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Biochemical and functional characterization of the interaction between liprin-α1 and GIT1: implications for the regulation of cell motility.

Asperti C, Astro V, Pettinato E, Paris S, Bachi A, de Curtis I - PLoS ONE (2011)

Bottom Line: Here we have addressed the involvement of the liprin-α1 partner GIT1 in liprin-α1-mediated effects on cell spreading and migration.No cumulative effects were observed after depletion of both liprin-α1 and GIT1, suggesting that the two proteins belong to the same signaling network in the regulation of cell spreading.These findings and the possible competition of liprin-α1 with paxillin for binding to GIT1 suggest that alternative binding of GIT1 to either liprin-α1 or paxillin plays distinct roles in different phases of the protrusive activity in the cell.

View Article: PubMed Central - PubMed

Affiliation: Division of Neuroscience, San Raffaele Scientific Institute and San Raffaele University, Milano, Italy.

ABSTRACT
We have previously identified the scaffold protein liprin-α1 as an important regulator of integrin-mediated cell motility and tumor cell invasion. Liprin-α1 may interact with different proteins, and the functional significance of these interactions in the regulation of cell motility is poorly known. Here we have addressed the involvement of the liprin-α1 partner GIT1 in liprin-α1-mediated effects on cell spreading and migration. GIT1 depletion inhibited spreading by affecting the lamellipodia, and prevented liprin-α1-enhanced spreading. Conversely inhibition of the formation of the liprin-α1-GIT complex by expression of liprin-ΔCC3 could still enhance spreading, although to a lesser extent compared to full length liprin-α1. No cumulative effects were observed after depletion of both liprin-α1 and GIT1, suggesting that the two proteins belong to the same signaling network in the regulation of cell spreading. Our data suggest that liprin-α1 may compete with paxillin for binding to GIT1, while binding of βPIX to GIT1 was unaffected by the presence of liprin-α1. Interestingly, GIT and liprin-α1 reciprocally regulated their subcellular localization, since liprin-α1 overexpression, but not the GIT binding-defective liprin-ΔCC3 mutant, affected the localization of endogenous GIT at peripheral and mature central focal adhesions, while the expression of a truncated, active form of GIT1 enhanced the localization of endogenous liprin-α1 at the edge of spreading cells. Moreover, GIT1 was required for liprin-α1-enhanced haptotatic migration, although the direct interaction between liprin-α1 and GIT1 was not needed. Our findings show that the functional interaction between liprin-α1 and GIT1 cooperate in the regulation of integrin-dependent cell spreading and motility on extracellular matrix. These findings and the possible competition of liprin-α1 with paxillin for binding to GIT1 suggest that alternative binding of GIT1 to either liprin-α1 or paxillin plays distinct roles in different phases of the protrusive activity in the cell.

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Liprin-α1 affects the subcellular localization of endogenous GIT.(A) Overexpression of liprin-α1 affects the localization of endogenous GIT at peripheral FAs. COS7 cells overexpressing either FLAG-liprin-α1 or FLAG-βgalactosidase were plated for 1 h on FN and immunostained for the transfected protein and for endogenous GIT. Scale bar, 20 µm. Right panel: four-fold enlargement of the boxed field; liprin-α1 overexpression (cell with asterisk) reduces the accumulation of GIT at newly formed FAs at the edge of transfected cells (arrowheads). Scale bar, 5 µm. (B) Cells transfected with FLAG-βgalactosidase, FLAG-liprin-α1, or FLAG-liprin-ΔCC3 were plated for 1 h on FN before fixation and staining for the transfected protein and for endogenous GIT and FAK proteins. Scale bar, 20 µm. (C) High magnification of the edge of transfected cells showing that endogenous GIT overlaps well with FAK at peripheral FAs of FLAG-liprin-ΔCC3 transfected cells, while poor overlap between endogenous GIT and FAK is seen at peripheral FAs of FLAG-liprin-α1 expressing cells. Scale bar, 10 µm. Panels on the right are 3-fold enlargements of the areas indicated by arrowheads in the corresponding images on the left.
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pone-0020757-g003: Liprin-α1 affects the subcellular localization of endogenous GIT.(A) Overexpression of liprin-α1 affects the localization of endogenous GIT at peripheral FAs. COS7 cells overexpressing either FLAG-liprin-α1 or FLAG-βgalactosidase were plated for 1 h on FN and immunostained for the transfected protein and for endogenous GIT. Scale bar, 20 µm. Right panel: four-fold enlargement of the boxed field; liprin-α1 overexpression (cell with asterisk) reduces the accumulation of GIT at newly formed FAs at the edge of transfected cells (arrowheads). Scale bar, 5 µm. (B) Cells transfected with FLAG-βgalactosidase, FLAG-liprin-α1, or FLAG-liprin-ΔCC3 were plated for 1 h on FN before fixation and staining for the transfected protein and for endogenous GIT and FAK proteins. Scale bar, 20 µm. (C) High magnification of the edge of transfected cells showing that endogenous GIT overlaps well with FAK at peripheral FAs of FLAG-liprin-ΔCC3 transfected cells, while poor overlap between endogenous GIT and FAK is seen at peripheral FAs of FLAG-liprin-α1 expressing cells. Scale bar, 10 µm. Panels on the right are 3-fold enlargements of the areas indicated by arrowheads in the corresponding images on the left.

