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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 is required for liprin-α1-enhanced COS7 cell migration.(A) Transfected cells were replated on 10 µg/ml FN for 50 min to allow spreading, and then monitored for motility for 2.5 h by taking one frame every 5 min. The upper panels show cell tracks from cells transfected with the indicated constructs. The lower panel shows the quantification (mean values ±SEM) of different parameters of random migration including cell tracks (path), Euclidean distance (displ.), path rate (Vp), Euclidean rate (Vd) and persistence of migration (persist = path/displ.). N = 18–20 cells per experimental condition; *P<0.05. (B) Transwell migration assays with cells transfected with GFP, GFP-liprin-α1, or GFP-liprin-ΔCC3. Bars are normalized means ± SEM (n = 4); *P<0.05; **P<0.01. (C) Transwell migration assays with cells cotransfected with the indicated combinations of siRNAs and plasmids. Bars are normalized means ± SEM (n = 4); *P<0.05.
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pone-0020757-g005: GIT1 is required for liprin-α1-enhanced COS7 cell migration.(A) Transfected cells were replated on 10 µg/ml FN for 50 min to allow spreading, and then monitored for motility for 2.5 h by taking one frame every 5 min. The upper panels show cell tracks from cells transfected with the indicated constructs. The lower panel shows the quantification (mean values ±SEM) of different parameters of random migration including cell tracks (path), Euclidean distance (displ.), path rate (Vp), Euclidean rate (Vd) and persistence of migration (persist = path/displ.). N = 18–20 cells per experimental condition; *P<0.05. (B) Transwell migration assays with cells transfected with GFP, GFP-liprin-α1, or GFP-liprin-ΔCC3. Bars are normalized means ± SEM (n = 4); *P<0.05; **P<0.01. (C) Transwell migration assays with cells cotransfected with the indicated combinations of siRNAs and plasmids. Bars are normalized means ± SEM (n = 4); *P<0.05.

Mentions: We have used a random migration assay to analyze the role of the liprin-α1/GIT1 complex in a different motility assay. COS7 cells were poorly motile when tested in a random migration assay on FN, while they became active after overexpression of liprin-α1 (Fig. S7). No differences were evident between cells expressing either GFP-liprin-α1 or the GIT1 binding-deficient mutant GFP-liprin-ΔCC3 (Fig. 5, A). Therefore, the interaction between liprin-α1 and GIT1 is not essential to regulate the random motility of COS7 cells. Similar results were obtained by using a haptotactic transwell migration assay, in which COS7 cell migration towards a FN-coated substrate was strongly enhanced by liprin-α1 overexpression, but also by the expression of the GIT1 binding-deficient mutant liprin-ΔCC3 (Fig. 5, B). On the other hand, we found that endogenous GIT1 was required for liprin-α1-enhanced migration (Fig. 5, C). Previous findings have shown that overexpression of GIT1 enhanced haptotactic COS7 cell migration [4] and CHO-K1 cell migration on FN [16], while GIT1 depletion prevented formyl-Met-Leu-Phe peptide-enhanced chemotaxis of rat basophilic leukaemia RBL cells [32]. Although silencing the endogenous GIT1 protein did not significantly affect basal cell migration, it prevented the potentiation of transwell migration induced by liprin-α1 overexpression (Fig. 5, C). Altogether these data indicate that the function of GIT1 is important for liprin-α1-mediated migration, although a direct interaction between the two proteins is not necessary.


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 is required for liprin-α1-enhanced COS7 cell migration.(A) Transfected cells were replated on 10 µg/ml FN for 50 min to allow spreading, and then monitored for motility for 2.5 h by taking one frame every 5 min. The upper panels show cell tracks from cells transfected with the indicated constructs. The lower panel shows the quantification (mean values ±SEM) of different parameters of random migration including cell tracks (path), Euclidean distance (displ.), path rate (Vp), Euclidean rate (Vd) and persistence of migration (persist = path/displ.). N = 18–20 cells per experimental condition; *P<0.05. (B) Transwell migration assays with cells transfected with GFP, GFP-liprin-α1, or GFP-liprin-ΔCC3. Bars are normalized means ± SEM (n = 4); *P<0.05; **P<0.01. (C) Transwell migration assays with cells cotransfected with the indicated combinations of siRNAs and plasmids. Bars are normalized means ± SEM (n = 4); *P<0.05.
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pone-0020757-g005: GIT1 is required for liprin-α1-enhanced COS7 cell migration.(A) Transfected cells were replated on 10 µg/ml FN for 50 min to allow spreading, and then monitored for motility for 2.5 h by taking one frame every 5 min. The upper panels show cell tracks from cells transfected with the indicated constructs. The lower panel shows the quantification (mean values ±SEM) of different parameters of random migration including cell tracks (path), Euclidean distance (displ.), path rate (Vp), Euclidean rate (Vd) and persistence of migration (persist = path/displ.). N = 18–20 cells per experimental condition; *P<0.05. (B) Transwell migration assays with cells transfected with GFP, GFP-liprin-α1, or GFP-liprin-ΔCC3. Bars are normalized means ± SEM (n = 4); *P<0.05; **P<0.01. (C) Transwell migration assays with cells cotransfected with the indicated combinations of siRNAs and plasmids. Bars are normalized means ± SEM (n = 4); *P<0.05.
Mentions: We have used a random migration assay to analyze the role of the liprin-α1/GIT1 complex in a different motility assay. COS7 cells were poorly motile when tested in a random migration assay on FN, while they became active after overexpression of liprin-α1 (Fig. S7). No differences were evident between cells expressing either GFP-liprin-α1 or the GIT1 binding-deficient mutant GFP-liprin-ΔCC3 (Fig. 5, A). Therefore, the interaction between liprin-α1 and GIT1 is not essential to regulate the random motility of COS7 cells. Similar results were obtained by using a haptotactic transwell migration assay, in which COS7 cell migration towards a FN-coated substrate was strongly enhanced by liprin-α1 overexpression, but also by the expression of the GIT1 binding-deficient mutant liprin-ΔCC3 (Fig. 5, B). On the other hand, we found that endogenous GIT1 was required for liprin-α1-enhanced migration (Fig. 5, C). Previous findings have shown that overexpression of GIT1 enhanced haptotactic COS7 cell migration [4] and CHO-K1 cell migration on FN [16], while GIT1 depletion prevented formyl-Met-Leu-Phe peptide-enhanced chemotaxis of rat basophilic leukaemia RBL cells [32]. Although silencing the endogenous GIT1 protein did not significantly affect basal cell migration, it prevented the potentiation of transwell migration induced by liprin-α1 overexpression (Fig. 5, C). Altogether these data indicate that the function of GIT1 is important for liprin-α1-mediated migration, although a direct interaction between the two proteins is not necessary.

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