Podosomes are adhesive and invasive structures which may play an important role in numerous physiological and pathological conditions including angiogenesis, atherosclerosis, and cancer metastasis. Recently, the cysteine protease m-calpain (m-Capn) has been shown to cleave cortactin, an integral component of the podosomal F-actin core, as well as various proteins found in the peripheral adhesive region leading to the disassembly of these dynamic structures. In this study, I investigated whether Capn plays a role in the formation of podosomes downstream of c-Src. I show that: 1) phorbol-12, 13-dibutyrate (PDBu) as well as c-Src-Y527F expression induces podosome formation in mouse embryonic fibroblasts; 2) PDBu- and constitutively active c-Src-induced podosome formation is inhibited by the knockout of the m- and µ-Capn small regulatory subunit Capn4 in mouse embryonic fibroblasts (Capn4-/-), but is partially restored by re-expression of Capn4; 3) Capn4 localizes to podosomes; and 4) Inhibition of m- and µ-Capn proteolytic activity by the cell permeable calpain inhibitors has little effect on the formation of podosomes downstream of active c-Src. I conclude that Capn4 may play a role in the assembly phase of podosomes independent of calpain proteolytic activity. Work done in collaboration to determine a possible mechanism of action for the role of Capn4 in podosome assembly indicates that a possible binding partner of Capn4, β-PIX, co-localizes with, and shows in vivo association with Capn4. Furthermore, β-PIX and Capn4 bind directly in vitro in the presence of Ca2+. We conclude that Capn4 plays a role in podosome assembly, and this role may be through direct interaction with β-PIX in a calcium-dependent manner. / Thesis (Master, Biochemistry) -- Queen's University, 2008-09-26 16:16:00.768
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:OKQ.1974/1494 |
| Date | 27 September 2008 |
| Creators | Dowler, THOMAS |
| Contributors | Queen's University (Kingston, Ont.). Theses (Queen's University (Kingston, Ont.)) |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English, English |
| Detected Language | English |
| Type | Thesis |
| Format | 6286532 bytes, application/pdf |
| Rights | This publication is made available by the authority of the copyright owner solely for the purpose of private study and research and may not be copied or reproduced except as permitted by the copyright laws without written authority from the copyright owner. |
| Relation | Canadian theses |
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