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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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Overexpression of IRS-58 in Swiss 3T3 cells. Serum-starved Swiss 3T3 cells were microinjected with cDNA encoding HA-tagged IRS-58 in a pXJ40 vector and left for 3 h before fixing and staining of cells. Anti-HA antibody was used to detect IRS-58–expressing cells and cells were also stained for F-actin with rhodamine-conjugated phalloidin as described in Materials and Methods. (A, C, and E) IRS-58; (B, D, and F) F-actin. Arrows mark injected cells in A, C, and E and the corresponding F-actin staining in B, D, and F. The insert in A shows a magnified version of part of the image. Arrowheads in the insert highlight particulate structures/protein aggregates. Arrows in panels indicate transfected cells. Some transfected cells are not marked. Bar, 10 μM.
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Figure 4: Overexpression of IRS-58 in Swiss 3T3 cells. Serum-starved Swiss 3T3 cells were microinjected with cDNA encoding HA-tagged IRS-58 in a pXJ40 vector and left for 3 h before fixing and staining of cells. Anti-HA antibody was used to detect IRS-58–expressing cells and cells were also stained for F-actin with rhodamine-conjugated phalloidin as described in Materials and Methods. (A, C, and E) IRS-58; (B, D, and F) F-actin. Arrows mark injected cells in A, C, and E and the corresponding F-actin staining in B, D, and F. The insert in A shows a magnified version of part of the image. Arrowheads in the insert highlight particulate structures/protein aggregates. Arrows in panels indicate transfected cells. Some transfected cells are not marked. Bar, 10 μM.

Mentions: Swiss 3T3 cells represent a model mammalian cell type which have been used to investigate the reorganization of both F-actin structures and the associated FCs. In Swiss 3T3 cells, factors such as bradykinin, lysophosphatidic acid, and PMA have been shown to induce the formation of peripheral actin microspikes, stress fibers, and membrane ruffles via Cdc42Hs (Kozma et al. 1995), RhoA (Ridley and Hall 1992), and Rac1 (Ridley et al. 1992), respectively. Each of these F-actin structures is associated with a morphologically unique FC (Nobes and Hall 1995). To investigate whether IRS-58 affected F-actin, it was overexpressed as an HA-tagged protein by DNA microinjection of Swiss 3T3 cells. HA staining of microinjected cells was punctate and revealed that IRS-58 localized to peripheral extensions where process formation (membrane extension and retraction) was induced (Fig. 4A and Fig. B). The punctate HA staining of IRS-58 may suggest its localization to an organelle or a large protein aggregate (Fig. 4 A). Similar observations of IRS-58 localization to protein aggregates were made by Yeh et al. 1998 and Okamura-Oho et al. 1999. Overexpression of IRS-58 caused a reorganization of the F-actin cytoskeleton. Stress fibers were completely disassembled under these conditions and the F-actin took on a punctate appearance (Fig. 4C and Fig. D). In some cells F-actin cluster formation could be seen (Fig. 4E and Fig. F). Fig. 4 shows the heterogeneity in morphology seen in expression of different levels of IRS-58 from low (A and B) to high (E and F).


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)

Overexpression of IRS-58 in Swiss 3T3 cells. Serum-starved Swiss 3T3 cells were microinjected with cDNA encoding HA-tagged IRS-58 in a pXJ40 vector and left for 3 h before fixing and staining of cells. Anti-HA antibody was used to detect IRS-58–expressing cells and cells were also stained for F-actin with rhodamine-conjugated phalloidin as described in Materials and Methods. (A, C, and E) IRS-58; (B, D, and F) F-actin. Arrows mark injected cells in A, C, and E and the corresponding F-actin staining in B, D, and F. The insert in A shows a magnified version of part of the image. Arrowheads in the insert highlight particulate structures/protein aggregates. Arrows in panels indicate transfected cells. Some transfected cells are not marked. Bar, 10 μM.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2195994&req=5

Figure 4: Overexpression of IRS-58 in Swiss 3T3 cells. Serum-starved Swiss 3T3 cells were microinjected with cDNA encoding HA-tagged IRS-58 in a pXJ40 vector and left for 3 h before fixing and staining of cells. Anti-HA antibody was used to detect IRS-58–expressing cells and cells were also stained for F-actin with rhodamine-conjugated phalloidin as described in Materials and Methods. (A, C, and E) IRS-58; (B, D, and F) F-actin. Arrows mark injected cells in A, C, and E and the corresponding F-actin staining in B, D, and F. The insert in A shows a magnified version of part of the image. Arrowheads in the insert highlight particulate structures/protein aggregates. Arrows in panels indicate transfected cells. Some transfected cells are not marked. Bar, 10 μM.
Mentions: Swiss 3T3 cells represent a model mammalian cell type which have been used to investigate the reorganization of both F-actin structures and the associated FCs. In Swiss 3T3 cells, factors such as bradykinin, lysophosphatidic acid, and PMA have been shown to induce the formation of peripheral actin microspikes, stress fibers, and membrane ruffles via Cdc42Hs (Kozma et al. 1995), RhoA (Ridley and Hall 1992), and Rac1 (Ridley et al. 1992), respectively. Each of these F-actin structures is associated with a morphologically unique FC (Nobes and Hall 1995). To investigate whether IRS-58 affected F-actin, it was overexpressed as an HA-tagged protein by DNA microinjection of Swiss 3T3 cells. HA staining of microinjected cells was punctate and revealed that IRS-58 localized to peripheral extensions where process formation (membrane extension and retraction) was induced (Fig. 4A and Fig. B). The punctate HA staining of IRS-58 may suggest its localization to an organelle or a large protein aggregate (Fig. 4 A). Similar observations of IRS-58 localization to protein aggregates were made by Yeh et al. 1998 and Okamura-Oho et al. 1999. Overexpression of IRS-58 caused a reorganization of the F-actin cytoskeleton. Stress fibers were completely disassembled under these conditions and the F-actin took on a punctate appearance (Fig. 4C and Fig. D). In some cells F-actin cluster formation could be seen (Fig. 4E and Fig. F). Fig. 4 shows the heterogeneity in morphology seen in expression of different levels of IRS-58 from low (A and B) to high (E and F).

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