Limits...
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.

Show MeSH

Related in: MedlinePlus

Quantification of the effects of IRS-58 on N1E-115 neuroblastoma cells. Cells transfected with cDNAs were left for 18–24 h and then scored for (A) neurite outgrowth or (B) neurite complexity. (A) Lane 1, pXJ40; lane 2, no cDNA with 5% serum; lane 3, no cDNA serum-starved; lane 4, IRS-58; lane 5, IRS-58 (amino acids 1–390); and lane 6, IRS-58I269N. Neurite outgrowth was scored by evaluating the percentage of cells with neurites longer than one cell body length. (B) Lane 1, serum starvation; lane 2, IRS-58; and lane 3, IRS-58 (amino acids 1–390). In determining percentage complexity, neurites were examined for both filopodial number and lamellipodial area as follows: (a) 50 filopodia/neurite was arbitrarily set at a score of 100%; (b) the proportion of neurite length covered by lamellipodia was scored using 100% to represent coverage of the whole neurite length. Percentage complexity was then determined by adding values obtained from (a) and (b) and dividing by 2. In some instances (e.g., Fig. 9 A, panel C), where lamellipodia were absent, percentage complexity was based solely on (a) values. Numbers are averages ± SD; n = 21.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2195994&req=5

Figure 10: Quantification of the effects of IRS-58 on N1E-115 neuroblastoma cells. Cells transfected with cDNAs were left for 18–24 h and then scored for (A) neurite outgrowth or (B) neurite complexity. (A) Lane 1, pXJ40; lane 2, no cDNA with 5% serum; lane 3, no cDNA serum-starved; lane 4, IRS-58; lane 5, IRS-58 (amino acids 1–390); and lane 6, IRS-58I269N. Neurite outgrowth was scored by evaluating the percentage of cells with neurites longer than one cell body length. (B) Lane 1, serum starvation; lane 2, IRS-58; and lane 3, IRS-58 (amino acids 1–390). In determining percentage complexity, neurites were examined for both filopodial number and lamellipodial area as follows: (a) 50 filopodia/neurite was arbitrarily set at a score of 100%; (b) the proportion of neurite length covered by lamellipodia was scored using 100% to represent coverage of the whole neurite length. Percentage complexity was then determined by adding values obtained from (a) and (b) and dividing by 2. In some instances (e.g., Fig. 9 A, panel C), where lamellipodia were absent, percentage complexity was based solely on (a) values. Numbers are averages ± SD; n = 21.

Mentions: We also examined the effect of overexpression of three IRS-58 COOH-terminal deletions (IRS-58Δ510–534, IRS-58Δ390–534, and IRS-58Δ143–534) on neurite outgrowth. As in Swiss 3T3 cells, IRS-58Δ510–534 had a more dramatic effect on cell morphology than wild-type in N1E-115 cells (Fig. 9 A, panel D). Interestingly, IRS-58Δ390–534, which lacks the SH3 domain and the WW-binding domain, induced neurite extension; however, neurite and growth cone complexity were lost (Fig. 9 A, panel E). IRS-58Δ143–534 failed to induce morphological change, suggesting that the amino acids 143–390 are essential for neurite extension (data not shown). A quantification of the effects of IRS-58 transfection on neuroblastoma cells is shown in Fig. 10.


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)

Quantification of the effects of IRS-58 on N1E-115 neuroblastoma cells. Cells transfected with cDNAs were left for 18–24 h and then scored for (A) neurite outgrowth or (B) neurite complexity. (A) Lane 1, pXJ40; lane 2, no cDNA with 5% serum; lane 3, no cDNA serum-starved; lane 4, IRS-58; lane 5, IRS-58 (amino acids 1–390); and lane 6, IRS-58I269N. Neurite outgrowth was scored by evaluating the percentage of cells with neurites longer than one cell body length. (B) Lane 1, serum starvation; lane 2, IRS-58; and lane 3, IRS-58 (amino acids 1–390). In determining percentage complexity, neurites were examined for both filopodial number and lamellipodial area as follows: (a) 50 filopodia/neurite was arbitrarily set at a score of 100%; (b) the proportion of neurite length covered by lamellipodia was scored using 100% to represent coverage of the whole neurite length. Percentage complexity was then determined by adding values obtained from (a) and (b) and dividing by 2. In some instances (e.g., Fig. 9 A, panel C), where lamellipodia were absent, percentage complexity was based solely on (a) values. Numbers are averages ± SD; n = 21.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2195994&req=5

Figure 10: Quantification of the effects of IRS-58 on N1E-115 neuroblastoma cells. Cells transfected with cDNAs were left for 18–24 h and then scored for (A) neurite outgrowth or (B) neurite complexity. (A) Lane 1, pXJ40; lane 2, no cDNA with 5% serum; lane 3, no cDNA serum-starved; lane 4, IRS-58; lane 5, IRS-58 (amino acids 1–390); and lane 6, IRS-58I269N. Neurite outgrowth was scored by evaluating the percentage of cells with neurites longer than one cell body length. (B) Lane 1, serum starvation; lane 2, IRS-58; and lane 3, IRS-58 (amino acids 1–390). In determining percentage complexity, neurites were examined for both filopodial number and lamellipodial area as follows: (a) 50 filopodia/neurite was arbitrarily set at a score of 100%; (b) the proportion of neurite length covered by lamellipodia was scored using 100% to represent coverage of the whole neurite length. Percentage complexity was then determined by adding values obtained from (a) and (b) and dividing by 2. In some instances (e.g., Fig. 9 A, panel C), where lamellipodia were absent, percentage complexity was based solely on (a) values. Numbers are averages ± SD; n = 21.
Mentions: We also examined the effect of overexpression of three IRS-58 COOH-terminal deletions (IRS-58Δ510–534, IRS-58Δ390–534, and IRS-58Δ143–534) on neurite outgrowth. As in Swiss 3T3 cells, IRS-58Δ510–534 had a more dramatic effect on cell morphology than wild-type in N1E-115 cells (Fig. 9 A, panel D). Interestingly, IRS-58Δ390–534, which lacks the SH3 domain and the WW-binding domain, induced neurite extension; however, neurite and growth cone complexity were lost (Fig. 9 A, panel E). IRS-58Δ143–534 failed to induce morphological change, suggesting that the amino acids 143–390 are essential for neurite extension (data not shown). A quantification of the effects of IRS-58 transfection on neuroblastoma cells is shown in Fig. 10.

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
Related in: MedlinePlus