To migrate normally, a cell must establish morphological polarity and continuously protrude a single cell margin, termed the lamellipodium, polarised in the direction of migration. Previous data from our laboratory showed that actin filament disassembly was necessary for protrusion of the lamellipodium during fibroblast migration but was not required for non-polarised lamellipodial protrusion in non-migrating cells. DNase I staining of actin monomer levels in the lamellipodium showed that this was because actin monomer was highly limiting in the lamellipodium of polarised migrating cells. As ADF/cofilin (AC) proteins are essential for the catalysis of filament disassembly in cells, their role in polarised cell migration was assessed. The spatial distribution of AC and inactive, phosphorylated AC (pAC) was compared in the lamellipodium of polarised migrating cells. AC, but not pAC, localised to the lamellipodium. Adenoviral-mediated gene transfer was used to manipulate AC activity levels in cells. Locally maintaining active AC at the leading edge was required for maintaining cell polarity during fibroblast migration. When pAC was forced into the lamellipodium by introduction of a constitutively active form of LIM kinase, cells lost both their morphological polarity and their ability to migrate. This polarity loss could be prevented by expression of a non-phosphorylatable form of AC. Furthermore; AC activity was necessary for the acquisition of morphological polarity. Fibroblasts polarised in a distinct series of sub-steps. The first step in polarity acquisition was organisation of actin from a circumferential organisation to an oriented array. This was required to specify position of the cell tail. Both jasplakinolide treatment and introduction of either constitutively active LIM kinase or dominant negative AC blocked formation of oriented actin bundles; actin remained circumferentially oriented and the cell failed to polarise. Blocking AC and actin filament disassembly did not affect later steps in acquisition of polarity. Stabilisation of the cell tail was dependent on myosin II. Blocking myosin using either methyl-blebbistatin or Y-27632 produced abnormally crescent-shaped cells as the tail encroached into the cell body. Microtubules were not required for polarity acquisition, however blocking microtubule dynamics led to de-stabilisation of the lamellipodium and a loss of migratory capability.

No description available.

Eph receptor tyrosine kinases and their ligands, the ephrins, are versatile regulators of cell migration and tissue morphogenesis. Previous studies have shown that endothelial expression of EphB4 and its ligand ephrin-B2 are essential for angiogenic remodelling of blood vessels in the early embryo. However, ephrin-B2 is also expressed on mural cells (vascular smooth muscle cells and pericytes), which wrap around the endothelium to form stable and functional vessels. To gain further insight into the role of ephrin-B2 on mural cells, I have analysed tissue-specific mutant mice that lack ephrin-B2 on both vascular smooth muscle cells (vSMCs) and pericytes. This has revealed that ephrin-B2 is essential for the proper association of mural cells with endothelium. Using ephrin-B2-deficient (Δephrin-B2) and control immortalised vSMCs, I have found that loss of ephrin-B2 leads to striking changes in cell morphology. Ephrin-B2-deficient cells appear elongated and insufficiently spread, show numerous lamellipodial protrusions, and have problems detaching at the rear. Live video microscopy has revealed that Δephrin-B2 cells are unable to stabilise their lamellipodia despite forming functional focal adhesions and move in an erratic nonpolarised fashion. To further investigate the mechanism by which the loss of ephrin-B2 leads to defects in vSMCs, I’ve carried out affymetrix chip microarray. This has revealed that control and ephrin-B2 deficient cells display distinct expression profiles and identified differentially expressed genes involved in cell adhesion, matrix deposition, and Rho GTPase Activity. Most interesting is the finding that Tiam1 (T-lymphoma invasion and metastasis), a specific guanine-exchange factor (GEF) for Rac1, is down-regulated 49-fold in cells lacking ephrin-B2.

Most multicellular organisms have body plans that exhibit an element of symmetry; either radial or bilateral symmetry. Bilateral symmetry refers to organisms which have a single plane of symmetry with each side a mirror image of the other. Although externally these species appear symmetrical, internally, the organs are positioned in an asymmetric manner. This left-right axis is the last axis established after the A-P axis and D- V axis and recent research has analysed how this asymmetry is established. In the mouse it has been shown that monocilia on the organiser region, the mouse ventral node, rotate and generate a leftward fluid flow across the node. This flow, termed nodal flow, is essential for the left-sided localised expression of nodal, lefty and pitx2 and subsequent asymmetric patterning of organs. Nodal flow is highly conserved in the vertebrates but, although the conservation of asymmetric gene expression extends into the invertebrates, whether this is established through directional flow remains unknown. This research has investigated the extent of nodal flow conservation in a basal chordate, the urochordate, Ciona intestinalis. Here I have shown, through Electron Microscopy and immunofluorescence, that cilia are present, at the posterior of each cell of the Ciona intestinal is embryo, at the time point indicative to a role in the establishment of left-right asymmetry. They do not appear to be homologous to the cilia of the mouse ventral node because they are not motile and have a disorganised microtubule structure. However, the cilia may still have an important role in the development of left-right asymmetry in a sensory capacity or provide the site for H+/K +-ATPase channels already known to be involved in Ciona intestinal is left-right asymmetry.

