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The aim of this project was to investigate the effects of a novel mouse cytoplasmic dynein mutation; Abnormal rear leg (Arl). Cytoplasmic dynein is a microtubule (MT) based motor protein important for diverse cellular processes including Golgi maintenance and retrograde transport of organelles. Arl is a mouse point mutation in the heavy chain subunit of dynein (Dync1h1). Homozygous Dync1h1Arl/Arl die at embryonic day 10. Dync1h1Arl/+ heterozygotes have a normal life span, but exhibit abnormal gait and hindlimb clasping during tail suspension, typical of neuronal dysfunction. Protein purification from wildtype and heterozygous brain tissue showed increased MT binding in Dync1h1Arl/+ compared to wildtype. Delayed endosomal trafficking was observed in EGF stimulated Dync1h1Arl/+ mouse embryonic fibroblasts (MEFs) compared to wildtype, in both fixed cells and using live cell imaging. Similarly, a delay in the reassembly of the Golgi complex after disruption with a MT depolymerisation agent, nocodazole, was observed in Dync1h1Arl/+ MEFs compared to wildtype. In addition, the Golgi complex was observed as being structurally perturbed in Dync1h1Arl/+ lumbar spinal cord neurons using transmission electron microscopy (TEM) compared to the wildtype. TEM also revealed that the mitochondria were structurally perturbed in Dync1h1Arl/+ lumbar spinal cord neurons compared to wildtype, and O2 consumption assays investigating their function showed the Dync1h1Arl/+ mitochondria to have increased respiration rates compared to wildtype. Thus, these data highlight the Arl mouse as an invaluable model for studying the mechanism of dynein function and the subsequent outcomes when they are compromised.

Maintenance of the intracellular NADH/NAD+ redox poise is vital for energy generation in cells. Gram-positive bacteria, including the antibiotic-producing organism, Streptomyces coelicolor, have evolved a regulatory protein Rex that both senses this ratio and mediates an adaptive response to changes in it. Rex is a dimeric redox-sensitive transcriptional repressor. It is capable of binding to both NAD+ and NADH, although only NADH is an effector, causing dissociation of the protein from operator (ROP) sites. As NADH levels rise during oxygen limitation Rex dissociates from its target genes allowing expression, which helps to restore the NADH/NAD+ ratio. Microarray-based expression studies had suggested that Rex regulated only a small number of genes. In this work, however, ChIP-on-chip analyses revealed 38 genes that are potential regulon members. Analysis of the Rex binding sites in S. coelicolor revealed new insights into the mode of binding and show that Rex can bind with low affinity to incomplete half sites. This work also focused on characterising two key Rex targets, ndh and nuoA-N, that encode non-proton-translocating and proton translocating NADH dehydrogenases, respectively. Whereas nuoAN is not essential and was not expressed in liquid media, ndh was essential for growth. Depletion of NDH from growing cells led to the induction of Rex target genes confirming that ndh and Rex play key roles in maintaining redox homeostasis. Structure-based dissection of Rex, via a close homologue in Thermus aquaticus, identified a key interaction between the NADH- and DNAbinding domains of Rex. An R29-D203' salt-bridge, that traverses the NADH binding and DNA binding domains of Rex, appeared to stabilise the DNA-bound form of Rex, but is ‘broken' in the presence of NADH. In the NADH-bound form of Rex, D203 alternatively interacts with Y111, which in turn interacts with the nicotinamide ring of NADH. In order to assess the importance of individual subunits in the dimeric Rex, a single-chain derivative was constructed and the NADH binding and DNA binding domains individually disrupted.

Culturing C2C12 myoblasts and myotubes with a combination of LPS, TNF-α, IFN-γ and IL1β for 18 hours was used to determine the effects of endotoxic shock on possible causes of the dysregulation of glucose homeostasis associated with the syndrome. The in vitro model was confirmed by the significant production of NO in both myoblasts and myotubes following treatment. The treatment resulted in significantly different results between both myocyte preparations with regards to the regulation of glycogen synthesis. In the myoblasts, the treatment significantly increased myoblast glycogen synthesis, in a NO-independent manner, as seen by the inclusion of the NO synthase inhibitor L-NAME. This stimulation was unlikely to be due to a change in either GS or Phosphorylase activity. However it may have been caused by a significant increase in glucose transport induced by the treatment. This latter increase was also NO-independent, as well as not requiring reactive oxygen species. Insulin-induced myoblast protein synthesis was impaired by the treatment, which is likely due to an impairment of insulin-stimulated ERK1/2 phosphorylation. In the myotubes the case was different, as the treatment significantly reduced glycogen synthesis in a NO-dependent manner. This correlated with a NO-dependent increase in GS phosphorylation, indicating it was less active, however measurements of GS fractional activity failed to confirm this. Insulin stimulation of myotube glycogen synthesis was impaired by the treatment in a NO-independent manner, which may have involved an impairment of the insulin signal to ERK1/2. However the latter impairment was NO-dependent, suggesting other contributory mechanisms. Endotoxic treatment significantly increased myoblast protein content, but failed to do so in myotubes. This effect in the myoblasts may be explained by a significant increase in protein synthesis between 6 and 12 hours of treatment. None of the effects observed in the study were due to the treatment compromising cell viability.

Meiotic recombination is initiated by self-inflicted DNA breaks and primarily involves homologous chromosomes, whereas mitotic recombination involves sister chromatids. Whilst the mitotic recombinase Rad51 exists during meiosis, its activity is suppressed in favour of the meiosis-specific recombinase, Dmc1, thus establishing a meiosis-specific mode of homologous recombination (HR). A key contributor to the suppression of Rad51 activity is the synaptonemal complex (SC), a meiosis-specific chromosomal structure that adheres homologous chromosomes along their entire lengths. Here, in budding yeast, we show that two major cell cycle kinases, Dbf4-dependent Cdc7 kinase (DDK) and Polo-kinase (Cdc5), collaborate to link the mode change of HR to the meiotic cell cycle by. This regulation of HR is through the SC. During prophase I, DDK is shown to maintain SC integrity and thus inhibition of Rad51. Cdc5, which is produced during the prophase I/metaphase I transition, interacts with DDK to cooperatively destroy the SC and remove Rad51 inhibition. By enhancing the interaction between DDK and Cdc5 or depleting DDK at late prophase I, meiotic DNA breaks are repaired even in the absence of Dmc1 by utilising Rad51. We propose that the interplay between DDK and Polo-kinase reactivates mitotic HR mechanisms to ensure complete repair of DNA breaks before meiotic chromosomem segregation.