No description available.

No description available.

No description available.

The centrosome is a non-membraneous organelle composed of two centrioles surrounded by pericentrolar material. The primary function of the centrosome is to act as the dominant microtubule organizing centre in animal cells. It therefore contributes to formation of both the interphase cytoskeleton and bipolar mitotic spindle. Both centrosome structure and microtubule organization are controlled in a cell cycle dependant manner by protein phosphorylation. The centrosomal kinase Nek2 regulates centrosome organization, mitotic progression and bipolar spindle assembly. However, the only core centrosomal substrate of this kinase so far identified is C-Napl, a structural protein required for centriolar cohesion. The aims of this project were therefore to isolate a Xenopus laevis homologue of C-Napl in order to study its function using Xenopus based cell free assays and identify and characterize novel centrosomal substrates of Nek2. By database screening, we identified a Xenopus protein of high similarity to C-Napl called rootletin, while using the yeast two hybrid system we identified the Xenopus Nip protein as a novel substrate of Nek2. Human Nip is a recently characterized centrosomal protein involved in microtubule organization and which is regulated by another centrosomal kinase, Plkl. Antibodies were raised to Xenopus Nip and used to confirm subcellular localization to the centrosome in Xenopus cells. Further localization and expression studies revealed that Nip is a mother centriole specific protein that is displaced from the centrosome, but not degraded, during mitosis. These data suggest that Nip is involved in interphasic microtubule anchorage. By transfection into Xenopus and human cells, we found that although Nek2 and Plkl phosphorylate Nip at distinct sites, they can both trigger Nip displacement from the centrosome at the onset of mitosis. Finally, data were obtained raising the possibility that Nek2 may act as a novel priming kinase for recruitment of Plkl to its substrate Nip.

Membrane fission is the cellular process by which organelles divide or release vesicles, and it is integral to cell functions such as membrane trafficking, organelle partitioning during mitosis, and mitochondrial dynamics. Membrane fission appears to rely on multiple mechanisms. Many fission events (typically, clathrin-dependent endocytosis) are driven by the large GTPase dynamin; others, instead, require C-terminal-binding-protein l -short form/brefeldin A (BFA) ADP-ribosylated substrate (CtBP1 -S/BARS; henceforth referred as BARS). Indeed, BARS can dramatically enhance the fission of Golgi-derived membranous tubules in vitro and also regulate several trafficking steps in vivo. These include the fission of basolaterally directed post-Golgi carriers and COPI vesicles, growth-factor-stimulated macropinocytosis, and fission of the Golgi ribbon during mitosis. The fission-inducing property of BARS was proposed to be dependent on an intrinsic lysophosphatidic acid acyltransferase (LP AA 1) activity responsible for phosphatidic acid production. Along with the closely metabolically interrelated lipids lysophosphatidic acid and diacylglycerol, phosphatidic acid has been suggested to mediate the rapid lipid geometry changes that might induce fission. Later work, however, showed that this activity is not intrinsic to BARS, but is due to an unknown associated protein involved in phosphatidic acid metabolism. Following from these combined considerations, the aim of the present study was to determine whether the fission-inducing property of BARS involves an interaction with a phosphatidic-acidproducing enzyme. BARS is shown to associate with two Golgi-localised LPAAT enzymes, LPAAT3 and LPAAT4. These LPAATs are involved in membrane trafficking, and treatments that block their catalytic activity result in impairment of membrane trafficking.

No description available.

This thesis presents an investigation into the development of plasmodesmata (PD), which are specialised pores in plant cell walls through which the cytosol and membranes of neighbouring cells are linked. Modification of PD from their initial single-tube (‘simple’) structures to branched (‘complex’) structures is an important part of tissue maturation as it allows cells to restrict the movement of syplasmically mobile molecules including hormones, RNAs and proteins. Conversion of PD from simple to complex is co-ordinated across large populations of cells to produce symplasmic domains, transport barriers, and preferential transport pathways. The development of PD is therefore intrinsic to the wider development and morphogenesis of cells, tissues, and organs. The aim of this project was to investigate the development of PD from simple to complex, particularly during the predictable, large-scale conversion of PD structure that accompanies the leaf transition from sink state to source. To study this I used transgenic plants expressing a GFP-tagged viral protein which accumulates specifically in complex PD, while leaving simple PD unlabelled. The project follows the development of complex PD from the early stages of leaf development to maturity using a range of microscopy techniques. Structured illumination microscopy was used to view labelled PD at super resolution, which gave new structural details about complex PD using a breakthrough technology. Conventional and high-throughput confocal and electron microscopy were used to localise PD within tissues in a broad survey of PD location in leaves to identify patterns of PD development. An imaging chamber was developed that allowed the development of complex PD to be viewed in real time and identified conditions that can trigger structural conversion of PD. Finally, a high-throughput microscopy study was performed to identify how hormones, sugar availability, environmental stresses, defence responses and inhibitors can affect PD development.

