Thesis (Ph. D.)--University of Adelaide, Dept. of Plant Physiology, 1985. / Includes bibliographical references (leaves 245-271).

Polar body emission (PBE), a form of asymmetric division, occurs twice during vertebrate oocyte maturation and is required to produce a haploid egg for sexual reproduction. Our lab elucidated parts of the mechanism that regulates PBE in Xenopus laevis oocytes. Cdc42 and RhoA, two GTPases, were shown to mediate membrane protrusion and the contractile ring, respectively. It is believed that cdc42 is mediating the protrusion by regulating actin polymerization. However, it is not clear what upstream signaling pathway regulates cdc42 activation during PBE. One possibility is calcium signaling, which occurs at fertilization, and is required for second PBE. Interestingly, the fertilization calcium transient also regulates cortical granule exocytosis/membrane retrieval, a process that also involves cdc42-mediated actin assembly. Furthermore, active cdc42 and RhoA are found in non-overlapping concentric zones in single-cell wound healing; their activation requires calcium signaling. To determine possible calcium transients during polar body emission, we employed the calcium-binding C2 domain of PKCβ in live cell imaging. Surprisingly, the most prominent C2 signal was seen after cdc42 activation and membrane protrusion. Co-localization experiments indicated that the C2 signal appeared at the cortical area marked by the contractile ring component anillin, and after partial constriction of the ring. Injection of the calcium chelator, dibromo-BAPTA, abolished the C2 signal, suggesting that it is indeed depicting a calcium transient. Dibromo-BAPTA injection also inhibited polar body abscission, as assessed by a novel abscission assay developed in our lab. We have for the first time detected a calcium signal during PBE that is essential to the last step of cytokinesis—abscission.

Premature and excessive leaf abscission during flowering time in the late avocado (Persea americana Mill.) cultivar ‘Ryan’ is a considerable problem for avocado growers. They are especially concerned that premature and excessive leaf abscission will have a negative effect on yield. No previous investigations have been performed where premature and excessive leaf abscission in avocado has been studied in detail. This study therefore aimed to investigate the pattern of premature and excessive leaf abscission in ‘Ryan’, and compare it with two other important commercial cultivars, ‘Fuerte’ and ‘Hass’, which do not display this phenomenon. Time course studies of leaf abscission in the orchard were performed during 2006 and 2007 to determine the pattern of leaf abscission on ‘Ryan’, Fuerte’ and ‘Hass’. This also included anatomical studies to determine the time of leaf abscission zone formation. Possible stress factors, which accelerate leaf abscission were also investigated, namely unfavourable climatic conditions (temperature, solar radiation, rainfall, relative humidity and evapotranspiration), nutrient imbalances, excessive flowering and leaf area. The possible impact leaf abscission may have on yield was then assessed by determining levels of reserve carbohydrates in the bark of the tree. In addition, practical solutions, i.e. the application of fertilizers, plant growth regulators (PGRs) and kaolin, were investigated in order to reduce or eliminate premature and excessive leaf abscission. This study was carried out over the period 2005 until 2007, with experiments being modified on an annual basis as information was gathered on the phenomenon. Experiments began in 2005 with a study on the pattern of leaf abscission in ‘Ryan’, which revealed an increase in leaf abscission just prior to flowering. However, this increase was not significant. During 2006, the leaf abscission pattern for ‘Ryan’ was compared with the leaf abscission patterns of ‘Fuerte’ and ‘Hass’. Leaf abscission for ‘Ryan’ was significantly higher than for ‘Fuerte’ and ‘Hass’ during 2006. During 2006 ‘Ryan’ displayed two periods of high leaf abscission, namely the spring flush between bud dormancy and bud swell, and a drastic increase in spring and summer flush leaf abscission between inflorescence development and full bloom. These periods of increased leaf abscission were absent during the 2007 season. In addition, ‘Fuerte’ and ‘Hass’ did not display these peaks of high leaf abscission, with leaf abscission occurring in these cultivars at higher rates from full bloom onwards. Premature and excessive leaf abscission is therefore not an annual event in ‘Ryan’ and is in all likelihood influenced by external factors. Anatomical studies did not reveal any results in terms of initiation of leaf abscission, with only the protective layer of the abscission zone being visible after leaf yellowing occurred. During 2006, two peaks of extremely low temperatures (<4°C) occurred just prior to the acceleration of leaf abscission. During the second period of low temperatures, the solar radiation:temperature-ratio was also considerably higher. These periods of low temperatures were absent during 2007, indicating that cold and light stress could be contributing to premature and excessive leaf abscission in ‘Ryan’ in 2006. In addition, ‘Ryan’ flowered excessively during 2006, which could have been triggered by low temperature stress just prior to flower initiation. A significant correlation was found between excessive flowering and excessive leaf abscission in ‘Ryan’ during 2006. The occurrence of reduced flowering in ‘Fuerte’ and ‘Hass’ may possibly be due to these two cultivars being more tolerant to stress, and it is possible that ‘Ryan’ is genetically more prone to excessive flowering than ‘Fuerte’ and ‘Hass’. Excessive flowering could accelerate leaf abscission by causing an unusually high demand for water, nutrients and carbohydrates, resulting in the acceleration of leaf abscission. No significant relationship between nutrient levels and excessive leaf abscission was found for either 2006 or 2007. In addition, no significant correlation could be found between leaf abscission on a branch and the total leaf area of that branch during the 2007 season. During 2007, leaf abscission was low and it is possible that a significant correlation could be found in a season with excessive leaf abscission. During 2005, chemical applications to reduce leaf abscission did not yield any significant improvement in leaf retention. In fact, the 50 g/tree Solubor® and 50 g/tree Solubor® in combination with 2 kg/tree dolomitic lime had a significant negative effect on fruit set, possibly because too high concentrations were applied too close to fruit set. Chemical applications during 2006 were therefore made at bud dormancy and bud swell, as it was found that leaf abscission occurred before flowering time. However, no effect was observed on leaf retention or fruit set. During 2007, emphasis was placed on treatments that might reduce stress, as it became evident that stress could be responsible for premature and excessive leaf abscission in ‘Ryan’. Most treatments showed a slight positive effect on leaf retention, but no significant results were obtained possibly because that particular season was a season of low leaf abscission. Further research on application of stress-reducing treatments is therefore recommended. Best farm management practices including optimal fertilization and irrigation is therefore vital to prevent stress, accelerating leaf abscission. Copyright / Dissertation (MSc)--University of Pretoria, 2009. / Plant Science / unrestricted

