The aim of this study was to test whether or not the equilibration time of two different cryodiluents influences the post thaw qualities of epididymal African buffalo (Syncerus caffer') sperm. Diluents and equilibration times were compared by assessing the post thaw spermatozoal motility, longevity and the acrosomal integrity. African buffaloes belong to Africa's "Big Five" and are, therefore, popular animals amongst game farmers, hunters and tourists. They are also asymptomatic carriers of foot-and-mouth-disease (FMD) and considered to be a wildlife reservoir for this plague. Other diseases, that are carried and can be transmitted from the African buffalo (Syncerus caffer') to livestock include tuberculosis, brucellosis and theileriosis or corridor disease (CD). Therefore, the transportation of African buffaloes is highly regulated. Disease-free buffalo populations are currently derived from a small genetic 8 pool and are smaller in their trophy size than the free-ranging animals from the diseased areas of the Kruger National Park (KNP) and the Hluhluwe/Umfolozi National Park. Hence there is a special interest in bringing new genetic material into the disease-free populations. Epididymal sperm from 11 mature African buffalo bulls was collected, diluted with two different semen extenders (TriiadylTM [Tris egg yolk extender] and AndroMed® [synthetic extender, i.e. fully defined medium]) and frozen. Pre-freezing equilibration times of 2 and 9 hours were tested. Total and progressive motilities, longevities and acrosomal integrity were measured and compared. Results show that there were no differences in post-thaw sperm quality when equilibration times between 2 and 9 hr were used. The use of the egg yolk containing extender (TriiadlyTM) resulted in higher percentage of post-thaw motilities than the use of the synthetic AndroMed®. Because a high percentage of progressive motile spermatozoa is one of the prerequisites for successful AI it must be concluded that TriladylTM is superior to AndroMed®. As I believe the advantages of higher motility to be bigger than the hygiene risks of a yolk containing extender I conclude that epididymal buffalo sperm should rather be frozen with TriiadylTM than with AndroMed®. / Dissertation (MSc (Production Animal Science))--University of Pretoria, 2003. / Production Animal Studies / unrestricted

Skeletal muscle fatigue is defined as a loss in the force/velocity generating capacity of a muscle. A portion of the loss in function is attributable to effects of acidosis (i.e. low pH) on the regulatory proteins, troponin and tropomyosin (Tm), which regulate the binding of myosin and actin in a calcium (Ca++) dependent manner. However, the relative role of troponin and Tm on myosin-actin function during acidosis is not clear, nor are the mechanisms underlying these effects. PURPOSE: To determine the role of Tm in the acidosis-induced depression of muscle function using isolated muscle proteins in an in vitro motility assay. METHODS: Three mutant constructs of Tm were expressed by replacing the two amino acid (histidine) residues most likely affected by low pH with alanine residues (H153A, H276A, H153A/H276A). These mutant constructs were compared to wild-type Tm (wt-Tm) in order to test whether the acidosis-induced charge change of the histidine amino acid governs the pH-dependent alteration of tropomyosin and therefore the decrease in maximal RTF velocity and Ca++-sensitivity. The effect of acidosis on regulated thin filament (RTF) function was determined by assessing the impact of low pH (pH 6.8) versus neutral pH (pH 7.4) on myosin’s ability to move RTFs in the motility assay as a function of increasing levels of Ca++. This was done separately for the wt-Tm and each structural variant. RESULTS: A two-way ANOVA (pH x Tm construct) revealed that acidosis significantly (p<0.05) depressed the maximal sliding velocity of the RTFs across all versions of Tm, but that the magnitude of the depression was similar among the wt and all of the Tm mutants. Acidosis did not significantly depress the sensitivity to Ca++ under the unloaded conditions of this assay (p>0.05). CONCLUSIONS: These data suggest that the histidine residues in tropomyosin do not mediate the acidosis-induced depression in contraction velocity observed during muscle fatigue. However, since these residues may be more important in mediating the depression of force, we are currently testing the impact of the three mutant Tm constructs on the acidosis-induced depression in Ca++-sensitivity using a loaded in vitro motility assay.

