Studies of cell signalling using bacterial toxins and organic electronic devices

Abstract

One of the most versatile biological signalling entities is the calcium ion, Ca2+. With a characteristic spatial and temporal signalling pattern, this universal second-messenger directs diverse cellular functions, e.g. secretion of neurotransmitters at the synaptic cleft to regulation of transcription and fertilization. The temporal feature of Ca2+signal can be described as oscillations occurring with periodicities ranging from μs to h.

The bacterial exotoxin alpha-haemolysin (Hly) is a pathophysiologically relevant protein known to induce Ca2+ oscillations that leads to production of pro-inflammatory cytokines in target cells. A role for this uropathogenic E.coli-encoded virulence factor was recently identified, as Hly was shown to modulate inflammatory responses in vivo. In this thesis, we investigate the molecular details of signalling pathways activated by Hly. All functional domains of Hly are required for cell signalling, and we suggest that the Ca2+ binding domain interacts with the membrane-bound receptor glycophorin. Components of the LPS-recognition system are essential to recruit Hly to the cell membrane, where signalling is initiated in specialized microdomains termed lipid rafts. We show that the signalling pathway involves the small GTPase RhoA, the linker protein ezrin, and the cytoskeleton. Ca2+ oscillations are generated by concerted actions of Hly-activated voltage-operated Ca2+ channels in the plasma membrane and generation of the second-messenger IP3, which causes release of Ca2+ from the ER via the IP3-receptor.

The tool-kit available to study the dynamic range of Ca2+ signals is limited. We envisaged that a powerful tool for such studies would be a device that is able to induce ion signalling with specified temporal and spatial resolution. To this end, we developed an organic electronic ion pump as a bio-interface to electronically induce Ca2+ signalling in neuronal cells. We show that the conjugated polymer PEDOT:PSS is biocompatible. A device is designed that is able to transport ions, e.g. H+, K+ and Ca2+, with high degree of electronic control and ON/OFF ratios exceeding 300. By integrating this device with cells, electronic control of cellular Ca2+ influx is achieved, showing that an electronically controlled ion conductor circuit can trigger a biological output. A prototype is manufactured demonstrating that the ion pump can be used to achieve controlled oscillations of an ion concentration. We believe that these novel devices have great potential as tools to study Ca2+ signalling patterns with specific frequencies in biological specimens. But most importantly, this work shows a successful integration of two research fields, which has paved the path for the novel research area termed Organic bioelectronics .