This thesis investigates the mechanical properties of the elastic proteins isolated by cyanogen bromide digestion from lamprey cartilages and compares them with the mammalian protein, elastin. Thermomechanical testing and measurements of the effects of hydrophobic solvents on mechanics are used to determine the energetic and entropic contributions to the mechanical properties and the role of solvent interactions. Raman microspectrometry is shown to be a valuable tool in determining the secondary structure of the proteins, their interactions with water and molecular-level effects of mechanical strain. The supramolecular structure of the proteins matrices are investigated using nonlinear microscopy and X-ray diffraction. The mechanical properties of fibrous elastin agreed with those previously reported with elastic moduli in the region of 0.2-0.4 MPa. Elastic moduli decrease by approximately 25% with increased temperature, which was accompanied by a small decrease in hysteresis loss. In agreement with earlier findings, an entropic mechanism of elasticity became dominant only at high temperatures with a major contribution from interactions with solvent water. The lamprey proteins can be divided into two broad groups, the 'soft' branchial and pericardial cartilages resembling elastin, with linear stress-strain behaviour over a range of strains, elastic moduli in the range 0.13 MPa to 0.35 MPa, breaking strains of up to 50% and low hysteresis. Annular and piston proteins showed a very different response having much higher elastic moduli (0.27 MPa to 0.75 MPa), higher breaking strains and large hysteresis. Similarities between elastin and the lamprey matrix proteins extended to their thermomechanical behaviour with a decrease in elastic moduli and a drive towards entropic elasticity at high temperatures, although the annulus and piston were less thermally stable. Raman spectroscopy was able to detect differences between the various proteins and between elastin fibres and fragmentation products. Although no vibrational modes associated with cross-linking of the fibres could be identified, the secondary structure of dehydrated fibrous elastin was significantly different from \alpha -elastin. The former differed from previous experimental measurements, but was close to the theoretical predictions with 36% \beta -structures, 46% unordered and 18% \alpha -helix. \alpha -Elastin contained 29% \beta -structures, 53% unordered and 18% \alpha -helix. Strains of up to 60% in ligament fibre bundles resulted in no significant shifts in peak positions or in secondary structure. Polarization measurements revealed that the peptide bonds and several of the bulky side-chains re-orientated closer to the fibre axis with strain. Heating nuchal elastin fibres to 60^{\circ} C to increase the energetic component of the elasticity was associated with a 30% increase in the proportion of \beta -structures in the amide I band, a 50% increase in the amide III band, and a 50% reduction in the signal from bound water. The Raman spectra of the lamprey matrix proteins are similar both to each other and when compared to fibrous elastin. Only small differences could be detected in side-chain modes consistent with reported biochemical differences. Decomposition of the amide I band indicated that the secondary structures were also very similar to that of elastin, with a preponderance of unordered structures which probably confer the high degree of conformational flexibility necessary for entropy elasticity. Piston and annular proteins, like elastin, showed a strong interaction with water, suggesting a greater role of hydrophobic interactions in their mechanics compared to the branchial and pericardial proteins. Elastin is well known to exhibit autofluorescence. However, only the branchial protein has been reported to autofluoresce. This study shows that all four lamprey matrix proteins investigated exhibit strong autofluorescence which was subsequently exploited to image these tissues using multiphoton microscopy. Microscopic investigations revealed that the architecture of lamprey proteins differ from that of elastin. Nuchal elastin forms bundles of fibres running predominantly parallel to the direction of applied force. The arrangement in lamprey cartilage is very different forming honeycomb structures, which in the case of annular and piston cartilages, is surrounded by a dense sheath of matrix material. Dye injections revealed that the branchial and pericardial form open systems whereas in piston and annular cartilages a closed system exists. These variations in architecture are reflected in their different mechanical properties and in vivo functions.

Centriole and basal bodies are related nine-fold symmetric microtubule-based eukaryotic organelles central to the organisation of cilia/flagella and centrosomes. Mechanisms of eukaryotic centriole and basal body assembly are mainly based on studies in animal systems. To understand which centriolar proteins are the universally important ones in the assembly across eukaryotes, a bioinformatic survey presented here investigates the distribution of centriolar and cilia-associated proteins across a diverse range of eukaryotes. This analysis showed also that the basal body function is ancestral to eukaryotes, whereas centrosomal components are specific to Holozoa (which include animals). It also suggested that the ancestor of all eukaryotes possessed a cilium/cilia not only with motility function but also with a sensory role. The most frequently conserved proteins in extant ciliated eukaryotes found in this analysis included SAS-6, SAS-4 and WDR16. To test whether these proteins are also important for basal body assembly in distantly-related species to metazoan and other model organisms where the proteins have been studied to date, the proteins were investigated in Trypanosoma brucei. I used a combination of genetic tools and microscopy techniques to demonstrate that SAS-6 but not SAS-4 is essential for basal body assembly in T. brucei. I showed that WDR16 is a stably integrated component of the transition zone and axoneme but not the basal body. Furthermore, I identified a novel SAS-6 like protein which localises to a position consistent with the basal plate and has the capacity to form into filaments. This thesis provides new insights into the evolution of centrioles and basal bodies, and into the function of conserved centriole proteins in T. brucei, a distantly-related organism to animals.