The primary cilium, a solitary hair-like structure that extends out of the apical membrane of many cell types, has been investigated here. The current paradigm suggests that the cilium behaves as a cantilever-like object which bends in a continuous manner along the length of the cilium. This thesis aims to investigate whether this paradigm is appropriate. Here, primary cilia are imaged by atomic force microscopy and light microscopy in live and fixed states. Using atomic force microscopy to do force spectroscopy, it is shown by buckling the primary cilium that the cilium has a Young's modulus of 3.5 ± 1.3 MPa, a value which places it an order of magnitude stiffer than previously recognised. Calcium imaging has been used to identify the presence of calcium fluxes in response to fluid flow and actuation of the primary cilium with the atomic force microscope (AFM). A new technique, coined Vertical Deflection Mapping, has been developed, whereby a defined force is applied by the AFM at a known distance from the base of the cilium; it was observed that the cilium had a spring constant of (3.9 ± 2) X 10-5 Nm-1, approximately an order of magnitude more sensitive than previously recognised. It was found that the spring constant decreased towards the tip of the cilium as a function of the reciprocal of the length squared. This relationship is best represented by a rigid body bending from the base. These results were compared to results collected on a cantilever, which displayed results that tended towards the reciprocal of the length cubed. Preliminary work has been completed on combining light microscopy and AFM with a patch-clamp electrophysiology set-up allowing all three systems to be used simultaneously. This will allow a new method of delving into the functioning of mechanosensitive ion channels in the primary cilium and other structures.

Kinesin-1 is a molecular motor that transports cellular cargo along microtubules by completing hundreds of consecutive steps of its two head domains. Questions remain over what conformational changes drive this ATP-powered motility, and how actions of the two heads are coordinated. Photonic Force Microscopy allows optical trapping and 3D position detection of particles, including their axial position, which is often ignored in similar experimental set-ups. Using this technique, the energy landscape of a bead, tethered to a microtubule by a single full-length kinesin molecule on the bead surface, was analysed in the presence and absence of nucleotides. The mechanical properties of kinesin molecules were extracted from these data and were found to alter significantly depending on whether no nucleotide or the ATP analogue AMP-PNP was present in the experimental chamber. The effective axial stiffness of the molecule away from the microtubule increased on AMP-PNP binding. A further increase in this stiffness associated with a decrease in the volume accessible to the bead along the kinesin axis was observed and attributed to a change from 1- to 2-headed microtubule binding. Simulations of a model kinesin structure, consisting of rigid rods and flexible hinges, undergoing thermal fluctuations were performed. They suggest a change in angular stiffness and orientation of the neck domain would explain the change in axial stiffness observed. Neck- linker docking on AMP-PNP binding and inter-head strain caused by two-headed microtubule attachment can account for both these structural changes. Taken together, the experimental and simulated results suggest that when the rear kinesin head has its neck linker docked, and both heads are microtubule-bound, the neck and lower half of the kinesin stalk become aligned parallel to the microtubule, reducing the cargo's height above the surface by 20 nm. Further analysis of the processive motion of kinesin in a saturating concentration of ATP, as is present in physiological conditions, together with these static results led to a full description of the motor movement with respect to chemical and conformational states. This description provides a novel explanation of how the molecule might detect useful movements of its cargo, caused by thermal forces, and use these to increase its efficiency. In this way the motor acts like a mechanical feedback loop, amplifying neck linker dynamics. Similarly, positive movements of its cargo caused by the pull of another molecule motor could be used to aid procession. This additionally explains cooperativity between molecular motors. The model developed also incorporates the presence of a state in which a kinesin head is weakly associated to a microtubule by what is thought to be an electrostatic attraction when the head has ADP bound. This state was unambiguously documented experimentally in this work for the first time, and allows a kinesin head to slide along a microtubule protofilament while only weakly feeling the periodic potential of the ab-tubulin subunits. The significance of this weak association is discussed in terms of how it might aid kinesin motility, including the procession of single-headed constructs, and how it may play a role in the regulation of kinesin function in a cell according to chemical stimuli or spatial position.

