved by electrophoresis and immunoblotted with antibodies against total SYT (pSYT), lamin-A/C, EEA-1, RhoGDI, and the β1 integrin subunit.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2714072&req=5

pone-0006455-g002: Cytosolic SYT.(A) Rat lung fibroblasts (RFL) and 3T3 fibroblasts were stained with pSYT antibody, and U2OS cells were stained with pSYT or SYT/L-specific antibodies. Bar = 10 µm. (B) Mouse retina was stained for SYT/L. Nuclear signal was seen in ganglion cells (GC) and cells of the inner (INL) and outer (ONL) nuclear layers and within the choroid (C). Prominent cytoplasmic signal was seen within the inner (IPL) and outer (OPL) plexiform layers, the photoreceptors (PR), and the choroid. (C) Total lysate (T) of U2OS cells was separated by centrifugation into cytoplasmic supernatant (S1) and nuclear pellet (P1). The S1 cytosolic fraction was separated further by ultracentrifugation, yielding supernatant (S2) and pellet (P2). Samples were resolved by electrophoresis and immunoblotted with antibodies against total SYT (pSYT), lamin-A/C, EEA-1, RhoGDI, and the β1 integrin subunit.
Mentions: Immunofluorescence with either SYT antibody verified nuclear signal in both cells and tissue (Figure 2A,B); however, signal for SYT protein was also seen in the cytosol of all cells examined, typically in a filamentous pattern (Figure 2A). In addition to U2OS, RLF, and 3T3 cells, we detected filamentous cytosolic SYT in human primary keratinocytes, human HaCaT keratinocytes, human diploid fibroblasts (HDF), human primary fibroblasts (IMR90), human cervical carcinoma cells (HeLa), and monkey kidney fibroblasts (CV-1 and Cos-1) (not shown). Furthermore, prominent signal for cytosolic SYT was detected in all adult mouse tissues examined, including all cell layers of the retina (Figure 2B), intestine, lung, skin, cardiac muscle, spleen, brain, uterus, and testis, among others (not shown). Thus, the cytoplasmic distribution of SYT was seen across tissues, cell types, and species.

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