α-actinin-4 promotes adhesion-mediated Shp2 activation at Focal Adhesions

• Main Authors: Yu-Chia Shih, 施又嘉
• Other Authors: Hsiao-Hui Lee
• Format: Others
• Language: zh-TW
• Published: 2018
• Online Access: http://ndltd.ncl.edu.tw/handle/7x7ab2

碩士 === 國立陽明大學 === 生命科學系暨基因體科學研究所 === 106 === The interactions of adherent cells with the extracellular matrix (ECM) mediated by integrins are essential for many cellular processes, including proliferation and migration. Focal adhesions (FAs) provide the linkages between actin cytoskeleton and ECM at the site of integrin binding. Previously, our lab had demonstrated that Shp2, a protein tyrosine phosphatase, is important for FA maturation and cellular force organization in mouse embryonic fibroblasts (MEFs). We further found that α-actinin-4 facilities Shp2 recruitment at FAs. We then generated α-actinin-4 knockout (ACTN4-/-) MEF clones by CRISPR/Cas9 method to study the role of α-actinin-4 in regulating Shp2 activation. In this thesis, I determined the genotyping by DNA sequencing of the ACTN4 gRNA targeting region. Four individual lines were characterized as deletion or insertion that resulting gene silencing. The expression of α-actinin-4 protein was also confirmed by Western blotting. To know whether α-actinin-4 plays a role in FA maturation, wild type (WT) and ACTN4-/- MEFs were seeded on fibronectin (FN)-coated coverslips for immunofluorescence staining with anti-paxillin antibody. I found that ACTN4-/- cells exhibited smaller FA compared with WT cells when they were spreading. Expression of mApple-α-actinin-4 rescued FA maturation in ACTN4-/- MEFs, suggesting the positive role of α-actinin-4 in FA maturation. I then used a fluorescence resonance energy transfer (FRET)-based Shp2 SWAP biosensor to evaluate Shp2 activation and found that Shp2 activity was significant decreased in ACTN4-/- cells compared with WT cells. Expression of mApple-α-actinin-4 rescued Shp2 activation in ACTN4-/- MEFs. Of note, expression Shp2 SWAP biosensor did not significant affect FA size. Taken together, our results suggest that α-actinin-4 is important for the adhesion-mediated Shp2 activation that may contribute to the regulation of FA maturation in fibroblasts.