Monypenny, James Edward; (2003) The development of quantitative live cell imaging techniques and their applications in the study of inter-cellular communication and sarcoma cell motility. Doctoral thesis (Ph.D), UCL (University College London).

Text The_development_of_quantitativ.pdf Download (31MB)

Abstract

The aim of the project was to develop and apply quantitative light microscopy and image analysis techniques to the study of two essential aspects of cell behaviour: gap junction- mediated intercellular communication and cell motility and chemotaxis. Gap junction-mediated intercellular communication is essential for the regulated diffusion of small molecules and ions between adjacent cells. Mutations in connexin proteins, the main components of gap junctions, result in a variety of diseases that include skin disorders, neuropathy, and deafness. In this study a combination of microinjection, multi-channel time- lapse microscopy, and image processing was used to investigate in vitro the effects of different disease associated connexin 31 mutations on intercellular communication and cell survival. This study revealed that a deafness/peripheral neuropathy mutation and a mutation previously reported as a polymorphic variant of connexin 31 were associated with impaired intercellular communication, while mutations linked to skin disorders were associated with cell death. Cell motility and chemotaxis are believed to play an important role in the metastatic spread of cancer. In order to investigate the role of cytoskeletal signalling molecules in the motility and chemotaxis of metastatic sarcoma cells, the Dunn chemotaxis chamber was applied in combination with microinjection and time-lapse microscopy. The study focused on the Rho GTPases Cdc42, and Tc10 and their effectors PAK1 and N-WASP, proteins previously identified as regulators of polarity in other cell systems. This study revealed that PAK1 was required for efficient chemotaxis and motility of the sarcoma cells. While Cdc42, Tc10 and N-WASP were all necessary for efficient motility they were not found to be important for chemotaxis. Comparative studies in fibroblasts confirmed the importance of Cdc42 in motility but not directionality. In additional studies the novel fluorescence localisation after photobleaching technique revealed increased actin dynamics in metastatic sarcoma cells compared to non-metastatic cells. Finally, the subcellular interaction of Cdc42 with PAK1 and N-WASP was explored using fluorescence lifetime imaging microscopy in order to reveal the dynamics of Cdc42/effector interactions in single cells. Cdc42 and N-WASP associated constitutively in the majority of cells studied, and this interaction was largely confined to a perinuclear region. In contrast, Cdc42 and PAK1 association was inducible, requiring growth factor stimulation.

Download activity - last month

Export as XMLJSONCSV

Download activity - last 12 months

Export as CSVJSONXML

Downloads by country - last 12 months

Export as XMLJSONCSV

Archive Staff Only

View Item