La souris "dystoniamusculorum" (Dstdt-j) résulte d'une mutation spontanée autosomale récessive d'un gène codant pour la dystonine, protéine du cytosquelette qui s'exprime normalement dans les neurones sensitifs du système nerveux périphérique, mais aussi dans de nombreuses structures du système nerveux central. Elle est caractérisée par une dégénérescence des fibres nerveuses affectant principalement les voies afférentes sensitives du système nerveux périphérique et les afférences secondaires cérébelleuses et thalamiques. Des troubles posturaux et une sévère ataxie apparaissent avec les dégénérescences. Une altération des muscles squelettiques est associée. Afin d'étudier l'incidence des troubles du transport axonal sur l'activité métabolique de la fibre nerveuse et l'impact de la dégénérescence sensitive sur les fonctions motrices, une analyse histopathologique et biochimique comparative du système nerveux central et des muscles squelettiques est effectuée sur des souris de souche B6C3Fea /a-Dstdt-j, âgées de 42 à 54 jours réparties un groupe de souris homozygotes Dstdt-j et un groupe contrôle, formé de souris homozygotes normales et hétérozygotes. En plus des dégénérescences afférentes périphériques, l'étude histopathologique montre des altérations centrales au niveau de centres segmentaires et de différentes structures du système extra-pyramidal comme le cervelet, le noyau rouge et les noyaux du pont. Une évaluation quantitative de l'activité métabolique régionale par marquage histochimique de la cytochrome oxydase (enzyme du complexe IV de la phosphorylation oxydative) est effectuée sur l'ensemble de l'encéphale et de la moelle épinière cervicale. Elle met en évidence une hyperactivité métabolique des voies de la proprioception inconsciente, affectant principalement les structures du tronc cérébral et du cervelet, et plus spécifiquement des structures motrices impliquées dans l'équilibre, le tonus musculaire et les réflexes posturaux. Une étude de l'innervation cholinergique est effectuée par marquage histochimique de l'acétylcholinestérase (AChE). Des augmentations importantes de l'activité AChE sont observées dans les structures cibles du noyau cholinergique tegmental pontique, à savoir le thalamus servant à l'activation corticale, le striatum et certains noyaux mésencéphaliques connectés au striatum, impliqués dans le contrôle des mouvements. Des altérations structuro-fonctionnelles des trois muscles observés (langue, muscle masséter et flexordigitorumsuperficialis) illustrent un tableau clinique d'hypotonicité et d'hypoactivité musculaire. Une dégénérescence partielle des fibres musculaires semble indiquer que ces modifications sont conséquentes de la lésion nerveuse plutôt que d'une atteinte intrinsèque des fibres musculaires. La présente étude sert à comprendre la physiopathologie 1) des neuropathies sensitives humaines présentant des atteintes similaires comme l'ataxie de Friedreich, ou la forme infantile de l'ataxie spino-cérébélleuse, 2) de déafférentations trigéminales pouvant survenir par compressions iatrogènes lors d'actes chirurgicaux / The "dystoniamusculorum" (Dstdt-J) mouse is the result of a recessive autosomal mutation of the dystonin (dst) gene responsible for the expression of a protein of the cytoskeleton, normally present in sensory peripheral neurons and numerous central nervous structures. The mutation is characterised by primary degeneration of peripheral afferent fibers followed by degenerative processes in secondary cerebellar and thalamic afferents. Postural reflex dysfunction and a severe ataxia occurat the same time as peripheral neurodegenerative processes. To study the impact of axonal transport alteration on neuronal activity as well as sensory degeneration on motorfunctions, histopathatological and biochemical studies have were performed on brain and skeletal muscles of B6C3Fea /a-Dstdt-j mice displayed in two groups, homozygotes Dstdt-J and controls. In addition to sensory defects, histopathological evaluation showed central alterations affecting the segmentar nuclei and motor control regions, such as cerebellum, red nucleus, and pontine nuclei. Regional metabolism assessed by cytochrome oxidase histochemistry showed hypermetabolic activity in cerebellum and motor regions of the brain stem involved in equilibrium, muscle tonicity, and postural reflexes. Brain cholinergic innervation was performed by histochemical labelling of acetylcholinesterase (AChE) activity. Enzymatic hyperactivity was observed in target structures of the cholinergic pedunculo-pontine tegmental nucleus, the thalamus, and the basal ganglia. The morphological and functional alterations observed in the three skeletal muscles (masseteric, flexor digitorum superficialis and tongue) were characterized by muscle hypotonicity and hypoactivity. A partial muscular fiber degeneration was observed, probably as a result of sensory denervation rather than an intrinsic defect of muscle fibers. Several sensory neuropathies (Friedreich's ataxia or infant-onset spinocerebellar ataxia) have similar clinicopathological features. The present studies are of interest in understanding the physiopathology of these lesions. They can also contribute in the study of the pathological effects of trigeminal deafferentation induced by compression