Mentions: As previously reported [4], [16], we found that endogenous GIT1 localized with paxillin to peripheral and central FAs in COS7 cells (Fig. 2, B). Intriguingly, endogenous GIT1 was relocalized following overexpression of liprin-α1 (Fig. 3, A). The localization of GIT1 was decreased both at the newly formed small FAs at the edge of spreading cells, as well as at central, mature FAs (Fig. 3, B–C). Liprin-α1 overexpression caused the specific loss of endogenous GIT from FAs, while endogenous FAK (Fig. 3, B–C) and paxillin (data not shown) remained at FAs. Also in HeLa cells, the effect of liprin-α1 overexpression was the specific removal of GIT from FAs, while the localization at FAs of paxillin and talin was not affected (Fig. S6). Interestingly, liprin-ΔCC3 expression did not affect the localization of endogenous GIT1 at peripheral FAs in COS7 cells (Fig. 3, B–C). In fact, while overexpression of the full length liprin-α1 caused a reduction of the localization of endogenous GIT1 at FAK-positive FAs, leaving a diffuse cytoplasmic signal for endogenous GIT, in cells expressing liprin-ΔCC3 GIT1 remained at peripheral FAK-positive FAs (Fig. 3, B–C). These data indicate that the direct interaction of liprin-α1 with GIT1 is required for the removal of GIT1 from FAs.


Biochemical and functional characterization of the interaction between liprin-α1 and GIT1: implications for the regulation of cell motility.

Asperti C, Astro V, Pettinato E, Paris S, Bachi A, de Curtis I - PLoS ONE (2011)

Liprin-α1 affects the subcellular localization of endogenous GIT.(A) Overexpression of liprin-α1 affects the localization of endogenous GIT at peripheral FAs. COS7 cells overexpressing either FLAG-liprin-α1 or FLAG-βgalactosidase were plated for 1 h on FN and immunostained for the transfected protein and for endogenous GIT. Scale bar, 20 µm. Right panel: four-fold enlargement of the boxed field; liprin-α1 overexpression (cell with asterisk) reduces the accumulation of GIT at newly formed FAs at the edge of transfected cells (arrowheads). Scale bar, 5 µm. (B) Cells transfected with FLAG-βgalactosidase, FLAG-liprin-α1, or FLAG-liprin-ΔCC3 were plated for 1 h on FN before fixation and staining for the transfected protein and for endogenous GIT and FAK proteins. Scale bar, 20 µm. (C) High magnification of the edge of transfected cells showing that endogenous GIT overlaps well with FAK at peripheral FAs of FLAG-liprin-ΔCC3 transfected cells, while poor overlap between endogenous GIT and FAK is seen at peripheral FAs of FLAG-liprin-α1 expressing cells. Scale bar, 10 µm. Panels on the right are 3-fold enlargements of the areas indicated by arrowheads in the corresponding images on the left.
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Related In: Results  -  Collection