Les kinésines sont des protéines moteur liées au cytosquelette de microtubules. Elles convertissent l’énergie provenant de l’hydrolyse de l’ATP en un travail mécanique. Leur fonction typique est de se déplacer le long du microtubule pour véhiculer des charges. La plupart des kinésines sont des dimères. Elles comprennent un domaine moteur, qui porte à la fois les sites de liaison du nucléotide et du microtubule, un domaine intermédiaire de dimérisation et une partie dite « queue » qui confère la spécificité des charges à transporter. Mon objectif est d’établir le mécanisme moléculaire à la base de la motilité, avec un intérêt particulier pour la détermination des variations structurales du domaine moteur de la kinésine le long de son cycle mécano-chimique. Au cours de ma thèse, mon objet d’étude principal a été la kinésine-1 humaine, encore appelée kinésine conventionnelle.J’ai étudié plus particulièrement deux aspects du cycle mécano-chimique de la kinésine-1, en combinant des approches de biologie structurale et l’étude de mutants. Les deux aspects concernent l’étude de la fixation de la kinésine-ADP au microtubule, conduisant à l’éjection du nucléotide et à une liaison forte de la kinésine au microtubule. Dans un premier temps, j’ai déterminé la structure du domaine moteur de la kinésine-1, dépourvue de nucléotide, et sous forme d’un complexe avec la tubuline. La tubuline est la protéine constitutive des microtubules. Cette structure était la donnée principale qui nous manquait dans le cycle structural de la kinésine. En comparant cette structure avec celle de la kinésine dans un état ATP, on peut rendre compte des changements de conformation de la kinésine selon le mouvement de trois sous-domaines du domaine moteur. Cette analyse explique notamment le lien entre la fixation de l’ATP et l’ouverture d’une poche hydrophobe distante de 28 Å du site du nucléotide. Cette cavité va accommoder le premier résidu du neck linker, conduisant à la stabilisation de ce peptide situé en partie C-terminale du domaine moteur. En s’ordonnant, le neck linker va faire avancer la charge ainsi que l’autre domaine moteur de la kinésine dimérique. Il lie ainsi la fixation de l’ATP au mouvement. L’étude de l’effet de mutations du neck linker montre aussi comment, réciproquement, le neck linker bloque la kinésine dans la conformation active pour l’hydrolyse de l’ATP. Ceci diminue la probabilité que l’ATP soit hydrolysé avant que l’étape mécanique se soit produite; cet aspect est essentiel pour rendre compte de la processivité de la kinésine-1.Ces données structurales suggèrent également comment la fixation de la kinésine-ADP au microtubule accélère l’éjection de l’ADP. Pour étudier cet aspect plus en détail, j’ai étudié l’effet de mutations sur la vitesse de largage de l’ADP. L’idée était de mimer à l’aide de mutations la fixation au microtubule. J’ai identifié ainsi deux séries de mutants qui présentent une vitesse accélérée de largage spontané de l’ADP, ce qui suggère deux voies pour interférer avec la fixation du nucléotide. J’ai ensuite déterminé la structure de deux de ces mutants dépourvus de nucléotide, ainsi que celle de la kinésine de départ également dans une forme apo, obtenue par digestion de l’ADP. En absence de microtubule, la kinésine dépourvue de nucléotide adopte une conformation soit à l’image de celle de la kinésine-ADP, ou proche de celle de la kinésine-apo liée à la tubuline. Dans un contexte naturel, seule la deuxième conformation est compatible avec la fixation au microtubule. L’ensemble de ces résultats suggère que le microtubule accélère l’éjection du nucléotide par un double mécanisme : en interférant avec la liaison du magnésium et en déstabilisant le motif P-loop de liaison du nucléotide. / Kinesins are a family of microtubule-interacting motor proteins that convert the chemical energy from ATP hydrolysis into mechanical work. Many kinesins are motile, walking along microtubules to fulfill different functions. Most kinesins are dimers, the monomer comprising a motor domain, a dimerizing stalk domain, and a tail domain. The motor domain contains both the nucleotide-binding site and the microtubule-binding site. I am interested in the molecular mechanism of kinesin's motility. In particular I want to establish the structural variations of the kinesin motor domain along with the mechanochemical cycle of this motor protein. During my thesis, I have focused my work on the human kinesin-1, also named conventional kinesin, which is the best characterized kinesin.I have studied two aspects of the kinesin mechanochemical cycle, by combining structural and mutational approaches. Both aspects rely on the binding of ADP-kinesin to a microtubule, which leads to the release of the nucleotide and to a tight kinesin-microtubule association. First I determined the crystal structure of nucleotide-free kinesin-1 motor domain in complex with a tubulin heterodimer, which is the building block of microtubule. This structure represented the main missing piece of the structural cycle of kinesin. Three subdomains in the kinesin motor domain can be identified through the comparison of my structure with ATP-analog kinesin-1-tubulin structure. The relative movements of these subdomains explain how ATP binding to apo-kinesin bound to microtubule triggers the opening of a hydrophobic cavity, 28 Å distant from the nucleotide-binding site. This cavity accommodates the first residue of the “neck linker”, a short peptide that is C-terminal to the motor domain, allowing the neck linker to dock on the motor domain. The docking of the neck linker is proposed to trigger the mechanical step, i.e. the displacement of the cargo and the stepping of the dimeric kinesin. By studying mutants of the neck linker, I have shown that, reciprocally, this peptide locks kinesin in the ATP state, which is also the conformation efficient for ATP hydrolysis. Doing so, it prevents the motor domain from switching back to the apo-state. It prevents also an untimely hydrolysis of ATP, before the mechanical step has occurred. These features are required for movement and processivity.Second, these structural data also suggest how the binding of ADP-kinesin to tubulin enhances nucleotide release from kinesin. To further study this step of the kinesin cycle, I studied the effect of kinesin-1 mutations. These mutations were designed in isolated kinesin to mimic the state when kinesin is bound to a microtubule. I identified two groups of mutations leading to a high spontaneous ADP dissociation rate, suggesting that there are two ways to interfere with ADP binding. Then I determined the crystal structures of the apo form of two mutants as well as that of the nucleotide-depleted wild type kinesin. It showed that apo-kinesin adopts either and ADP-like conformation or a tubulin-bound apo-like one. In the natural context, the second one is stabilized upon microtubule binding. Overall, the mutational and structural data suggest that microtubules accelerate ADP dissociation in kinesin by two main paths, by interfering with magnesium binding and by destabilizing the nucleotide-binding P-loop motif.