Flagella are complex structures with important roles in bacterial motility, virulence and protein secretion. As of yet, little work has been undertaken to characterise the function and regulation of flagella in Clostridium botulinum, an organism which produces an extremely potent neurotoxin which is the causative agent of botulism and is utilised for numerous pharmaceutical purposes. Genes encoding structural components of the flagellar basal body, hook and filament were inactivated using the ClosTron insertional mutation method, resulting in reduced swarming motility and reduced neurotoxin release from the cell into culture supernatant as determined by an ELISA method. To determine regulatory factors affecting flagellar assembly and secretion, sigD, the gene encoding δD, an alternative sigma factor that has been shown to be responsible for the expression of late flagellar genes in various bacterial species, was inactivated. The mutant strain was analysed by microarray and quantitative real-time reverse-transcription PCR methods. It was shown that δD positively regulates the expression of multiple genes including the late flagellar genes, the clostridiolysin S genes and an operon of secreted proteases. Phenotypic assays were performed on the sigD mutant and confirmed that it was null for motility and that proteolysis by secreted proteases was reduced. This reduced proteolysis phenotype was complemented back towards wild-type levels by the introduction of a plasmid containing the sigD gene from Clostridium sporogenes under the control of its native Pft9B promoter. To aid future work, a method for inducing and selecting for double homologous recombination events in C. botulinum was developed for the purpose of creating a strain in which the chromosomal botA gene is converted to a toxoid encoding sequence by allelic exchange. This work has therefore implicated flagella in the secretion of the neurotoxin, characterised the regulatory roles of δD and developed a novel method for allelic exchange in clostridia.

In recent years, an increasing number of inherited diseases, of previously unknown aetiology, have been shown to arise from defects in the structure and function of primary cilia. Cilia play important roles in many physiological processes, including cell and fluid movement, development and chemo-, . mechano-, and photosensation. Ciliopathies now comprise a broad set of developmental and adult phenotypes including polycystic kidneys, CNS anomalies such as hydrocephalus and some forms of retinal dystrophy. Functional candidates for Meckel-Gruber syndrome were screened in a cohort using high resolution melt curve analysis and the utility of the technique is further demonstrated by genotyping a rare polymorphism in RPGRlPlL in ciliopathy patients. Mutations in TMEM216 are shown to cause Meckel-Gruber syndrome type 2 and it is demonstrated that cilia are lost in patients with these mutations. It is also shown that, like other ciliopathy proteins, TMEM216 localises to the primary cilium and interacts with key players of the noncanonical Wnt signalling pathway. Further work aimed to elucidate the molecular genetics of Ivemark syndrome, a possible ciliopathy in which patients are affected by disorders of symmetry.

Ephrin ligands and their Eph receptor tyrosine kinases both are surface tethered proteins that control cell shape and movements through direct cell-cell contact. Their binding, and subsequent clustering, triggers bidirectional signalling pathways, with signals transduced from the receptor (forward) and the ligand (reverse), that regulate the behaviour of both Eph- and ephrin- expressing cells. Recent evidence suggests that reverse ephrin-B2 signalling controls endothelial cell sprout outgrowth and tip elongation, and smooth muscle cell shape changes and behaviour. In addition, misregulation of ephrin-B2 expression is observed in various tumour types and high expression of this ligand is correlated with increased tumour vascularisation and tendency to metastasise. To investigate how ephrin-B2 "reverse" signalling pathways direct changes during angiogenesis and how the expression level of ephrin ligands influences changes in cell behaviour and cell mot motility, I have used Human Umbilical Vein Endothelial Cells (HUVECs) overexpressing ephrin ligands as a model system.

The bacterial flagellar motor is one of the few rotary motors found in nature, and an excellent example of a complex molecular machine. Flagellar motors in the model organism Escherichia coli are products of the coordinated expression of ∼50 different genes. The E. coli flagellar motor is powered by the proton-motive force (pmf), an electrochemical gradient across the cell membrane. Motor torque is gen- erated by proton flow through membrane-embedded stator units which bind to the basal body of the motor. This thesis aimed to investigate the relationship between the pmf and the flag- ellar motor. A novel pmf control system was developed, based on the light-driven proton pump proteorhodopsin (pR). This system enabled pmf -dependent changes in motor behaviour to be precisely monitored in vivo. Expression of pR in E. coli was shown to be sufficient to drive the flagellar motor at wild-type speeds. Using the pR-based pmf control system, the motor was shown to respond to changes in pmf on a timescale of milliseconds. Surprisingly, motor speed increase was observed when pmf was increased above the physiological norm. Reduction of pmf to low levels enabled individual steps in motor rotation to be observed. Motor response to loss of pmf was investigated. Motors were shown to exhibit a two-stage speed decrease after disruption of pmf , with motor speed falling to ∼20 % of its initial value within milliseconds, reaching a complete stop after 1 s. Extended periods of pmf loss was shown to lead to disengagement of stators from the motor, with motor speed increasing in a stepwise fashion after pmf restoration. The integrity of the motor at different pmf levels was investigated by using TIRF microscopy to directly image positioning of fluorescently tagged motor components. The stator protein MotB was shown to physically leave and rejoin the motor after pmf disruption and restoration, with MotB dispersal following motor stop.