Limits...
Delineating the Tes Interaction Site in Zyxin and Studying Cellular Effects of Its Disruption.

Hadzic E, Catillon M, Halavatyi A, Medves S, Van Troys M, Moes M, Baird MA, Davidson MW, Schaffner-Reckinger E, Ampe C, Friederich E - PLoS ONE (2015)

Bottom Line: Similar to zyxin, Tes harbors three highly conserved LIM domains of which the LIM1 domain directly interacts with zyxin.Using different zyxin variants in pull-down assays and ectopic recruitment experiments, we identified the Tes binding site in zyxin and showed that four highly conserved amino acids are crucial for its interaction with Tes.Quantitative analysis showed that the loss of interaction between zyxin and Tes affects the process of cell spreading.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cytoskeleton and Cell Plasticity, Life Sciences Research Unit, University of Luxembourg, Luxemburg, Luxembourg.

ABSTRACT
Focal adhesions are integrin-based structures that link the actin cytoskeleton and the extracellular matrix. They play an important role in various cellular functions such as cell signaling, cell motility and cell shape. To ensure and fine tune these different cellular functions, adhesions are regulated by a large number of proteins. The LIM domain protein zyxin localizes to focal adhesions where it participates in the regulation of the actin cytoskeleton. Because of its interactions with a variety of binding partners, zyxin has been proposed to act as a molecular scaffold. Here, we studied the interaction of zyxin with such a partner: Tes. Similar to zyxin, Tes harbors three highly conserved LIM domains of which the LIM1 domain directly interacts with zyxin. Using different zyxin variants in pull-down assays and ectopic recruitment experiments, we identified the Tes binding site in zyxin and showed that four highly conserved amino acids are crucial for its interaction with Tes. Based upon these findings, we used a zyxin mutant defective in Tes-binding to assess the functional consequences of abrogating the zyxin-Tes interaction in focal adhesions. Performing fluorescence recovery after photobleaching, we showed that zyxin recruits Tes to focal adhesions and modulates its turnover in these structures. However, we also provide evidence for zyxin-independent localization of Tes to focal adhesions. Zyxin increases focal adhesion numbers and reduces focal adhesion lifetimes, but does so independent of Tes. Quantitative analysis showed that the loss of interaction between zyxin and Tes affects the process of cell spreading. We conclude that zyxin influences focal adhesion dynamics, that it recruits Tes and that this interaction is functional in regulating cell spreading.

No MeSH data available.


Related in: MedlinePlus

The VGEI sequence in full-length zyxin is necessary for the interaction with Tes LIM1.(A) and (C) expression levels of GFP-fusion proteins in HeLa cells were verified by Western blot using an anti-GFP antibody (A), an anti-zyxin antibody (C) and an anti-β-actin antibody (control) (A, C). (B) Western blot analysis of GST pull-down experiments performed with GST-Tes LIM1 immobilized on glutathione sepharose resin, and extracts of HeLa cells transfected with Zyx 51-63-GFP, Zyx 51–63 MT-GFP or GFP (negative control). The presence of zyxin-GFP variants was analyzed in bound “B” or non-bound “NB” fractions using an anti-GFP antibody. (D) Zyx FL WT-GFP and Zyx FL MT-GFP extracts from transfected HeLa cells, were analyzed in bound (B) and non-bound (NB) fractions using an anti-zyxin antibody. Note the presence of endogenous zyxin (zyxin endo) in the bound fraction.
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pone.0140511.g004: The VGEI sequence in full-length zyxin is necessary for the interaction with Tes LIM1.(A) and (C) expression levels of GFP-fusion proteins in HeLa cells were verified by Western blot using an anti-GFP antibody (A), an anti-zyxin antibody (C) and an anti-β-actin antibody (control) (A, C). (B) Western blot analysis of GST pull-down experiments performed with GST-Tes LIM1 immobilized on glutathione sepharose resin, and extracts of HeLa cells transfected with Zyx 51-63-GFP, Zyx 51–63 MT-GFP or GFP (negative control). The presence of zyxin-GFP variants was analyzed in bound “B” or non-bound “NB” fractions using an anti-GFP antibody. (D) Zyx FL WT-GFP and Zyx FL MT-GFP extracts from transfected HeLa cells, were analyzed in bound (B) and non-bound (NB) fractions using an anti-zyxin antibody. Note the presence of endogenous zyxin (zyxin endo) in the bound fraction.

Mentions: To biochemically confirm the importance of the VGEI sequence, we performed pull-down assays with GST-Tes LIM1 (amino acids 234–299) produced in E. coli and zyxin-GFP fusion proteins from HeLa cell extracts (Fig 4A and 4C, showing equal expression levels of zyxin WT and MT). Western blot analysis using a GFP-specific antibody revealed the interaction between Tes LIM1 and Zyx 51-63-GFP. However, in the case of the variant Zyx 51–63 MT-GFP, no interaction could be detected (Fig 4B). We were able to pull down Zyx FL WT-GFP as well as endogenous zyxin with GST-Tes LIM1, this in contrast to Zyx FL MT-GFP (Fig 4D).


