Dissecting the roles of the Dectin-1R in the bladder innate defences

Ph. D. ThesisPrevious work using immortalised cells transfected with a NF-κB reporter and challenged with zymosan, to mimic a fungal infection, suggested possible co-operation between Dectin-1 and TLR5 receptors in urogenital tissues. These data were interesting as research describing Dectin-1R functioning relates specifically to myeloid derived cells and TLR5, classically, is known to function as a homodimer. The aim of the research reported in this thesis was therefore to focus on the Dectin-1 and TLR5 receptors in the bladder urothelium, with the objective of investigating Dectin-1 receptor functioning, Dectin1/TLR5 receptor co-operation and cell signalling events following a zymosan (β-glucan) challenge. Western analyses of cell lysates from a bladder biopsy and immortalised RT4 bladder cells using a Dectin-1R antibody (R&D systems, AF1859) revealed the synthesis of proteins representing full-length and truncated (stalk-less) Dectin-1 receptors. These data supported the use of the immortalised RT4 cells in all subsequent analyses exploring Dectin-1R functioning in the urothelial tissues. Following challenge of the cells with the yeast cell wall extract zymosan (50ug/ml) increased synthesis of an array of host defence agents including hBD2, IL-8 and LCN2 were observed (p<0.05). Furthermore IL-8 effector synthesis was significantly reduced following CLEC7A (Dectin-1R) gene knockdown (p<0.05) and antibody blocking of the Dectin-1R (p<0.01). These data supported synthesis of the Dectin-1R in urothelial cells and a role in defending the bladder against a fungal challenge. Knocking-down the TLR5 gene in RT4 cells (80% knockdown) and challenging the cells with zymosan, resulted in a significant reduction in hBD2, IL8 and LCN2 synthesis (p<0.05). These data suggested co-operation between the Dectin-1 and TLR5 receptors in bladder cells, which was supported by immunostaining and a proximity ligation assay approach. In addition these analyses supported clustering of the Dectin-1 and TLR5 receptors following activation. Experiments to explore roles for the encoded Dectin-1 receptor isoforms, 1, 2 and 4, in the TLR5 co-operation events involved engineering a suite of stable cell lines each over-expressing a Dectin-1 receptor isoform and either a TLR5 full-length or TLR5 truncated receptor. This approach exploited the pVitro2-neo-mcs vector, which is able to co-express two cDNA sequences simultaneously. However, the resultant cell lines did not over-express the Dectin-1 and TLR5 receptors. It was hypothesised that the increased number of receptors synthesised by the cells over-loaded the urothelial cell membranes causing gene silencing and/or cell death. The signalling mechanisms activating the transcription of host effector genes following a zymosan challenge of RT4 bladder cells were explored using western analyses and an antibody to Syk-P (Cell Signalling, C87C1), SYK gene knockdown and piceatannol (100 ug/ml) a Syk inhibitor followed by ELISA to measure LCN2 and IL-8 media concentrations. No phosphorylation of Syk was observed and the knock-down/inhibition approaches had no significant effects on the media concentrations of either IL-8 or LCN2. However, knockdown of the RAF1 gene resulted in decreased secretion of the host defence molecules LCN2 and IL-8 (p<0.01). Additionally western blot analyses showed phosphorylation and degradation of the NF-κB inhibitor IκBα. These data suggested that Dectin-1R activation in response to zymosan in bladder cells involved Raf-1 signalling, which supported a non-canonical signalling pathway. In summary, Dectin-1 receptors were shown to be synthesized in RT4 bladder epithelial cells and were activated in response to zymosan (fungal) challenge resulting in the production of host defence effector molecules. Data showed potential co-operation between Dectin-1 and TLR5 receptors in mediating the bladder cell response. Dectin-1R signalling in urothelial cells was also orchestrated via a non-canonical signalling pathway involving Raf-1. These data support a novel host defence mechanism, involving Dectin-1 and TLR5 receptors functioning co-operatively, to defend urothelial from fungal infections.Dr William Edmund Harker Foundatio

N-terminal acetylation and arginylation of actin determines the architecture and assembly rate of linear and branched actin networks

