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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 is required for integrin induced Rac activation upon adhesion to LN5. (A) Growth factor–starved keratinocytes were seeded on a LN5 matrix. After 0, 5, and 30 min, cells were lysed and Rac activity was determined. A representative experiment is shown and quantified in the histogram, which represents the Rac activation (relative to the total Rac levels) in WT and Tiam1−/− cells. (B) Growth factor–starved keratinocytes were seeded on a LN5 matrix. After 0, 1, and 3 h, cells were lysed and Rac activity was determined and quantified as described in A. (C) Growth factor–starved keratinocytes were seeded on LN5-, FN-, VN-, or PLL-coated plates for 45 min. Subsequently, cells were lysed, and Rac activity was determined. The histogram represents the average Rac activity after 45 min in both WT and Tiam1−/− cells determined in three independent experiments. Error bars represent the SD.
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fig6: Tiam1 is required for integrin induced Rac activation upon adhesion to LN5. (A) Growth factor–starved keratinocytes were seeded on a LN5 matrix. After 0, 5, and 30 min, cells were lysed and Rac activity was determined. A representative experiment is shown and quantified in the histogram, which represents the Rac activation (relative to the total Rac levels) in WT and Tiam1−/− cells. (B) Growth factor–starved keratinocytes were seeded on a LN5 matrix. After 0, 1, and 3 h, cells were lysed and Rac activity was determined and quantified as described in A. (C) Growth factor–starved keratinocytes were seeded on LN5-, FN-, VN-, or PLL-coated plates for 45 min. Subsequently, cells were lysed, and Rac activity was determined. The histogram represents the average Rac activity after 45 min in both WT and Tiam1−/− cells determined in three independent experiments. Error bars represent the SD.

Mentions: We then studied the activation of Rac upon adhesion of keratinocytes to exogenous LN5 substrate. In WT cells, a small increase in active GTP-bound Rac could be measured 5 min after seeding, and this increase was much more pronounced after 30 min (Fig. 6 A). Rac activity remained elevated for at least 1 h and returned to basal levels within 3 h (Fig. 6 B). In contrast, in Tiam1−/− keratinocytes we did not detect any Rac activation upon adhesion to LN5 after 5 and 30 min, or even after 3 h (Fig. 6, A and B), indicating that α3β1-mediated Rac signaling is impaired in Tiam1−/− cells.


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 is required for integrin induced Rac activation upon adhesion to LN5. (A) Growth factor–starved keratinocytes were seeded on a LN5 matrix. After 0, 5, and 30 min, cells were lysed and Rac activity was determined. A representative experiment is shown and quantified in the histogram, which represents the Rac activation (relative to the total Rac levels) in WT and Tiam1−/− cells. (B) Growth factor–starved keratinocytes were seeded on a LN5 matrix. After 0, 1, and 3 h, cells were lysed and Rac activity was determined and quantified as described in A. (C) Growth factor–starved keratinocytes were seeded on LN5-, FN-, VN-, or PLL-coated plates for 45 min. Subsequently, cells were lysed, and Rac activity was determined. The histogram represents the average Rac activity after 45 min in both WT and Tiam1−/− cells determined in three independent experiments. Error bars represent the SD.
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Related In: Results  -  Collection

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fig6: Tiam1 is required for integrin induced Rac activation upon adhesion to LN5. (A) Growth factor–starved keratinocytes were seeded on a LN5 matrix. After 0, 5, and 30 min, cells were lysed and Rac activity was determined. A representative experiment is shown and quantified in the histogram, which represents the Rac activation (relative to the total Rac levels) in WT and Tiam1−/− cells. (B) Growth factor–starved keratinocytes were seeded on a LN5 matrix. After 0, 1, and 3 h, cells were lysed and Rac activity was determined and quantified as described in A. (C) Growth factor–starved keratinocytes were seeded on LN5-, FN-, VN-, or PLL-coated plates for 45 min. Subsequently, cells were lysed, and Rac activity was determined. The histogram represents the average Rac activity after 45 min in both WT and Tiam1−/− cells determined in three independent experiments. Error bars represent the SD.
Mentions: We then studied the activation of Rac upon adhesion of keratinocytes to exogenous LN5 substrate. In WT cells, a small increase in active GTP-bound Rac could be measured 5 min after seeding, and this increase was much more pronounced after 30 min (Fig. 6 A). Rac activity remained elevated for at least 1 h and returned to basal levels within 3 h (Fig. 6 B). In contrast, in Tiam1−/− keratinocytes we did not detect any Rac activation upon adhesion to LN5 after 5 and 30 min, or even after 3 h (Fig. 6, A and B), indicating that α3β1-mediated Rac signaling is impaired in Tiam1−/− cells.

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