The theory that fruit abscission may be the result of a reduction in metabolites available to the young fruit was addressed by this study. Shade cloth or terbacil, a photosynthetic inhibitor, were applied to limbs and whole trees to examine the influence of treatment and time of application on fruit nonstructural carbohydrates and abscission. 'Stayman' apple limbs shaded with 92% shade material from 5-15, 10-20, 15-25, 20-30, and 25-35 days after full bloom (April 22) had lower fruit retention than unshaded controls on 11 June. On 18 June, fruit diameter was greater on limbs shaded between 5-25 days after full bloom (DAFB) than on unshaded limbs. At 15, 20, 25, and 30 DAFB, fruit from limbs shaded for 10 days had lower total nonstructural carbohydrates (TNC), total sugars, and reducing sugars (% dry wt) than fruit from limbs shaded for 0 or 5 days. Terbacil (3-tert-butyl-5-chloro-6-methyluracil) was applied at 0, 50, 100, and 200 ppm to whole nine-year-old 'Redchief Delicious' apple trees at 15 DAFB. Terbacil markedly inhibited Pn; recovery occurred by 9 and 26 DAA for the 50 and 100 ppm rates, respectively. Phytotoxicity prevented the determination of Pn in the 200 ppm treated trees. Fruit dry weight, TNC, total sugars, and reducing sugars (% dry wt and mg/fruit) declined with increasing rates of terbacil. Total fruit abscission was observed 12 DAA for the 100 and 200 ppm treatments, while the 0 and 50 ppm applications retained 4.6 and 1.4 fruit per cm² limb cross sectional area (LCSA) at 35 DAA, respectively. Compared to the control, 50 ppm terbacil decreased fruit number and weight at harvest, but increased fruit weight. Terbacil at 75 ppm and 92% shade material were applied to whole, 3-year-old 'Redchief Delicious' trees at 18, 23, and 28 DAFB. Generally, fruit dry weight, total sugars, and reducing sugars were lowered by both shade and terbacil treatments. In most cases, fruit from shaded trees were lower in dry weight and measured nonstructural carbohydrates than fruit from terbacil-treated trees after 5 or 10 days of treatment. Shading for 5 or 10 days resulted in total fruit drop. Terbacil at 75 ppm resulted in 0.8 as opposed to 2.9 fruit per cm² LCSA on the controls at 54 DAFB. / M.S.

