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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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Rho activity upon adhesion to LN5. (A) Wt and Tiam1−/− keratinocytes were seeded on LN5-coated coverslips. After 10, 30, and 60 min, cells were stained with phalloidin. Bar, 20 μM. (B) Growth factor–starved keratinocytes were seeded on a LN5 matrix. After 0, 5, and 30 min, cells were lysed and Rho activity was determined. A representative experiment is shown and quantified in the histogram, which represents the Rho activation (relative to the total Rho levels) in WT and Tiam1−/− cells. (C) Growth factor–starved keratinocytes were seeded on a LN5 matrix as described in B. After 0, 1, and 3 h, cells were lysed and Rho activity was determined and quantified as described in B.
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fig7: Rho activity upon adhesion to LN5. (A) Wt and Tiam1−/− keratinocytes were seeded on LN5-coated coverslips. After 10, 30, and 60 min, cells were stained with phalloidin. Bar, 20 μM. (B) Growth factor–starved keratinocytes were seeded on a LN5 matrix. After 0, 5, and 30 min, cells were lysed and Rho activity was determined. A representative experiment is shown and quantified in the histogram, which represents the Rho activation (relative to the total Rho levels) in WT and Tiam1−/− cells. (C) Growth factor–starved keratinocytes were seeded on a LN5 matrix as described in B. After 0, 1, and 3 h, cells were lysed and Rho activity was determined and quantified as described in B.

Mentions: Although Tiam1−/− keratinocytes spread less well on exogenous LN5 matrix than WT cells (Fig. 7 A), they are able to spread without any detectable change in Rac activity. Earlier studies have demonstrated that cross talk exists between Rac and Rho GTPases, and that changes in the balance between the activities of these proteins can influence cell morphology (van Leeuwen et al., 1997; Kodama et al., 1999; Sander et al., 1999). Therefore, we hypothesized that spreading of Tiam1−/− keratinocytes might be regulated by a change in Rho activity, rather than Rac activity. In WT cells, a small decrease (10–15%) in Rho activity could be detected 5, 30, and 60 min after seeding on LN5 (Fig. 7, B and C). However, in Tiam1−/− cells a much larger decrease in Rho activity was found 5, 30, and 60 min after seeding (45, 55, and 60%, respectively). In both WT and Tiam1−/− cells, the decrease in Rho activity was observed during at least 1 h and returned to basal levels within 3 h (Fig. 7, B and C). This suggests that spreading of Tiam1−/− keratinocytes is caused by a substantial decrease in Rho activity, which leads to cytoskeletal relaxation. In this manner, in Tiam1−/− keratinocytes Rac activity can be relatively increased, as compared with Rho activity, upon adhesion to exogenous LN5, thereby allowing cell spreading.


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)

Rho activity upon adhesion to LN5. (A) Wt and Tiam1−/− keratinocytes were seeded on LN5-coated coverslips. After 10, 30, and 60 min, cells were stained with phalloidin. Bar, 20 μM. (B) Growth factor–starved keratinocytes were seeded on a LN5 matrix. After 0, 5, and 30 min, cells were lysed and Rho activity was determined. A representative experiment is shown and quantified in the histogram, which represents the Rho activation (relative to the total Rho levels) in WT and Tiam1−/− cells. (C) Growth factor–starved keratinocytes were seeded on a LN5 matrix as described in B. After 0, 1, and 3 h, cells were lysed and Rho activity was determined and quantified as described in B.
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fig7: Rho activity upon adhesion to LN5. (A) Wt and Tiam1−/− keratinocytes were seeded on LN5-coated coverslips. After 10, 30, and 60 min, cells were stained with phalloidin. Bar, 20 μM. (B) Growth factor–starved keratinocytes were seeded on a LN5 matrix. After 0, 5, and 30 min, cells were lysed and Rho activity was determined. A representative experiment is shown and quantified in the histogram, which represents the Rho activation (relative to the total Rho levels) in WT and Tiam1−/− cells. (C) Growth factor–starved keratinocytes were seeded on a LN5 matrix as described in B. After 0, 1, and 3 h, cells were lysed and Rho activity was determined and quantified as described in B.
Mentions: Although Tiam1−/− keratinocytes spread less well on exogenous LN5 matrix than WT cells (Fig. 7 A), they are able to spread without any detectable change in Rac activity. Earlier studies have demonstrated that cross talk exists between Rac and Rho GTPases, and that changes in the balance between the activities of these proteins can influence cell morphology (van Leeuwen et al., 1997; Kodama et al., 1999; Sander et al., 1999). Therefore, we hypothesized that spreading of Tiam1−/− keratinocytes might be regulated by a change in Rho activity, rather than Rac activity. In WT cells, a small decrease (10–15%) in Rho activity could be detected 5, 30, and 60 min after seeding on LN5 (Fig. 7, B and C). However, in Tiam1−/− cells a much larger decrease in Rho activity was found 5, 30, and 60 min after seeding (45, 55, and 60%, respectively). In both WT and Tiam1−/− cells, the decrease in Rho activity was observed during at least 1 h and returned to basal levels within 3 h (Fig. 7, B and C). This suggests that spreading of Tiam1−/− keratinocytes is caused by a substantial decrease in Rho activity, which leads to cytoskeletal relaxation. In this manner, in Tiam1−/− keratinocytes Rac activity can be relatively increased, as compared with Rho activity, upon adhesion to exogenous LN5, thereby allowing cell spreading.

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