Limits...
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.

Show MeSH

Related in: MedlinePlus

Adhesion and signaling of WT and Tiam1−/− keratinocytes. (A) Keratinocytes were seeded on an exogenous LN5 or LN1 matrix. After 30 min, the number of adherent cells was quantified in an enzymatic assay. Phase-contrast images were taken before cell lysis. The values in the histogram are means ± SD. Note that both WT and Tiam1−/− keratinocytes hardly adhere to and spread on LN1. Bar, 50 μm. (B) Growth factor–starved WT and Tiam1−/− keratinocytes were detached from the culture dish and reseeded on a LN5-coated surface in growth factor–free medium. A sample of the cells in suspension was lysed and the attached cells were lysed after 10, 30, 60, and 180 min. Lysates were immunoblotted for phosphoY397-FAK. β-Actin was used as a loading control. (C) Cells seeded as described in B were lysed after 5, 15, and 45 min. Lysates were immunoblotted for phospho-Src, -Stat3, and -ERK1/2. β-Actin was used as a loading control.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2171282&req=5

fig5: Adhesion and signaling of WT and Tiam1−/− keratinocytes. (A) Keratinocytes were seeded on an exogenous LN5 or LN1 matrix. After 30 min, the number of adherent cells was quantified in an enzymatic assay. Phase-contrast images were taken before cell lysis. The values in the histogram are means ± SD. Note that both WT and Tiam1−/− keratinocytes hardly adhere to and spread on LN1. Bar, 50 μm. (B) Growth factor–starved WT and Tiam1−/− keratinocytes were detached from the culture dish and reseeded on a LN5-coated surface in growth factor–free medium. A sample of the cells in suspension was lysed and the attached cells were lysed after 10, 30, 60, and 180 min. Lysates were immunoblotted for phosphoY397-FAK. β-Actin was used as a loading control. (C) Cells seeded as described in B were lysed after 5, 15, and 45 min. Lysates were immunoblotted for phospho-Src, -Stat3, and -ERK1/2. β-Actin was used as a loading control.

Mentions: It has been well established that the α3β1 integrin, and not the α6β4 integrin, is essential for cell spreading on LN5. Keratinocytes expressing α6β4 that were isolated from α3- mice retained their ability to attach to LN5, but did not spread properly (DiPersio et al., 1997; Hodivala-Dilke et al., 1998). This suggests that Tiam1 acts downstream of α3β1 rather than of α6β4. Indeed, α3-deficient cells contain more thick actin bundles than WT keratinocytes and display robust peripheral focal adhesions (Hodivala-Dilke et al., 1998). Both phenotypes are also found in Tiam1−/− keratinocytes, suggesting a role for Tiam1 in α3β1-mediated adhesion and signaling, rather than in the mechanisms of α6β4. To further substantiate this hypothesis, WT and Tiam1−/− keratinocytes were seeded on LN1, which is a ligand for α6β4, but not for α3β1 (Delwel et al., 1993; Rousselle and Aumailley, 1994). As expected, both WT and Tiam1−/− keratinocytes adhered to LN1, but they were unable to spread on this substrate (Fig. 5 A). In general, Tiam1−/− cells adhered even better to LN1 than WT cells, indicating that adhesion through α6β4 is not impaired in Tiam1−/− cells (Fig. 5 A). In accordance with the notion that adhesion of keratinocytes to LN5 is mediated by both α3β1 and α6β4, both genotypes adhered much better to LN5 than to LN1 (Fig. 5 A). The cells spread on LN5 using α3β1, although the spreading of the Tiam1−/− keratinocytes was still reduced when compared with that of WT keratinocytes. Consistent with the adhesion results, Western blotting and FACS analysis revealed a similar level of expression of the α3 and β1 integrin subunits in the keratinocytes of both genotypes (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200509172/DC1). In addition, no differences were found in the expression level of the α6β4 integrin in WT and Tiam1−/− cells, although a proportion of the WT cells lacked α6β4. These results confirm that the observed defects in adhesion and spreading of Tiam1−/− cells are not caused by changes in the expression of the LN5-binding integrins.


