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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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Related in: MedlinePlus

Tiam1−/− keratinocytes spread less well on exogenous Col IV, show more stress fibers, larger focal adhesions, and less Rac activity than WT cells. (A) Lysates were subjected to immunoprecipitation and analyzed for Tiam1 expression. SV40 LT expression was determined by immunoblotting the same lysates. (B) Cells were seeded on a Col IV matrix for 24 h. Bar, 20 μm. (C) Cells were seeded on Col IV–coated coverslips for 48 h, fixed, and stained for F-actin fibers (phalloidin) and focal adhesions (paxillin). Bar, 10 μm. (D) Cells were seeded on a Col IV matrix for 48 h and lysed, and Rac activity was determined. The histogram represents the average Rac activation (relative to the total Rac levels) in both WT and Tiam1−/− cells determined in four independent experiments. Error bars represent the SD.
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fig1: Tiam1−/− keratinocytes spread less well on exogenous Col IV, show more stress fibers, larger focal adhesions, and less Rac activity than WT cells. (A) Lysates were subjected to immunoprecipitation and analyzed for Tiam1 expression. SV40 LT expression was determined by immunoblotting the same lysates. (B) Cells were seeded on a Col IV matrix for 24 h. Bar, 20 μm. (C) Cells were seeded on Col IV–coated coverslips for 48 h, fixed, and stained for F-actin fibers (phalloidin) and focal adhesions (paxillin). Bar, 10 μm. (D) Cells were seeded on a Col IV matrix for 48 h and lysed, and Rac activity was determined. The histogram represents the average Rac activation (relative to the total Rac levels) in both WT and Tiam1−/− cells determined in four independent experiments. Error bars represent the SD.

Mentions: To investigate the role of Tiam1 in Rac-dependent adhesion and cell spreading, we isolated keratinocytes from WT and Tiam1−/− mice. The absence of Tiam1 in cells derived from the knockout mice was confirmed by immunoprecipitation (Fig. 1 A). Primary, as well as immortalized WT and Tiam1−/−, keratinocytes were used in further studies.


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 spread less well on exogenous Col IV, show more stress fibers, larger focal adhesions, and less Rac activity than WT cells. (A) Lysates were subjected to immunoprecipitation and analyzed for Tiam1 expression. SV40 LT expression was determined by immunoblotting the same lysates. (B) Cells were seeded on a Col IV matrix for 24 h. Bar, 20 μm. (C) Cells were seeded on Col IV–coated coverslips for 48 h, fixed, and stained for F-actin fibers (phalloidin) and focal adhesions (paxillin). Bar, 10 μm. (D) Cells were seeded on a Col IV matrix for 48 h and lysed, and Rac activity was determined. The histogram represents the average Rac activation (relative to the total Rac levels) in both WT and Tiam1−/− cells determined in four independent experiments. Error bars represent the SD.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2171282&req=5

fig1: Tiam1−/− keratinocytes spread less well on exogenous Col IV, show more stress fibers, larger focal adhesions, and less Rac activity than WT cells. (A) Lysates were subjected to immunoprecipitation and analyzed for Tiam1 expression. SV40 LT expression was determined by immunoblotting the same lysates. (B) Cells were seeded on a Col IV matrix for 24 h. Bar, 20 μm. (C) Cells were seeded on Col IV–coated coverslips for 48 h, fixed, and stained for F-actin fibers (phalloidin) and focal adhesions (paxillin). Bar, 10 μm. (D) Cells were seeded on a Col IV matrix for 48 h and lysed, and Rac activity was determined. The histogram represents the average Rac activation (relative to the total Rac levels) in both WT and Tiam1−/− cells determined in four independent experiments. Error bars represent the SD.
Mentions: To investigate the role of Tiam1 in Rac-dependent adhesion and cell spreading, we isolated keratinocytes from WT and Tiam1−/− mice. The absence of Tiam1 in cells derived from the knockout mice was confirmed by immunoprecipitation (Fig. 1 A). Primary, as well as immortalized WT and Tiam1−/−, keratinocytes were used in further studies.

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