The theory that fruit abscission may be the result of a reduction in metabolites available to the young fruit was addressed by this study. Shade cloth or terbacil, a photosynthetic inhibitor, were applied to limbs and whole trees to examine the influence of treatment and time of application on fruit nonstructural carbohydrates and abscission. 'Stayman' apple limbs shaded with 92% shade material from 5-15, 10-20, 15-25, 20-30, and 25-35 days after full bloom (April 22) had lower fruit retention than unshaded controls on 11 June. On 18 June, fruit diameter was greater on limbs shaded between 5-25 days after full bloom (DAFB) than on unshaded limbs. At 15, 20, 25, and 30 DAFB, fruit from limbs shaded for 10 days had lower total nonstructural carbohydrates (TNC), total sugars, and reducing sugars (% dry wt) than fruit from limbs shaded for 0 or 5 days. Terbacil (3-tert-butyl-5-chloro-6-methyluracil) was applied at 0, 50, 100, and 200 ppm to whole nine-year-old 'Redchief Delicious' apple trees at 15 DAFB. Terbacil markedly inhibited Pn; recovery occurred by 9 and 26 DAA for the 50 and 100 ppm rates, respectively. Phytotoxicity prevented the determination of Pn in the 200 ppm treated trees. Fruit dry weight, TNC, total sugars, and reducing sugars (% dry wt and mg/fruit) declined with increasing rates of terbacil. Total fruit abscission was observed 12 DAA for the 100 and 200 ppm treatments, while the 0 and 50 ppm applications retained 4.6 and 1.4 fruit per cm² limb cross sectional area (LCSA) at 35 DAA, respectively. Compared to the control, 50 ppm terbacil decreased fruit number and weight at harvest, but increased fruit weight. Terbacil at 75 ppm and 92% shade material were applied to whole, 3-year-old 'Redchief Delicious' trees at 18, 23, and 28 DAFB. Generally, fruit dry weight, total sugars, and reducing sugars were lowered by both shade and terbacil treatments. In most cases, fruit from shaded trees were lower in dry weight and measured nonstructural carbohydrates than fruit from terbacil-treated trees after 5 or 10 days of treatment. Shading for 5 or 10 days resulted in total fruit drop. Terbacil at 75 ppm resulted in 0.8 as opposed to 2.9 fruit per cm² LCSA on the controls at 54 DAFB. / M.S.

No description available.