The regulation and maintenance of normal cell movements and shape play a vital role in the normal development and health of every living thing. The characterization of 6 zebrafish Zipper Interacting Protein Kinase homolog has helped to better understand how changes in cell cytoskeletal elements can lead to changes in cell shape and movement. Zebrafish are ideal model organisms for studying ZIPK because it has been previously shown that zebrafish ZIPK has closer sequence homology to human ZIPK than rodent ZIPK, and because zebrafish embryos are ideal for studying cell shape and movement in vivo. Using Whole Mount In Situ Hybridization we found that the zebrafish ZIPK is expressed during all stages of embryonic development, but most importantly during gastrulation when cell motility and changes in cell shape can best be studied. To determine where zebrafish ZIPK is expressed at the sub-cellular level, GFP-ZIPK and Flag-ZIPK clones were created and used for transfecting into Hek293T cells and Hela Cells. From these transfections, cell counterstaining and confocal microscopy we found that ZIPK is expressed ubiquitously throughout the cell, although mainly cytoplasmic. To study the effects on cell shape various ZIPK mutants were created through site-directed mutagenesis. These mutants were made to study the effects of the kinase domain of the protein, or other functional domains within the protein. From these studies it was shown that ZIPK does affect cell shape through changes in the actomyosin cytoskeleton resulting in aberrant cytoskeletal structures. Finally, we have also shown through phosphorylation assays that ZIPK phosphorylates and thus regulates MYPT-1, a scaffolding protein of the myosin protein phosphate complex and directly phosphorylates myosin light chain, both of which play a role in changes in cell shape and movement.

No description available.

No description available.

