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

Vascular smooth muscle cell migration is a significant contributor to many aspects of heart disease, and specifically atherosclerosis. Tissue damage in the arteries can result in the formation of a fatty streak. Smooth muscle cells (SMC) can then migrate to this site to form a fibrous cap, stabilizing the fatty plaque. Since cardiovascular disease is the leading cause of death in developed countries, this function of SMC is an essential area of study. The formation of lamellipodia and circular dorsal ruffles were studied in this project as indicators that cell migration is occurring. The roles of the proteins p53, Rac, caldesmon and PTEN were investigated with regards to these actin-based structures. The tumour suppressor p53 is often reported to cause apoptosis, senescence or cell cycle arrest when stress is placed on a cell, but has recently been shown to regulate cell migration as well. It was determined in this project that p53 could inhibit the formation of both lamellipodia and circular dorsal ruffles. It was also shown that this could occur directly through an inhibition of the GTPase Rac. Previous studies have shown that p53 can upregulate caldesmon, a protein which is known to bind to and stabilize actin filaments while inhibiting Arp2/3-mediated branching. It was confirmed that p53 could upregulate caldesmon, and that caldesmon could inhibit the formation of lamellipodia and circular dorsal ruffles. The phosphorylation of caldesmon by p21-associated kinase (PAK) or extracellular signal-related kinase (Erk) was shown to effectively reverse the ability of caldesmon to inhibit these structures. The role of phosphatase and tensin homologue deleted on chromosome 10 (PTEN) was also studied with regards to this signalling pathway. PTEN was shown to inhibit lamellipodia and circular dorsal ruffles through its lipid phosphatase activity. It was concluded that p53 can inhibit the formation of lamellipodia and circular dorsal ruffles in vascular SMC, and that this occurs through Rac, caldesmon and PTEN. / Thesis (Master, Biochemistry) -- Queen's University, 2011-06-10 13:15:37.081

Cell-substrate interactions influence various cellular processes such as morphology, motility, proliferation and differentiation. Actin dynamics within cells have been shown to be influenced by substrate stiffness, as NIH 3T3 fibroblasts grown on stiffer substrates tend to exhibit more prominent actin stress fiber formation. Circular dorsal ruffles (CDRs) are transient actin-rich ring-like structures within cells, induced by various growth factors, such as the platelet-derived growth factor (PDGF). CDRs grow and shrink in size after cells are stimulated with PDGF, eventually disappearing ten of minutes after stimulation. As substrate stiffness affect actin structures and cell motility, and CDRs are actin structures which have been previously linked to cell motility and macropinocytosis, the role of substrate stiffness on the properties of CDRs in NIH 3T3 fibroblasts and how they proceed to affect cell behavior is investigated. Cells were seeded on Poly-dimethylsiloxane (PDMS) substrates of various stiffnesses and stimulated with PDGF to induce CDR formation. It was found that an increase in substrate stiffness increases the lifetime of CDRs, but did not affect their size. A mathematical model of the signaling pathways involved in CDR formation is developed to provide insight into this lifetime and size dependence, and is linked to substrate stiffness via Rac-Rho antagonism. CDR formation did not affect the motility of cells seeded on 10 kPa stiff substrates, but is shown to increase localized lamellipodia formation in the cell via the diffusion of actin from the CDRs to the lamellipodia. To further probe the influence of cell-substrate interactions on cell behavior and actin dynamics, a two dimensional system which introduces a dynamically changing, reversible and localized substrate stiffness environment is constructed. Cells are seeded on top of thin PDMS nano-membranes, and are capable of feeling through the thin layer, experiencing the stiffness of the polyacrylamide substrates below the nano-membrane. The membranes are carefully re-transplanted on top of other polyacrylamide substrates with differing stiffnesses. This reversible dynamic stiffness system is a novel approach which would help in the investigation of the influence of reversible dynamic stiffness environments on cell morphology, motility, proliferation and differentiation in various cells types.