The great diversity in actin network architectures and dynamics is exploited by cells to drive fundamental biological processes, including cell migration, endocytosis and cell division. While it is known that this versatility is the result of the many actin-remodeling activities of actin-binding proteins, such as Arp2/3 and Cofilin, recent work also implicates post-translational acetylation or arginylation of the actin N-terminus itself as an equally important regulatory mechanism. However, the molecular mechanisms by which acetylation and arginylation alter the properties of actin are not well understood. Here, we directly compare how processing and modification of the N-terminus of actin affects its intrinsic polymerization dynamics and its remodeling by actin-binding proteins that are essential for cell migration. We find that in comparison to acetylated actin, arginylated actin reduces intrinsic as well as formin-mediated elongation and Arp2/3-mediated nucleation. By contrast, there are no significant differences in Cofilin-mediated severing. Taken together, these results suggest that cells can employ these differently modified actins to regulate actin dynamics. In addition, unprocessed actin with an N-terminal methionine residue shows very different effects on formin-mediated-elongation, Arp2/3-mediated nucleation, and severing by Cofilin. Altogether, this study shows that the nature of the N-terminus of actin can promote distinct actin network dynamics, which can be differentially used by cells to locally finetune actin dynamics at distinct cellular locations, such as at the leading edge

Calponin-homology domain mediated bending of membrane-associated actin filaments

Actin filaments are central to numerous biological processes in all domains of life. Driven by the interplay with molecular motors, actin binding and actin modulating proteins, the actin cytoskeleton exhibits a variety of geometries. This includes structures with a curved geometry such as axon-stabilizing actin rings, actin cages around mitochondria and the cytokinetic actomyosin ring, which are generally assumed to be formed by short linear filaments held together by actin cross-linkers. However, whether individual actin filaments in these structures could be curved and how they may assume a curved geometry remains unknown. Here, we show that ‘curly’, a region from the IQGAP family of proteins from three different organisms, comprising the actin-binding calponin-homology domain and a C-terminal unstructured domain, stabilizes individual actin filaments in a curved geometry when anchored to lipid membranes. Although F-actin is semi-flexible with a persistence length of ~10 μm, binding of mobile curly within lipid membranes generates actin filament arcs and full rings of high curvature with radii below 1 μm. Higher rates of fully formed actin rings are observed in the presence of the actin-binding coiled-coil protein tropomyosin and when actin is directly polymerized on lipid membranes decorated with curly. Strikingly, curly induced actin filament rings contract upon the addition of muscle myosin II filaments and expression of curly in mammalian cells leads to highly curved actin structures in the cytoskeleton. Taken together, our work identifies a new mechanism to generate highly curved actin filaments, which opens a range of possibilities to control actin filament geometries, that can be used, for example, in designing synthetic cytoskeletal structures

Cytosolic actin isoforms form networks with different rheological properties that indicate specific biological function

Abstract The implications of the existence of different actins expressed in epithelial cells for network mechanics and dynamics is investigated by microrheology and confocal imaging. γ-actin predominately found in the apical cortex forms stiffer networks compared to β-actin, which is preferentially organized in stress fibers. We attribute this to selective interactions with Mg2+-ions interconnecting the filaments’ N-termini. Bundling propensity of the isoforms is different in the presence of Mg2+-ions, while crosslinkers such as α-actinin, fascin, and heavy meromyosin alter the mechanical response independent of the isoform. In the presence of myosin, β-actin networks show a large number of small contraction foci, while γ-actin displays larger but fewer foci indicative of a stronger interaction with myosin motors. We infer that subtle changes in the amino acid sequence of actin isoforms lead to alterations of the mechanical properties on the network level with potential implications for specific biological functions

Single-benzene-based clickable fluorophores for in vitro and in vivo bioimaging

A series of clickable single-benzene-based fluorophores derived from tetrafluoroterephthalonitrile (4F-2CN) is reported. Fluorophores based on a tetrahydroquinoxaline skeleton (2F-2CN-(β-NH2Ala)) exhibited improved photophysical properties owing to better planarity and conjugation over those with dihydro[1,4]thiazine skeleton (2F-2CN-Cys). These easily produced clickable fluorophores were successfully applied in in vitro and in vivo bioimaging after protein conjugation