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)

Adhesion and signaling of WT and Tiam1−/− keratinocytes. (A) Keratinocytes were seeded on an exogenous LN5 or LN1 matrix. After 30 min, the number of adherent cells was quantified in an enzymatic assay. Phase-contrast images were taken before cell lysis. The values in the histogram are means ± SD. Note that both WT and Tiam1−/− keratinocytes hardly adhere to and spread on LN1. Bar, 50 μm. (B) Growth factor–starved WT and Tiam1−/− keratinocytes were detached from the culture dish and reseeded on a LN5-coated surface in growth factor–free medium. A sample of the cells in suspension was lysed and the attached cells were lysed after 10, 30, 60, and 180 min. Lysates were immunoblotted for phosphoY397-FAK. β-Actin was used as a loading control. (C) Cells seeded as described in B were lysed after 5, 15, and 45 min. Lysates were immunoblotted for phospho-Src, -Stat3, and -ERK1/2. β-Actin was used as a loading control.
© Copyright Policy
Related In: Results  -  Collection

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

fig5: Adhesion and signaling of WT and Tiam1−/− keratinocytes. (A) Keratinocytes were seeded on an exogenous LN5 or LN1 matrix. After 30 min, the number of adherent cells was quantified in an enzymatic assay. Phase-contrast images were taken before cell lysis. The values in the histogram are means ± SD. Note that both WT and Tiam1−/− keratinocytes hardly adhere to and spread on LN1. Bar, 50 μm. (B) Growth factor–starved WT and Tiam1−/− keratinocytes were detached from the culture dish and reseeded on a LN5-coated surface in growth factor–free medium. A sample of the cells in suspension was lysed and the attached cells were lysed after 10, 30, 60, and 180 min. Lysates were immunoblotted for phosphoY397-FAK. β-Actin was used as a loading control. (C) Cells seeded as described in B were lysed after 5, 15, and 45 min. Lysates were immunoblotted for phospho-Src, -Stat3, and -ERK1/2. β-Actin was used as a loading control.
Mentions: It has been well established that the α3β1 integrin, and not the α6β4 integrin, is essential for cell spreading on LN5. Keratinocytes expressing α6β4 that were isolated from α3- mice retained their ability to attach to LN5, but did not spread properly (DiPersio et al., 1997; Hodivala-Dilke et al., 1998). This suggests that Tiam1 acts downstream of α3β1 rather than of α6β4. Indeed, α3-deficient cells contain more thick actin bundles than WT keratinocytes and display robust peripheral focal adhesions (Hodivala-Dilke et al., 1998). Both phenotypes are also found in Tiam1−/− keratinocytes, suggesting a role for Tiam1 in α3β1-mediated adhesion and signaling, rather than in the mechanisms of α6β4. To further substantiate this hypothesis, WT and Tiam1−/− keratinocytes were seeded on LN1, which is a ligand for α6β4, but not for α3β1 (Delwel et al., 1993; Rousselle and Aumailley, 1994). As expected, both WT and Tiam1−/− keratinocytes adhered to LN1, but they were unable to spread on this substrate (Fig. 5 A). In general, Tiam1−/− cells adhered even better to LN1 than WT cells, indicating that adhesion through α6β4 is not impaired in Tiam1−/− cells (Fig. 5 A). In accordance with the notion that adhesion of keratinocytes to LN5 is mediated by both α3β1 and α6β4, both genotypes adhered much better to LN5 than to LN1 (Fig. 5 A). The cells spread on LN5 using α3β1, although the spreading of the Tiam1−/− keratinocytes was still reduced when compared with that of WT keratinocytes. Consistent with the adhesion results, Western blotting and FACS analysis revealed a similar level of expression of the α3 and β1 integrin subunits in the keratinocytes of both genotypes (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200509172/DC1). In addition, no differences were found in the expression level of the α6β4 integrin in WT and Tiam1−/− cells, although a proportion of the WT cells lacked α6β4. These results confirm that the observed defects in adhesion and spreading of Tiam1−/− cells are not caused by changes in the expression of the LN5-binding integrins.

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