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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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GIT1 and LAR depletion inhibit cell spreading and prevent enhanced spreading by liprin-α1 overexpression.(A) Specific and control (Luc = luciferase) siRNA duplexes were used to downregulate the expression of endogenous GIT1, GIT2, liprin-α1 and LAR in COS7 cells. Cells were lysed 2 days after transfection with siRNAs. After SDS-PAGE and blotting of 50 µg of each lysate, filters were incubated with antibodies for the indicated proteins. For each specific siRNA, we could only detect the downregulation of the specific target proteins with respect to the other endogenous proteins tested as controls. For GIT1 and GIT2, a monoclonal antibody recognizing both proteins was used here. (B) The signal for endogenous GIT (red) is strongly decreased at paxillin-positive (green) focal adhesions following transfection with siRNA for either GIT1 (top) or LAR (bottom) when compared to control cells (middle). Scale bar, 5 µm. (C) COS7 cells were trypsinized 2 days after co-transfection with the indicated siRNAs and βgalactosidase (βGal), and plated 1 h on FN before immunostaining. Scale bar, 20 µm. (D, E) Quantification of spreading after replating 1 h on FN of cells co-transfected for 2 days with siRNAs (D: means ±SEM; n = 100 cells per condition), or with siRNAs and plasmids for either βgalactosidase or liprin-α1 (E: means ±SEM, n = 80–90 cells per condition from 2 experiments). **P<0.01.
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pone-0020757-g002: GIT1 and LAR depletion inhibit cell spreading and prevent enhanced spreading by liprin-α1 overexpression.(A) Specific and control (Luc = luciferase) siRNA duplexes were used to downregulate the expression of endogenous GIT1, GIT2, liprin-α1 and LAR in COS7 cells. Cells were lysed 2 days after transfection with siRNAs. After SDS-PAGE and blotting of 50 µg of each lysate, filters were incubated with antibodies for the indicated proteins. For each specific siRNA, we could only detect the downregulation of the specific target proteins with respect to the other endogenous proteins tested as controls. For GIT1 and GIT2, a monoclonal antibody recognizing both proteins was used here. (B) The signal for endogenous GIT (red) is strongly decreased at paxillin-positive (green) focal adhesions following transfection with siRNA for either GIT1 (top) or LAR (bottom) when compared to control cells (middle). Scale bar, 5 µm. (C) COS7 cells were trypsinized 2 days after co-transfection with the indicated siRNAs and βgalactosidase (βGal), and plated 1 h on FN before immunostaining. Scale bar, 20 µm. (D, E) Quantification of spreading after replating 1 h on FN of cells co-transfected for 2 days with siRNAs (D: means ±SEM; n = 100 cells per condition), or with siRNAs and plasmids for either βgalactosidase or liprin-α1 (E: means ±SEM, n = 80–90 cells per condition from 2 experiments). **P<0.01.

Mentions: Liprin-α1 is a regulator of cell motility required for the efficient integrin-mediated spreading of COS7 cells [11]. COS7 cells express mainly GIT1, and very little GIT2 (Fig. 1, D). We depleted endogenous GIT1 by specific siRNAs (short interfering RNAs) to analyze the effects on cell spreading. GIT1 silencing caused both a strong decrease of the endogenous protein (Fig. 2, A; Fig. S2), and loss of GIT signal from FAs (Fig. 2, B). It has been previously shown that GIT1 silencing by siRNA inhibits the rate of protrusion, while enhancing the stability and reducing the turnover of FAs [17]. Here, we show that GIT1 depletion inhibited COS7 cell spreading on FN (fibronectin) by negatively affecting the formation of lamellipodia and of paxillin-positive FAs at the cell edge (Fig. 2, C and Fig. S2), as previously observed after liprin-α1 silencing [11]. Quantitative analysis showed similar effects on spreading after depletion of either or both proteins (Fig. 2, D). Interestingly, no additive inhibitory effects on spreading were detected after double knockdown of liprin-α1 and GIT1 (Fig. 2, C–D), suggesting that these proteins participate into the same signaling pathway for the regulation of cell edge dynamics. In contrast to the positive effect of GIT1 in COS7 cell spreading, silencing of GIT2 causes an increase in spreading in HeLa cells, indicating that GIT2, but not GIT1, is an essential inhibitor of cell spreading and FA turnover in these cells [29]. GIT2 also inhibits cell migration, since its silencing results in a dramatic increase of transwell migration [29].


