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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 cell spreading when seeded on an inert surface. (A) Wt and Tiam1−/− keratinocytes were seeded on glass coverslips or on LN5-, Col IV-, FN-, VN-, or PLL-coated coverslips. After 8 h, the number of adherent cells was quantified with an enzymatic assay using NPAG as a substrate. The values in the histogram are means ± SD. (B) Wt, Tiam1−/−, and Tiam1−/− keratinocytes stably expressing full-length Tiam1 or RacV12 were seeded for 12 h on glass coverslips and on coverslips coated with Col IV. Cells were washed and phase-contrast images were taken. Bar, 50 μm. (C) Cells were seeded as in B and the number of adherent cells was quantified with an enzymatic assay using NPAG as a substrate. The values in the histogram are means ± SEM (n = 3). (D) Cell lysates of WT, Tiam1−/−, and Tiam1−/− cells infected with full-length Tiam1 or RacV12 viruses were immunoblotted for Tiam1 and myc-tagged RacV12.
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fig2: Tiam1 is required for cell spreading when seeded on an inert surface. (A) Wt and Tiam1−/− keratinocytes were seeded on glass coverslips or on LN5-, Col IV-, FN-, VN-, or PLL-coated coverslips. After 8 h, the number of adherent cells was quantified with an enzymatic assay using NPAG as a substrate. The values in the histogram are means ± SD. (B) Wt, Tiam1−/−, and Tiam1−/− keratinocytes stably expressing full-length Tiam1 or RacV12 were seeded for 12 h on glass coverslips and on coverslips coated with Col IV. Cells were washed and phase-contrast images were taken. Bar, 50 μm. (C) Cells were seeded as in B and the number of adherent cells was quantified with an enzymatic assay using NPAG as a substrate. The values in the histogram are means ± SEM (n = 3). (D) Cell lysates of WT, Tiam1−/−, and Tiam1−/− cells infected with full-length Tiam1 or RacV12 viruses were immunoblotted for Tiam1 and myc-tagged RacV12.

Mentions: The capacity of WT and Tiam1−/− keratinocytes to adhere to a LN5-, FN-, vitronectin (VN)-, or Col IV-coated surface was not significantly different (Fig. 2, A–C), although spreading of Tiam1−/− cells was consistently found to be slightly reduced (Fig. 1 C and not depicted). If no exogenous matrix is available, adhesion and spreading of keratinocytes depends on the ability of these cells to secrete and deposit their own LN5 matrix. To investigate how the loss of Tiam1 affects keratinocyte adhesion under such conditions, Tiam1−/− and WT cells were seeded on an inert glass surface. After 12 h, 75% of the WT cells had adhered to and spread on the glass. In contrast, only 5–10% of the Tiam1−/− keratinocytes had adhered to the glass (Fig. 2, B and C). The few adherent cells were rounded and refractile, as if they were blocked in an early stage of spreading (Fig. 2 B). Similar results were found when cells were seeded on plastic (not depicted).


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 cell spreading when seeded on an inert surface. (A) Wt and Tiam1−/− keratinocytes were seeded on glass coverslips or on LN5-, Col IV-, FN-, VN-, or PLL-coated coverslips. After 8 h, the number of adherent cells was quantified with an enzymatic assay using NPAG as a substrate. The values in the histogram are means ± SD. (B) Wt, Tiam1−/−, and Tiam1−/− keratinocytes stably expressing full-length Tiam1 or RacV12 were seeded for 12 h on glass coverslips and on coverslips coated with Col IV. Cells were washed and phase-contrast images were taken. Bar, 50 μm. (C) Cells were seeded as in B and the number of adherent cells was quantified with an enzymatic assay using NPAG as a substrate. The values in the histogram are means ± SEM (n = 3). (D) Cell lysates of WT, Tiam1−/−, and Tiam1−/− cells infected with full-length Tiam1 or RacV12 viruses were immunoblotted for Tiam1 and myc-tagged RacV12.
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Related In: Results  -  Collection

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fig2: Tiam1 is required for cell spreading when seeded on an inert surface. (A) Wt and Tiam1−/− keratinocytes were seeded on glass coverslips or on LN5-, Col IV-, FN-, VN-, or PLL-coated coverslips. After 8 h, the number of adherent cells was quantified with an enzymatic assay using NPAG as a substrate. The values in the histogram are means ± SD. (B) Wt, Tiam1−/−, and Tiam1−/− keratinocytes stably expressing full-length Tiam1 or RacV12 were seeded for 12 h on glass coverslips and on coverslips coated with Col IV. Cells were washed and phase-contrast images were taken. Bar, 50 μm. (C) Cells were seeded as in B and the number of adherent cells was quantified with an enzymatic assay using NPAG as a substrate. The values in the histogram are means ± SEM (n = 3). (D) Cell lysates of WT, Tiam1−/−, and Tiam1−/− cells infected with full-length Tiam1 or RacV12 viruses were immunoblotted for Tiam1 and myc-tagged RacV12.
Mentions: The capacity of WT and Tiam1−/− keratinocytes to adhere to a LN5-, FN-, vitronectin (VN)-, or Col IV-coated surface was not significantly different (Fig. 2, A–C), although spreading of Tiam1−/− cells was consistently found to be slightly reduced (Fig. 1 C and not depicted). If no exogenous matrix is available, adhesion and spreading of keratinocytes depends on the ability of these cells to secrete and deposit their own LN5 matrix. To investigate how the loss of Tiam1 affects keratinocyte adhesion under such conditions, Tiam1−/− and WT cells were seeded on an inert glass surface. After 12 h, 75% of the WT cells had adhered to and spread on the glass. In contrast, only 5–10% of the Tiam1−/− keratinocytes had adhered to the glass (Fig. 2, B and C). The few adherent cells were rounded and refractile, as if they were blocked in an early stage of spreading (Fig. 2 B). Similar results were found when cells were seeded on plastic (not depicted).

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