Delineating the Tes Interaction Site in Zyxin and Studying Cellular Effects of Its Disruption.

Hadzic E, Catillon M, Halavatyi A, Medves S, Van Troys M, Moes M, Baird MA, Davidson MW, Schaffner-Reckinger E, Ampe C, Friederich E - PLoS ONE (2015)

The VGEI sequence in full-length zyxin is necessary for the interaction with Tes LIM1.(A) and (C) expression levels of GFP-fusion proteins in HeLa cells were verified by Western blot using an anti-GFP antibody (A), an anti-zyxin antibody (C) and an anti-β-actin antibody (control) (A, C). (B) Western blot analysis of GST pull-down experiments performed with GST-Tes LIM1 immobilized on glutathione sepharose resin, and extracts of HeLa cells transfected with Zyx 51-63-GFP, Zyx 51–63 MT-GFP or GFP (negative control). The presence of zyxin-GFP variants was analyzed in bound “B” or non-bound “NB” fractions using an anti-GFP antibody. (D) Zyx FL WT-GFP and Zyx FL MT-GFP extracts from transfected HeLa cells, were analyzed in bound (B) and non-bound (NB) fractions using an anti-zyxin antibody. Note the presence of endogenous zyxin (zyxin endo) in the bound fraction.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4624954&req=5

pone.0140511.g004: The VGEI sequence in full-length zyxin is necessary for the interaction with Tes LIM1.(A) and (C) expression levels of GFP-fusion proteins in HeLa cells were verified by Western blot using an anti-GFP antibody (A), an anti-zyxin antibody (C) and an anti-β-actin antibody (control) (A, C). (B) Western blot analysis of GST pull-down experiments performed with GST-Tes LIM1 immobilized on glutathione sepharose resin, and extracts of HeLa cells transfected with Zyx 51-63-GFP, Zyx 51–63 MT-GFP or GFP (negative control). The presence of zyxin-GFP variants was analyzed in bound “B” or non-bound “NB” fractions using an anti-GFP antibody. (D) Zyx FL WT-GFP and Zyx FL MT-GFP extracts from transfected HeLa cells, were analyzed in bound (B) and non-bound (NB) fractions using an anti-zyxin antibody. Note the presence of endogenous zyxin (zyxin endo) in the bound fraction.
Mentions: To biochemically confirm the importance of the VGEI sequence, we performed pull-down assays with GST-Tes LIM1 (amino acids 234–299) produced in E. coli and zyxin-GFP fusion proteins from HeLa cell extracts (Fig 4A and 4C, showing equal expression levels of zyxin WT and MT). Western blot analysis using a GFP-specific antibody revealed the interaction between Tes LIM1 and Zyx 51-63-GFP. However, in the case of the variant Zyx 51–63 MT-GFP, no interaction could be detected (Fig 4B). We were able to pull down Zyx FL WT-GFP as well as endogenous zyxin with GST-Tes LIM1, this in contrast to Zyx FL MT-GFP (Fig 4D).

Bottom Line: Similar to zyxin, Tes harbors three highly conserved LIM domains of which the LIM1 domain directly interacts with zyxin.Using different zyxin variants in pull-down assays and ectopic recruitment experiments, we identified the Tes binding site in zyxin and showed that four highly conserved amino acids are crucial for its interaction with Tes.Quantitative analysis showed that the loss of interaction between zyxin and Tes affects the process of cell spreading.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cytoskeleton and Cell Plasticity, Life Sciences Research Unit, University of Luxembourg, Luxemburg, Luxembourg.

ABSTRACT
Focal adhesions are integrin-based structures that link the actin cytoskeleton and the extracellular matrix. They play an important role in various cellular functions such as cell signaling, cell motility and cell shape. To ensure and fine tune these different cellular functions, adhesions are regulated by a large number of proteins. The LIM domain protein zyxin localizes to focal adhesions where it participates in the regulation of the actin cytoskeleton. Because of its interactions with a variety of binding partners, zyxin has been proposed to act as a molecular scaffold. Here, we studied the interaction of zyxin with such a partner: Tes. Similar to zyxin, Tes harbors three highly conserved LIM domains of which the LIM1 domain directly interacts with zyxin. Using different zyxin variants in pull-down assays and ectopic recruitment experiments, we identified the Tes binding site in zyxin and showed that four highly conserved amino acids are crucial for its interaction with Tes. Based upon these findings, we used a zyxin mutant defective in Tes-binding to assess the functional consequences of abrogating the zyxin-Tes interaction in focal adhesions. Performing fluorescence recovery after photobleaching, we showed that zyxin recruits Tes to focal adhesions and modulates its turnover in these structures. However, we also provide evidence for zyxin-independent localization of Tes to focal adhesions. Zyxin increases focal adhesion numbers and reduces focal adhesion lifetimes, but does so independent of Tes. Quantitative analysis showed that the loss of interaction between zyxin and Tes affects the process of cell spreading. We conclude that zyxin influences focal adhesion dynamics, that it recruits Tes and that this interaction is functional in regulating cell spreading.

No MeSH data available.


Related in: MedlinePlus