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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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Rescue of Tiam1 deficiency by Rac effector mutants. Wt, Tiam1−/−, and Tiam1−/− keratinocytes expressing RacL61, RacL61A37, or RacL61C40 were seeded on glass coverslips. After 8 h, cells were washed and phase-contrast images were taken. Bar, 50 μm. The number of adherent cells was quantified in an enzymatic assay using NPAG as a substrate. The values in the histogram are means ± SD.
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fig3: Rescue of Tiam1 deficiency by Rac effector mutants. Wt, Tiam1−/−, and Tiam1−/− keratinocytes expressing RacL61, RacL61A37, or RacL61C40 were seeded on glass coverslips. After 8 h, cells were washed and phase-contrast images were taken. Bar, 50 μm. The number of adherent cells was quantified in an enzymatic assay using NPAG as a substrate. The values in the histogram are means ± SD.

Mentions: We next investigated the pathways controlled by Rac that are responsible for the adhesion and spreading defect. We used effector loop mutants of GTPases previously shown to differentially bind and activate downstream effectors (Lamarche et al., 1996). The constitutively active L61Y40C mutant of Rac1 has lost its ability to interact with p21-activated kinase (PAK)–1 and is unable to activate c-Jun NH2-terminal kinase activity, but it still induces F-actin polymerization and membrane ruffling. Conversely, the L61F37A mutant of Rac1 is unable to remodel the cytoskeleton, but interacts with p65PAK and activates c-Jun NH2-terminal kinase. As shown in Fig. 3, the expression of the L61Y40C mutant of Rac1, but not that of the L61F37A mutant, strongly increased the number of Tiam1−/− cells adhering to and spreading on glass.


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)

Rescue of Tiam1 deficiency by Rac effector mutants. Wt, Tiam1−/−, and Tiam1−/− keratinocytes expressing RacL61, RacL61A37, or RacL61C40 were seeded on glass coverslips. After 8 h, cells were washed and phase-contrast images were taken. Bar, 50 μm. The number of adherent cells was quantified in an enzymatic assay using NPAG as a substrate. The values in the histogram are means ± SD.
© Copyright Policy
Related In: Results  -  Collection

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

fig3: Rescue of Tiam1 deficiency by Rac effector mutants. Wt, Tiam1−/−, and Tiam1−/− keratinocytes expressing RacL61, RacL61A37, or RacL61C40 were seeded on glass coverslips. After 8 h, cells were washed and phase-contrast images were taken. Bar, 50 μm. The number of adherent cells was quantified in an enzymatic assay using NPAG as a substrate. The values in the histogram are means ± SD.
Mentions: We next investigated the pathways controlled by Rac that are responsible for the adhesion and spreading defect. We used effector loop mutants of GTPases previously shown to differentially bind and activate downstream effectors (Lamarche et al., 1996). The constitutively active L61Y40C mutant of Rac1 has lost its ability to interact with p21-activated kinase (PAK)–1 and is unable to activate c-Jun NH2-terminal kinase activity, but it still induces F-actin polymerization and membrane ruffling. Conversely, the L61F37A mutant of Rac1 is unable to remodel the cytoskeleton, but interacts with p65PAK and activates c-Jun NH2-terminal kinase. As shown in Fig. 3, the expression of the L61Y40C mutant of Rac1, but not that of the L61F37A mutant, strongly increased the number of Tiam1−/− cells adhering to and spreading on glass.

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