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Role of HECTD1 in regulating adhesion dynamics during cell movement

Shen, Xiaoli. Role of HECTD1 in regulating adhesion dynamics during cell movement. 2015, PhD Thesis, University of Basel, Faculty of Science.

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Official URL: http://edoc.unibas.ch/diss/DissB_11348

Abstract

E3 ubiqutin ligase for inhibin B receptor (HECTD1) has been demonstrated to play an indispensable role in embryonic development, including neural tube closure, placentation and eye formation, etc. The malformations caused by loss of HECTD1 are also found in humans, such as neural tube closure defects in HECTD1 knockout mouse model are comparable to anencephaly in humans. Therefore, the investigation of HECTD1 function in regulating neural tube closure and placental development may be helpful for understanding the underlying mechanisms of embryonic development and for the search of new approaches to prevent or treat the associated diseases. Normal embryonic development is the result of proper cell movement which is precisely regulated by complex cellular events. Although several mechanisms of HECTD1 function have been proposed recently, many aspects of the connection of HECTD1 with intracellular signaling pathways in cell movement are novel.
Using HECTD1 knockout mouse embryonic fibroblasts (MEFs) as the primary model, we first examined cell spreading and migration respectively. We found that loss of HECTD1 shortened the duration and reduced the area of cell spreading, while the velocity was enhanced and the directionality of cell migration impaired. Furthermore, as compared to wild-type cells, the cell adhesion proteins, paxillin and zyxin, were inhibited in maturing from focal complexes into focal adhesions in HECTD1 knockout cells. These defects in the formation of focal adhesions were associated with enhanced tyrosine phosphorylation of paxillin (paxillin-Y118), as well as higher activity of Rac1 and RhoA.
Screening with mass spectrometry has led to the identification of IQ motif containing GTPase activating protein 1 (IQGAP1) which is essential for the formation of cell polarity, stabilization of cell-cell adhesion and proper cell movement (Watanabe et al., 2004; Kuroda et al., 1998; Choi et al., 2013). Interestingly, we discovered that, in contrast to wild-type cells, IQGAP1 is overexpressed in HECTD1 knockout cells. Using co-immunoprecipitation and co-localization assay, we confirmed that IQGAP1 and HECTD1 physically interact with each other. Since HECTD1 is an E3 ubiquitin ligase, then we examined whether IQGAP1 acts as the substrate protein of HECTD1 by checking the ubiquitination and half life of IQGAP1. Our results confirmed that in HECTD1 knockout cells ubiquitination was reduced thereby prolonging the half life of IQGAP1.
To answer the question whether the changes in adhesion proteins and cell movement observed in HECTD1 knockout cells were caused by overexpression of IQGAP1, we mimicked the observed effects by overexpressing GFP-IQGAP1 in wild-type cells and by siRNA knockdown of IQGAP1 in HECTD1 knockout cells. Intriguingly, overexpression of GFP-IQGAP1 in wild-type cells resulted in similar impaired expression of focal adhesions in HECTD1 knockout cells, while siRNA knockdown of IQGAP1 in HECTD1 knockout cells significantly rescued the maturation of focal adhesions, activity of RhoA, duration of cell spreading and velocity of cell migration.
Taken all data together, our findings indicate that HECTD1 plays a regulatory role in ubiquitination of IQGAP1, which in turn impacts on dynamics of focal adhesions and regulates cell spreading and migration.
Advisors:Handschin, Christoph
Committee Members:De Geyter, Christian and Scheiffele, Peter
Faculties and Departments:03 Faculty of Medicine > Departement Biomedizin > Associated Research Groups > Pharmakologie (Handschin)
Item Type:Thesis
Thesis no:11348
Bibsysno:Link to catalogue
Number of Pages:119 S.
Language:English
Identification Number:
Last Modified:30 Jun 2016 10:58
Deposited On:06 Oct 2015 08:34

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