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The Rac activator Tiam1 is required for (alpha)3(beta)1-mediated laminin-5 deposition, cell spreading, and cell migration.

Hamelers IH, Olivo C, Mertens AE, Pegtel DM, van der Kammen RA, Sonnenberg A, Collard JG - J. Cell Biol. (2005)

Bottom Line: Both Tiam1 and V12Rac1 can rescue the defects of Tiam1-/- keratinocytes, indicating that these deficiencies are caused by impaired Tiam1-mediated Rac activation.Moreover, Tiam1 deficiency impairs keratinocyte migration in vitro and reepithelialization of excision wounds in mouse skin.Our studies indicate that Tiam1 is a key molecule in alpha3beta1-mediated activation of Rac, which is essential for proper production and secretion of LN5, a requirement for the spreading and migration of keratinocytes.

View Article: PubMed Central - PubMed

Affiliation: Division of Cell Biology, Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands.

ABSTRACT
The Rho-like guanosine triphosphatase Rac1 regulates various signaling pathways, including integrin-mediated adhesion and migration of cells. However, the mechanisms by which integrins signal toward Rac are poorly understood. We show that the Rac-specific guanine nucleotide exchange factor Tiam1 (T-lymphoma invasion and metastasis 1) is required for the integrin-mediated laminin (LN)-5 deposition, spreading, and migration of keratinocytes. In contrast to wild-type keratinocytes, Tiam1-deficient (Tiam1-/-) keratinocytes are unable to adhere to and spread on a glass substrate because they are unable to deposit their own LN5 substrate. Both Tiam1 and V12Rac1 can rescue the defects of Tiam1-/- keratinocytes, indicating that these deficiencies are caused by impaired Tiam1-mediated Rac activation. Tiam1-/- cells are unable to activate Rac upon alpha3beta1-mediated adhesion to an exogenous LN5 substrate. Moreover, Tiam1 deficiency impairs keratinocyte migration in vitro and reepithelialization of excision wounds in mouse skin. Our studies indicate that Tiam1 is a key molecule in alpha3beta1-mediated activation of Rac, which is essential for proper production and secretion of LN5, a requirement for the spreading and migration of keratinocytes.

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Tiam1−/− keratinocytes and cell migration. (A) Cells were seeded on Col IV–coated dishes and grown to confluency. The cultures were band stripped and allowed to migrate into the wounded area for 24 h in keratinocyte medium with defined growth supplement. Photographs show representative examples. The line indicates the wound edge at the start of the experiment (t = 0 h). Bar, 200 μm. (B) Cells were seeded and grown as described in A, band stripped, and allowed to migrate into the wounded area for 24 h in keratinocyte medium with defined growth supplement and 10% chelexed fetal calf serum. Photographs show representative examples. The line indicates the wound edge at the start of the experiment (t = 0 h). Bar, 200 μm. (C) Tiam1−/− keratinocytes were seeded on Col IV–coated dishes and grown to confluency. The cultures were band stripped and allowed to migrate into the wounded area for 3 h in keratinocyte medium with defined growth supplement. Cells were fixed and stained for F-actin and the LN5 γ2 chain. Bar, 40 μm. Note the removal of the LN5 substrate by scratching.
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fig4: Tiam1−/− keratinocytes and cell migration. (A) Cells were seeded on Col IV–coated dishes and grown to confluency. The cultures were band stripped and allowed to migrate into the wounded area for 24 h in keratinocyte medium with defined growth supplement. Photographs show representative examples. The line indicates the wound edge at the start of the experiment (t = 0 h). Bar, 200 μm. (B) Cells were seeded and grown as described in A, band stripped, and allowed to migrate into the wounded area for 24 h in keratinocyte medium with defined growth supplement and 10% chelexed fetal calf serum. Photographs show representative examples. The line indicates the wound edge at the start of the experiment (t = 0 h). Bar, 200 μm. (C) Tiam1−/− keratinocytes were seeded on Col IV–coated dishes and grown to confluency. The cultures were band stripped and allowed to migrate into the wounded area for 3 h in keratinocyte medium with defined growth supplement. Cells were fixed and stained for F-actin and the LN5 γ2 chain. Bar, 40 μm. Note the removal of the LN5 substrate by scratching.

Mentions: Next, we studied the migration of WT and Tiam1−/− keratinocytes into a scrape wound, a process also dependent on the ability of keratinocytes to produce and secrete LN5. Confluent monolayers of WT and Tiam1−/− keratinocytes, cultured on Col IV–coated surfaces in keratinocyte medium with defined growth factors but without ECM components, were scrape wounded and the migration of keratinocytes was investigated. As expected, WT keratinocytes migrated into the denuded area and closed the wound within 24 h (Fig. 4 A). In contrast, Tiam1−/− keratinocytes did not migrate into the wound (Fig. 4, A and C), where the LN5 and Col IV coating was removed by scraping (Fig. 4 C). However, when ECM components were provided by the addition of chelated fetal calf serum to the medium after scraping, the Tiam1−/− cells did migrate, albeit less efficiently than WT keratinocytes (Fig. 4 B). These data are consistent with our earlier conclusion that Tiam1−/− keratinocytes are unable to produce and secrete sufficient amounts of LN5 substrate, resulting in their inability to spread and migrate onto an uncoated surface.


