Identifying the interaction between N-WASP, an actin nucleation factor and vinculin, a focal adhesion protein

• Main Author: Ganaesh
• Other Authors: Thirumaran s/o Thanabalu
• Format: Final Year Project
• Language: English
• Published: 2015
• Subjects: DRNTU::Science
• Online Access: http://hdl.handle.net/10356/62233
• Institution: Nanyang Technological University

N-WASP belongs to the WASP family of proteins which regulate actin cytoskeleton remodeling via the activation of Arp2/3 complex. Arp2/3 complex interacts with vinculin, an intracellular focal adhesion protein. Knockdown of N-WASP have shown to have reduced the levels of vinculin patches which could lead to cell migration and cancer metastasis. However, it is not known if N-WASP directly binds vinculin or if it is mediated by Arp2/3 complex and other intermediates. Identifying these interactions may give good insights for future anti-cancer therapies. Yeast two hybrid assay was used to study the interaction between N-WASP and vinculin. Cloned N-WASP (298-503 residues) was used in the experiment. The results showed interaction between N-WASP and vinculin in PJ69. A mammalian pull down assay was also performed using HEK 293 cells transfected with N-WASP-His and Vinculin RFP. Although the two-hybrid assay showed interaction between N-WASP and vinculin in yeast cells, vinculin could not be detected in the pull down. This could be due to interactions being weaker than normal protein interactions or being a transient interaction. Future research can focus on identifying a sound interaction in mammalian cells between N-WASP and vinculin which can shed more light on focal adhesion