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Cdc42Hs facilitates cytoskeletal reorganization and neurite outgrowth by localizing the 58-kD insulin receptor substrate to filamentous actin.

Govind S, Kozma R, Monfries C, Lim L, Ahmed S - J. Cell Biol. (2001)

Bottom Line: In Swiss 3T3 cells and N1E-115 cells, IRS-58 colocalizes with F-actin in clusters and filopodia.An IRS-58(1267N) mutant unable to bind Cdc42Hs failed to localize with F-actin to induce neurite outgrowth or significant cytoskeletal reorganization.These results suggest that Cdc42Hs facilitates cytoskeletal reorganization and neurite outgrowth by localizing protein complexes via adaptor proteins such as IRS-58 to F-actin.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurochemistry, Institute of Neurology, London WC1N 1PJ, United Kingdom.

ABSTRACT
Cdc42Hs is involved in cytoskeletal reorganization and is required for neurite outgrowth in N1E-115 cells. To investigate the molecular mechanism by which Cdc42Hs regulates these processes, a search for novel Cdc42Hs protein partners was undertaken by yeast two-hybrid assay. Here, we identify the 58-kD substrate of the insulin receptor tyrosine kinase (IRS-58) as a Cdc42Hs target. IRS-58 is a brain-enriched protein comprising at least four protein-protein interaction sites: a Cdc42Hs binding site, an Src homology (SH)3-binding site, an SH3 domain, and a tryptophan, tyrptophan (WW)-binding domain. Expression of IRS-58 in Swiss 3T3 cells leads to reorganization of the filamentous (F)-actin cytoskeleton, involving loss of stress fibers and formation of filopodia and clusters. In N1E-115 cells IRS-58 induces neurite outgrowth with high complexity. Expression of a deletion mutant of IRS-58, which lacks the SH3- and WW-binding domains, induced neurite extension without complexity in N1E-115 cells. In Swiss 3T3 cells and N1E-115 cells, IRS-58 colocalizes with F-actin in clusters and filopodia. An IRS-58(1267N) mutant unable to bind Cdc42Hs failed to localize with F-actin to induce neurite outgrowth or significant cytoskeletal reorganization. These results suggest that Cdc42Hs facilitates cytoskeletal reorganization and neurite outgrowth by localizing protein complexes via adaptor proteins such as IRS-58 to F-actin.

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Analysis of IRS-58 F-actin cluster formation. Serum-starved Swiss 3T3 cells were microinjected with IRS-58 cDNA in pXJ40 as described in the legend to Fig. 3. A single cell with low levels of IRS-58 expression is examined in more detail by confocal analysis. (A) F-actin (panel A); HA stain (panel B); and HA stain and IRS-58 (green), with F-actin (red) staining with colocalization shown in blue (panel C); colocalization signal only (panel D). Inserts in A–D show a magnified region of the cell (position of magnification shown by dashed line box in panel C). Arrow in inserts show filopodia. Arrows in main figure show F-actin clusters. (B) Z-series with panel A bottom to panel H top. Green stain visualizing IRS-58; red stain, F-actin; and blue/purple stain, colocalization. Bars: (A) 10 μM; (B) 20 μM.
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Figure 5: Analysis of IRS-58 F-actin cluster formation. Serum-starved Swiss 3T3 cells were microinjected with IRS-58 cDNA in pXJ40 as described in the legend to Fig. 3. A single cell with low levels of IRS-58 expression is examined in more detail by confocal analysis. (A) F-actin (panel A); HA stain (panel B); and HA stain and IRS-58 (green), with F-actin (red) staining with colocalization shown in blue (panel C); colocalization signal only (panel D). Inserts in A–D show a magnified region of the cell (position of magnification shown by dashed line box in panel C). Arrow in inserts show filopodia. Arrows in main figure show F-actin clusters. (B) Z-series with panel A bottom to panel H top. Green stain visualizing IRS-58; red stain, F-actin; and blue/purple stain, colocalization. Bars: (A) 10 μM; (B) 20 μM.

Mentions: To determine whether IRS-58 colocalized with F-actin, we used colocalization software (LSM v4; ZEISS) with images obtained through a confocal microscope (Fig. 5). Swiss 3T3 cells with low levels of IRS-58 expression possessed filopodia, small F-actin clusters, and thin stress fibers (Fig. 5 A). In these types of cells IRS-58 expression was polarized within the cytoplasm and thin stress fibers were seen only where expression of IRS-58 was very low (Fig. 5 A). IRS-58 colocalizes with the filopodia (see insert, Fig. 5 A), punctate F-actin, and F-actin clusters (Fig. 5 A). IRS-58 was also localized in the cytoplasm as protein aggregates/complexes where there was no phalloidin staining (Fig. 5 B, see green in panels A and B). The organization of the F-actin clusters was examined further by obtaining a Z-series using a confocal microscope (Fig. 5 B, panels A–H, bottom to top). Colocalization of IRS-58 with F-actin occurred in microvilli-like structures that formed a ring around the nucleus. F-actin was found over the nucleus, whereas IRS-58 was excluded from this region of the cell (Fig. 5 B, panels C and D). The colocalization of IRS-58 with F-actin is seen most clearly on the roof of the ring (Fig. 5 B, panels G and H), a point in the Z-series where the intense F-actin ring is absent. This confocal analysis suggests that IRS-58 may be involved in microvilli formation.