The Rac activator Tiam1 is required for (alpha)3(beta)1-mediated laminin-5 deposition, cell spreading, and cell migration.

Hamelers IH, Olivo C, Mertens AE, Pegtel DM, van der Kammen RA, Sonnenberg A, Collard JG - J. Cell Biol. (2005)

Tiam1−/− keratinocytes and cell migration. (A) Cells were seeded on Col IV–coated dishes and grown to confluency. The cultures were band stripped and allowed to migrate into the wounded area for 24 h in keratinocyte medium with defined growth supplement. Photographs show representative examples. The line indicates the wound edge at the start of the experiment (t = 0 h). Bar, 200 μm. (B) Cells were seeded and grown as described in A, band stripped, and allowed to migrate into the wounded area for 24 h in keratinocyte medium with defined growth supplement and 10% chelexed fetal calf serum. Photographs show representative examples. The line indicates the wound edge at the start of the experiment (t = 0 h). Bar, 200 μm. (C) Tiam1−/− keratinocytes were seeded on Col IV–coated dishes and grown to confluency. The cultures were band stripped and allowed to migrate into the wounded area for 3 h in keratinocyte medium with defined growth supplement. Cells were fixed and stained for F-actin and the LN5 γ2 chain. Bar, 40 μm. Note the removal of the LN5 substrate by scratching.
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Related In: Results  -  Collection

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fig4: Tiam1−/− keratinocytes and cell migration. (A) Cells were seeded on Col IV–coated dishes and grown to confluency. The cultures were band stripped and allowed to migrate into the wounded area for 24 h in keratinocyte medium with defined growth supplement. Photographs show representative examples. The line indicates the wound edge at the start of the experiment (t = 0 h). Bar, 200 μm. (B) Cells were seeded and grown as described in A, band stripped, and allowed to migrate into the wounded area for 24 h in keratinocyte medium with defined growth supplement and 10% chelexed fetal calf serum. Photographs show representative examples. The line indicates the wound edge at the start of the experiment (t = 0 h). Bar, 200 μm. (C) Tiam1−/− keratinocytes were seeded on Col IV–coated dishes and grown to confluency. The cultures were band stripped and allowed to migrate into the wounded area for 3 h in keratinocyte medium with defined growth supplement. Cells were fixed and stained for F-actin and the LN5 γ2 chain. Bar, 40 μm. Note the removal of the LN5 substrate by scratching.
Mentions: Next, we studied the migration of WT and Tiam1−/− keratinocytes into a scrape wound, a process also dependent on the ability of keratinocytes to produce and secrete LN5. Confluent monolayers of WT and Tiam1−/− keratinocytes, cultured on Col IV–coated surfaces in keratinocyte medium with defined growth factors but without ECM components, were scrape wounded and the migration of keratinocytes was investigated. As expected, WT keratinocytes migrated into the denuded area and closed the wound within 24 h (Fig. 4 A). In contrast, Tiam1−/− keratinocytes did not migrate into the wound (Fig. 4, A and C), where the LN5 and Col IV coating was removed by scraping (Fig. 4 C). However, when ECM components were provided by the addition of chelated fetal calf serum to the medium after scraping, the Tiam1−/− cells did migrate, albeit less efficiently than WT keratinocytes (Fig. 4 B). These data are consistent with our earlier conclusion that Tiam1−/− keratinocytes are unable to produce and secrete sufficient amounts of LN5 substrate, resulting in their inability to spread and migrate onto an uncoated surface.

Bottom Line: Both Tiam1 and V12Rac1 can rescue the defects of Tiam1-/- keratinocytes, indicating that these deficiencies are caused by impaired Tiam1-mediated Rac activation.Moreover, Tiam1 deficiency impairs keratinocyte migration in vitro and reepithelialization of excision wounds in mouse skin.Our studies indicate that Tiam1 is a key molecule in alpha3beta1-mediated activation of Rac, which is essential for proper production and secretion of LN5, a requirement for the spreading and migration of keratinocytes.

View Article: PubMed Central - PubMed

Affiliation: Division of Cell Biology, Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands.

ABSTRACT
The Rho-like guanosine triphosphatase Rac1 regulates various signaling pathways, including integrin-mediated adhesion and migration of cells. However, the mechanisms by which integrins signal toward Rac are poorly understood. We show that the Rac-specific guanine nucleotide exchange factor Tiam1 (T-lymphoma invasion and metastasis 1) is required for the integrin-mediated laminin (LN)-5 deposition, spreading, and migration of keratinocytes. In contrast to wild-type keratinocytes, Tiam1-deficient (Tiam1-/-) keratinocytes are unable to adhere to and spread on a glass substrate because they are unable to deposit their own LN5 substrate. Both Tiam1 and V12Rac1 can rescue the defects of Tiam1-/- keratinocytes, indicating that these deficiencies are caused by impaired Tiam1-mediated Rac activation. Tiam1-/- cells are unable to activate Rac upon alpha3beta1-mediated adhesion to an exogenous LN5 substrate. Moreover, Tiam1 deficiency impairs keratinocyte migration in vitro and reepithelialization of excision wounds in mouse skin. Our studies indicate that Tiam1 is a key molecule in alpha3beta1-mediated activation of Rac, which is essential for proper production and secretion of LN5, a requirement for the spreading and migration of keratinocytes.

Show MeSH
Related in: MedlinePlus