Type IVa pili (T4aP) are long, retractile, filamentous, surface appendages involved in cellular surface adhesion, biofilm formation, DNA uptake, and a unique form of motility called ‘twitching’. They are a critical virulence factor in a number of bacteria, including the opportunistic pathogen Pseudomonas aeruginosa, a major cause of hospital-acquired infections. T4aP function is controlled by a number of different regulatory proteins and systems. A putative chemosensory system termed ‘Chp’, controls levels of the second messenger molecule cyclic adenosine monophosphate (cAMP). cAMP works with a cAMP receptor protein called Vfr to control expression of ~200 virulence genes, including those that are required to make T4aP. cAMP levels are regulated by proteins outside the Chp system, including the bitopic inner membrane protein, FimV. This study examines the role of the Chp system and FimV in T4aP regulation. Both proteins are required for regulation of cAMP levels, while the Chp system also has a cAMP-independent role in regulating twitching. FimV has been shown to regulate cAMP levels, possibly connecting to the Chp system through a scaffold protein, FimL. We present the structure of a conserved cytoplasmic region of FimV, and show that this region is required for connecting FimV to the Chp system. We also characterize the cAMP-independent role of FimV, confirming that it is distinct from that of the Chp system, and is involved in localizing T4P regulatory proteins. We also provide evidence that the cAMP-independent role of the Chp system is to mediate the balance between T4P extension and retraction, possibly through denoting the ‘front’ of a motile cell. Together, these data help to resolve the cAMP-independent and –dependent pathways controlling twitching motility. / Thesis / Doctor of Philosophy (PhD) / Pseudomonas aeruginosa is a bacterium that causes infection in people with weakened immune systems. One key factor it uses to cause infection is the type IVa pilus (T4aP), a filamentous appendage displayed on the cell surface. T4aP can repeatedly extend and retract, and are involved in attachment to host cells, and movement along surfaces. When T4aP cannot extend or retract, the bacteria cannot cause infection. Many proteins work together to control T4aP function – this study focuses on two of them. They have one overlapping function, controlling levels of a signalling molecule needed to make T4aP. We also show that they have a second, non-overlapping function. One is involved in controlling the extension/retraction balance, possibly by marking the front of a cell, while the other may localize pilus-related proteins within a cell. This work helps us understand how P. aeruginosa makes T4aP, and provides information helpful to understanding control of virulence.

No description available.

No description available.

Clostridium perfringens is a Gram-positive anaerobic pathogen of humans and animals. While lacking flagella, C. perfringens cells can still migrate across surfaces using a type of gilding motility that involves the formation of filaments of bacteria lined up in an end to end conformation. To discover the gene products that play a role in gliding, we developed a plasmid-based mariner transposon mutagenesis system that works effectively in C. perfringens. Twenty-four mutants with deficiency in gliding motility were identified and one gene, which encodes a homolog of the SagA cell wall-dependent endopeptidase, was further characterized. We also isolated and characterized two hypermotile variants of strain SM101. Compared to wide type cells, the hypermotile cells are longer and video microscopy of their gliding motility suggests they form long, thin filaments that move rapidly away from a colony, analogous to swarmer cells in bacteria with flagella. Whole genome sequencing analysis showed that both mutants have mutations in cell division genes. Complementation of these mutations with wild-type copies of each gene restored the normal motility phenotype. A model is presented explaining the principles underlying the hypermotility phenotype. Heat resistant spores are the major route for disease transmission for C. perfringens, which cause food poisoning. To elucidate the molecular mechanisms involved in spore germination as well as to identify attractive targets for development of germination inhibitors to kill spores, we combined 1D-SDS-PAGE and MALDI-TOF-MS/MS to map the whole spore inner membrane proteome, both from dormant and germinated spores. As the first comprehensive spore inner membrane proteome study, we identified 494 proteins in total and 119 are predicted to be membrane-associated proteins. Among those membrane-associated proteins, 71 changed at least two-fold in abundance after germination. This study provides the first comprehensive list of the spore inner membrane proteins that may be involved in germination of the C. perfringens spore and their relative levels during germination. / Ph. D.