Cdc42Hs facilitates cytoskeletal reorganization and neurite outgrowth by localizing the 58-kD insulin receptor substrate to filamentous actin.

Govind S, Kozma R, Monfries C, Lim L, Ahmed S - J. Cell Biol. (2001)

Analysis of IRS-58 F-actin cluster formation. Serum-starved Swiss 3T3 cells were microinjected with IRS-58 cDNA in pXJ40 as described in the legend to Fig. 3. A single cell with low levels of IRS-58 expression is examined in more detail by confocal analysis. (A) F-actin (panel A); HA stain (panel B); and HA stain and IRS-58 (green), with F-actin (red) staining with colocalization shown in blue (panel C); colocalization signal only (panel D). Inserts in A–D show a magnified region of the cell (position of magnification shown by dashed line box in panel C). Arrow in inserts show filopodia. Arrows in main figure show F-actin clusters. (B) Z-series with panel A bottom to panel H top. Green stain visualizing IRS-58; red stain, F-actin; and blue/purple stain, colocalization. Bars: (A) 10 μM; (B) 20 μM.
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Figure 5: Analysis of IRS-58 F-actin cluster formation. Serum-starved Swiss 3T3 cells were microinjected with IRS-58 cDNA in pXJ40 as described in the legend to Fig. 3. A single cell with low levels of IRS-58 expression is examined in more detail by confocal analysis. (A) F-actin (panel A); HA stain (panel B); and HA stain and IRS-58 (green), with F-actin (red) staining with colocalization shown in blue (panel C); colocalization signal only (panel D). Inserts in A–D show a magnified region of the cell (position of magnification shown by dashed line box in panel C). Arrow in inserts show filopodia. Arrows in main figure show F-actin clusters. (B) Z-series with panel A bottom to panel H top. Green stain visualizing IRS-58; red stain, F-actin; and blue/purple stain, colocalization. Bars: (A) 10 μM; (B) 20 μM.
Mentions: To determine whether IRS-58 colocalized with F-actin, we used colocalization software (LSM v4; ZEISS) with images obtained through a confocal microscope (Fig. 5). Swiss 3T3 cells with low levels of IRS-58 expression possessed filopodia, small F-actin clusters, and thin stress fibers (Fig. 5 A). In these types of cells IRS-58 expression was polarized within the cytoplasm and thin stress fibers were seen only where expression of IRS-58 was very low (Fig. 5 A). IRS-58 colocalizes with the filopodia (see insert, Fig. 5 A), punctate F-actin, and F-actin clusters (Fig. 5 A). IRS-58 was also localized in the cytoplasm as protein aggregates/complexes where there was no phalloidin staining (Fig. 5 B, see green in panels A and B). The organization of the F-actin clusters was examined further by obtaining a Z-series using a confocal microscope (Fig. 5 B, panels A–H, bottom to top). Colocalization of IRS-58 with F-actin occurred in microvilli-like structures that formed a ring around the nucleus. F-actin was found over the nucleus, whereas IRS-58 was excluded from this region of the cell (Fig. 5 B, panels C and D). The colocalization of IRS-58 with F-actin is seen most clearly on the roof of the ring (Fig. 5 B, panels G and H), a point in the Z-series where the intense F-actin ring is absent. This confocal analysis suggests that IRS-58 may be involved in microvilli formation.

Bottom Line: In Swiss 3T3 cells and N1E-115 cells, IRS-58 colocalizes with F-actin in clusters and filopodia.An IRS-58(1267N) mutant unable to bind Cdc42Hs failed to localize with F-actin to induce neurite outgrowth or significant cytoskeletal reorganization.These results suggest that Cdc42Hs facilitates cytoskeletal reorganization and neurite outgrowth by localizing protein complexes via adaptor proteins such as IRS-58 to F-actin.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurochemistry, Institute of Neurology, London WC1N 1PJ, United Kingdom.

ABSTRACT
Cdc42Hs is involved in cytoskeletal reorganization and is required for neurite outgrowth in N1E-115 cells. To investigate the molecular mechanism by which Cdc42Hs regulates these processes, a search for novel Cdc42Hs protein partners was undertaken by yeast two-hybrid assay. Here, we identify the 58-kD substrate of the insulin receptor tyrosine kinase (IRS-58) as a Cdc42Hs target. IRS-58 is a brain-enriched protein comprising at least four protein-protein interaction sites: a Cdc42Hs binding site, an Src homology (SH)3-binding site, an SH3 domain, and a tryptophan, tyrptophan (WW)-binding domain. Expression of IRS-58 in Swiss 3T3 cells leads to reorganization of the filamentous (F)-actin cytoskeleton, involving loss of stress fibers and formation of filopodia and clusters. In N1E-115 cells IRS-58 induces neurite outgrowth with high complexity. Expression of a deletion mutant of IRS-58, which lacks the SH3- and WW-binding domains, induced neurite extension without complexity in N1E-115 cells. In Swiss 3T3 cells and N1E-115 cells, IRS-58 colocalizes with F-actin in clusters and filopodia. An IRS-58(1267N) mutant unable to bind Cdc42Hs failed to localize with F-actin to induce neurite outgrowth or significant cytoskeletal reorganization. These results suggest that Cdc42Hs facilitates cytoskeletal reorganization and neurite outgrowth by localizing protein complexes via adaptor proteins such as IRS-58 to F-actin.

Show MeSH