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)

GIT1 and LAR depletion inhibit cell spreading and prevent enhanced spreading by liprin-α1 overexpression.(A) Specific and control (Luc = luciferase) siRNA duplexes were used to downregulate the expression of endogenous GIT1, GIT2, liprin-α1 and LAR in COS7 cells. Cells were lysed 2 days after transfection with siRNAs. After SDS-PAGE and blotting of 50 µg of each lysate, filters were incubated with antibodies for the indicated proteins. For each specific siRNA, we could only detect the downregulation of the specific target proteins with respect to the other endogenous proteins tested as controls. For GIT1 and GIT2, a monoclonal antibody recognizing both proteins was used here. (B) The signal for endogenous GIT (red) is strongly decreased at paxillin-positive (green) focal adhesions following transfection with siRNA for either GIT1 (top) or LAR (bottom) when compared to control cells (middle). Scale bar, 5 µm. (C) COS7 cells were trypsinized 2 days after co-transfection with the indicated siRNAs and βgalactosidase (βGal), and plated 1 h on FN before immunostaining. Scale bar, 20 µm. (D, E) Quantification of spreading after replating 1 h on FN of cells co-transfected for 2 days with siRNAs (D: means ±SEM; n = 100 cells per condition), or with siRNAs and plasmids for either βgalactosidase or liprin-α1 (E: means ±SEM, n = 80–90 cells per condition from 2 experiments). **P<0.01.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3113849&req=5

pone-0020757-g002: GIT1 and LAR depletion inhibit cell spreading and prevent enhanced spreading by liprin-α1 overexpression.(A) Specific and control (Luc = luciferase) siRNA duplexes were used to downregulate the expression of endogenous GIT1, GIT2, liprin-α1 and LAR in COS7 cells. Cells were lysed 2 days after transfection with siRNAs. After SDS-PAGE and blotting of 50 µg of each lysate, filters were incubated with antibodies for the indicated proteins. For each specific siRNA, we could only detect the downregulation of the specific target proteins with respect to the other endogenous proteins tested as controls. For GIT1 and GIT2, a monoclonal antibody recognizing both proteins was used here. (B) The signal for endogenous GIT (red) is strongly decreased at paxillin-positive (green) focal adhesions following transfection with siRNA for either GIT1 (top) or LAR (bottom) when compared to control cells (middle). Scale bar, 5 µm. (C) COS7 cells were trypsinized 2 days after co-transfection with the indicated siRNAs and βgalactosidase (βGal), and plated 1 h on FN before immunostaining. Scale bar, 20 µm. (D, E) Quantification of spreading after replating 1 h on FN of cells co-transfected for 2 days with siRNAs (D: means ±SEM; n = 100 cells per condition), or with siRNAs and plasmids for either βgalactosidase or liprin-α1 (E: means ±SEM, n = 80–90 cells per condition from 2 experiments). **P<0.01.
Mentions: Liprin-α1 is a regulator of cell motility required for the efficient integrin-mediated spreading of COS7 cells [11]. COS7 cells express mainly GIT1, and very little GIT2 (Fig. 1, D). We depleted endogenous GIT1 by specific siRNAs (short interfering RNAs) to analyze the effects on cell spreading. GIT1 silencing caused both a strong decrease of the endogenous protein (Fig. 2, A; Fig. S2), and loss of GIT signal from FAs (Fig. 2, B). It has been previously shown that GIT1 silencing by siRNA inhibits the rate of protrusion, while enhancing the stability and reducing the turnover of FAs [17]. Here, we show that GIT1 depletion inhibited COS7 cell spreading on FN (fibronectin) by negatively affecting the formation of lamellipodia and of paxillin-positive FAs at the cell edge (Fig. 2, C and Fig. S2), as previously observed after liprin-α1 silencing [11]. Quantitative analysis showed similar effects on spreading after depletion of either or both proteins (Fig. 2, D). Interestingly, no additive inhibitory effects on spreading were detected after double knockdown of liprin-α1 and GIT1 (Fig. 2, C–D), suggesting that these proteins participate into the same signaling pathway for the regulation of cell edge dynamics. In contrast to the positive effect of GIT1 in COS7 cell spreading, silencing of GIT2 causes an increase in spreading in HeLa cells, indicating that GIT2, but not GIT1, is an essential inhibitor of cell spreading and FA turnover in these cells [29]. GIT2 also inhibits cell migration, since its silencing results in a dramatic increase of transwell migration [29].

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