Les cellules épithéliales forment un tissu de cellules étroitement juxtaposées qui assure une barrière physique et chimique entre les compartiments internes et externes du corps. L’intégrité de ces tissus est donc essentielle. Au cours du développement et de la vie adulte, le tissu doit grandir ou se régénérer, ce qui implique de nombreuses divisions cellulaires. La dernière étape de la division, la cytodiérèse, met en jeu la formation d’un anneau contractile qui, en se fermant, va séparer les cellules sœurs. Une fois complètement fermé, il donne naissance au midbody, juste sous le niveau des jonctions adhérentes, au sein des jonctions septées, chez la drosophile. L’ultime étape, l’abscission, permet la séparation physique définitive et l’isolation cytoplasmique des cellules sœurs. Si de nombreuses études ont décrit ces processus dans les cellules isolées, peu de choses sont connues quant à la cytodiérèse des cellules épithéliales. Ce travail de thèse a permis de mettre en évidence que malgré le recrutement de tous les effecteurs et régulateurs de l’abscission, celle-ci est retardée dans les cellules épithéliales. Des expériences de photo-conversion de KAEDE ont montré que l’abscission est liée à l’entrée en mitose des cellules épithéliales. La question de l’intégrité du tissu et notamment de la barrière de perméabilité a ensuite été investigué. Nous avons montré que les cellules voisines formaient des protrusions de membrane restant connectées au midbody tout au long de sa lente migration vers le pôle basal des cellules. Les expériences de FRAP menées sur les jonctions bicellulaires et tri-cellulaires des jonctions septées ont permis de montrer que celles-ci se formaient juste sous les jonctions adhérentes et toujours au-dessus du midbody, participant ainsi à la migration de ce dernier vers le pôle basal. Les contacts maintenus avec les voisines ainsi que l’assemblage polarisé des jonctions septées participent au maintien de l’intégrité du tissu au cours des divisions de cellules épithéliales. / Epithelial cells are closely juxtaposed to form a tissue playing a physical and chemical barrier between external and internal body compartments. Thus, tissue integrity is essential. During development and adult life, epithelia has to growth and regenerate meaning a lot of divisions. At the end of cell division, cytokinesis occurs, implying the formation of a contractile ring which contracts to separate daughter cells. In Drosophila, once totally closed, the contractile ring gives rise to the midbody, just below adherens junctions, in the septate junctions layer. Last step of cytokinesis, abscission, permits the final cut and the cytoplasmic isolation of daughter cells. If cytokinesis is well described in isolated cells, little is known about epithelial cells cytokinesis. This work shows that whereas all abscission regulators and effectors are recruited, abscission is delayed in epithelial cells. KAEDE photo-conversion assays show that abscission is linked to epithelial cells mitosis entry. Then we investigate how permeability barrier is maintained during cell division. We show that neighboring cells present finger-like protrusions contacting the midbody all along the midbody is moving basally across septate junctions. FRAP experiments on bicellular and tricellular septate junctions show that they form just below adherens junctions and always above the midbody, leading to its basal migration. Contacts maintained with neighbors and polarized assembly of septate junctions participate to the maintenance of tissue integrity throughout epithelial cells divisions.