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pone-0020757-g003: Liprin-α1 affects the subcellular localization of endogenous GIT.(A) Overexpression of liprin-α1 affects the localization of endogenous GIT at peripheral FAs. COS7 cells overexpressing either FLAG-liprin-α1 or FLAG-βgalactosidase were plated for 1 h on FN and immunostained for the transfected protein and for endogenous GIT. Scale bar, 20 µm. Right panel: four-fold enlargement of the boxed field; liprin-α1 overexpression (cell with asterisk) reduces the accumulation of GIT at newly formed FAs at the edge of transfected cells (arrowheads). Scale bar, 5 µm. (B) Cells transfected with FLAG-βgalactosidase, FLAG-liprin-α1, or FLAG-liprin-ΔCC3 were plated for 1 h on FN before fixation and staining for the transfected protein and for endogenous GIT and FAK proteins. Scale bar, 20 µm. (C) High magnification of the edge of transfected cells showing that endogenous GIT overlaps well with FAK at peripheral FAs of FLAG-liprin-ΔCC3 transfected cells, while poor overlap between endogenous GIT and FAK is seen at peripheral FAs of FLAG-liprin-α1 expressing cells. Scale bar, 10 µm. Panels on the right are 3-fold enlargements of the areas indicated by arrowheads in the corresponding images on the left.
Mentions: As previously reported [4], [16], we found that endogenous GIT1 localized with paxillin to peripheral and central FAs in COS7 cells (Fig. 2, B). Intriguingly, endogenous GIT1 was relocalized following overexpression of liprin-α1 (Fig. 3, A). The localization of GIT1 was decreased both at the newly formed small FAs at the edge of spreading cells, as well as at central, mature FAs (Fig. 3, B–C). Liprin-α1 overexpression caused the specific loss of endogenous GIT from FAs, while endogenous FAK (Fig. 3, B–C) and paxillin (data not shown) remained at FAs. Also in HeLa cells, the effect of liprin-α1 overexpression was the specific removal of GIT from FAs, while the localization at FAs of paxillin and talin was not affected (Fig. S6). Interestingly, liprin-ΔCC3 expression did not affect the localization of endogenous GIT1 at peripheral FAs in COS7 cells (Fig. 3, B–C). In fact, while overexpression of the full length liprin-α1 caused a reduction of the localization of endogenous GIT1 at FAK-positive FAs, leaving a diffuse cytoplasmic signal for endogenous GIT, in cells expressing liprin-ΔCC3 GIT1 remained at peripheral FAK-positive FAs (Fig. 3, B–C). These data indicate that the direct interaction of liprin-α1 with GIT1 is required for the removal of GIT1 from FAs.

Bottom Line: Here we have addressed the involvement of the liprin-α1 partner GIT1 in liprin-α1-mediated effects on cell spreading and migration.No cumulative effects were observed after depletion of both liprin-α1 and GIT1, suggesting that the two proteins belong to the same signaling network in the regulation of cell spreading.These findings and the possible competition of liprin-α1 with paxillin for binding to GIT1 suggest that alternative binding of GIT1 to either liprin-α1 or paxillin plays distinct roles in different phases of the protrusive activity in the cell.

View Article: PubMed Central - PubMed

Affiliation: Division of Neuroscience, San Raffaele Scientific Institute and San Raffaele University, Milano, Italy.

ABSTRACT
We have previously identified the scaffold protein liprin-α1 as an important regulator of integrin-mediated cell motility and tumor cell invasion. Liprin-α1 may interact with different proteins, and the functional significance of these interactions in the regulation of cell motility is poorly known. Here we have addressed the involvement of the liprin-α1 partner GIT1 in liprin-α1-mediated effects on cell spreading and migration. GIT1 depletion inhibited spreading by affecting the lamellipodia, and prevented liprin-α1-enhanced spreading. Conversely inhibition of the formation of the liprin-α1-GIT complex by expression of liprin-ΔCC3 could still enhance spreading, although to a lesser extent compared to full length liprin-α1. No cumulative effects were observed after depletion of both liprin-α1 and GIT1, suggesting that the two proteins belong to the same signaling network in the regulation of cell spreading. Our data suggest that liprin-α1 may compete with paxillin for binding to GIT1, while binding of βPIX to GIT1 was unaffected by the presence of liprin-α1. Interestingly, GIT and liprin-α1 reciprocally regulated their subcellular localization, since liprin-α1 overexpression, but not the GIT binding-defective liprin-ΔCC3 mutant, affected the localization of endogenous GIT at peripheral and mature central focal adhesions, while the expression of a truncated, active form of GIT1 enhanced the localization of endogenous liprin-α1 at the edge of spreading cells. Moreover, GIT1 was required for liprin-α1-enhanced haptotatic migration, although the direct interaction between liprin-α1 and GIT1 was not needed. Our findings show that the functional interaction between liprin-α1 and GIT1 cooperate in the regulation of integrin-dependent cell spreading and motility on extracellular matrix. These findings and the possible competition of liprin-α1 with paxillin for binding to GIT1 suggest that alternative binding of GIT1 to either liprin-α1 or paxillin plays distinct roles in different phases of the protrusive activity in the cell.

Show MeSH
Related in: MedlinePlus