Au sein d'un organisme les cellules se divisent et assurent la croissance, la différentiation et l'homéostasie des tissus. Des travaux récents proposent qu'elles communiquent activement entre voisines au sein des organes solides pour coordonner leur propre division et la préservation de l'intégrité tissulaire. Nous proposons que la signalisation Eph-éphrine, acteur de la communication cellulaire locale, participe à cette coordination entre division cellulaire et cohésion du tissu. Au cours de ma thèse, j'ai démontré dans plusieurs modèles cellulaires que la signalisation Eph-éphrine contrôle la division cellulaire et peut induire des retards dans l'abscission et de la polyploïdie. J'ai prouvé par vidéomicrosocpie que ces défauts d'abscission dépendent du domaine catalytique du récepteur EphB2 et de l'activation de la protéine tyrosine kinase relais c-Src. En cascade, c-Src phosphoryle un régulateur clé de la stabilité du pont intercellulaire, la protéine citron kinase (CitK). J'ai également observé que CitK était anormalement localisé durant la cytocinése en aval de la voie Eph. Par des essais kinase in vitro, j'ai exclu une phosphorylation directe de CitK par le récepteur Eph et identifié c-Src comme capable de phosphoryler directement CitK. J'ai identifié les résidus tyrosines de CitK phosphorylés par c-Src, mutés deux d'entre eux et à l'aide d'analyses de sauvetage phénotypique, démontré que ces résidus étaient nécessaires et suffisants pour induire des défauts d'abscission. J'ai ensuite validé in vivo ce rôle original de la voie Eph-éphrine, dans le contexte du développement neuronal chez la souris. Plusieurs membres de la famille des Eph-éphrines sont exprimés dans les progéniteurs neuraux à l'origine des neurones corticaux et des auteurs ont montrés que CitK contrôle la cytocinèse de ces cellules. En utilisant un système Cre-lox, j'ai spécifiquement éteint la signalisation Eph dans ces progéniteurs et observé une modification de la ploïdie neuronale dans ces animaux. J'ai également observé dans les progéniteurs neuraux une co-localisation physiologique de résidus tyrosines phosphorylés et de la protéine CitK, qui adopte un enrichissement apical caractéristique. Ces résultats suggèrent notamment que la signalisation Eph-éphrine pourrait contrôler l'abscission des progéniteurs neuraux via la phosphorylation de CitK. La cytocinèse est aujourd'hui décrite comme un processus cellulaire autonome orchestré par la machinerie intracellulaire. Les résultats obtenus durant mon doctorat suggèrent que la cytocinèse est également régulée par l'environnement local de la cellule comme j'en ai fait la démonstration avec la signalisation Eph-éphrine. D'autre part, mes travaux suggèrent que la phosphorylation de CitK sert d'interrupteur moléculaire durant la progression à travers la division cellulaire et le contrôle de la ploïdie des neurones. / Cells within an organism successfully divide to ensure growth, differentiation and homeostasie. Recent work suggests that dividing cells actively communicate with neighbours thus spatially and temporally coordinating cell division while maintaining tissue cohesiveness. We hypothesized that Eph-ephrin signalling, a local cell-cell signalling pathway, could participate in coordinating cell division within a tissue. Using vertebrate and invertebrate cell culture models I showed that Eph-signalling controls cell division and induces delay in the abscission of nascent daughter cells as well as polyploidy. Using time-lapse imaging I proved that the Eph-mediated abscission failure depends on the catalytic activity of the receptor via the non receptor tyrosine kinase relay molecule c-Src. Downstream of Eph signalling c-Src phosphorylates the protein citron kinase (CitK) a well known regulator of intercellular bridge stability. I also observed that CitK was abnormally localized during cytokinesis when Eph signalling was active. Further, using in vitro kinase assays, I demonstrated that Eph does not directly phosphorylate CitK but that c-Src could do so. In addition, using Mass Spectrometry I mapped all tyrosine residues directly phosphorylated by c-Src. I mutated two of them located in the Rho binding domain of CitK and demonstrated that phosphorylation of those residues are necessary and sufficient to induce cytokinesis failure. I validated in vivo this novel role of Eph-ephrin signalling in a physiological context in the developing mouse neocortex. Members of the Eph/ephrin family are expressed in neural progenitors that give rise to neurons of the cortex upon neurogenic division. Importantly, CitK has been shown by others to control cytokinesis of these progenitor cells. Using the Cre-lox system, I specifically turned off Eph forward signalling in neural progenitor cells and observed an alteration of neuronal ploidy in these mutant animals. Further, I also observed that CitK which adopts a particular apical localisation in neural progenitors physiologically co-localized with phosphorylated tyrosine residues. Altogether, these results suggest that Eph-ephrin signalling controls abscission of neural progenitors by promoting phosphorylation of CitK. The textbook view of cytokinesis is that it is a cell autonomous event orchestrated by the intracellular machinery. Data obtained during my PhD suggest that cytokinesis is also regulated by local environment, here Eph/ephrin signalling, and that phosphorylation of CitK may represent a molecular switch in the normal progression of cell division and in the control of neuronal ploidy.

No description available.

La dernière étape de la cytocinèse, l'abscission, consiste en la coupure du pont intercellulaire reliant les deux cellules filles à la suite de la contraction de l'anneau acto-myosique. Comme toutes les étapes de la division cellulaire, l'abscission doit être régulée dans l'espace et dans le temps afin qu'elle intervienne au bon endroit et au bon moment. Mon travail de doctorat a porté sur l'étude de la régulation dans le temps de l'abscission par l'environnement des cellules filles, en particulier par les forces de traction exercées par les cellules sur le pont intercellulaire. En utilisant une combinaison d'approches permettant de contrôler le confinement spatial 2D des cellules filles, de mesurer les forces exercées par les cellules au cours de la cytocinèse et de micro-manipuler le pont intercellulaire, j'ai montré que, de façon contre-intuitive, une tension exercée au niveau du pont retardait l'abscission et qu'au contraire la relâche de cette tension induisait l'abscission. De plus, la régulation temporelle de l'abscission par les facteurs environnementaux des cellules filles implique les protéines des " Endosomal Sorting Complex Required for Transport III " (ESCRT-III), machinerie centrale de l'abscission. Enfin, des expériences préliminaires suggèrent que cette régulation serait importante pour le maintien de l'intégrité tissulaire et la morphogenèse au cours du développement.