We have developed a novel, sensitive and less time-consuming method to detect activity of the SecA-dependent protein-conducting channels. Nanogram levels of E. coli inverted membrane vesicles were injected into Xenopus oocytes, and ionic currents were recorded using the two-electrode voltage clamp. Currents were observed only in the presence of E. coli SecA in conjunction with E. coli membranes. The observed currents showed outward rectification in the presence of KCl as permeable ions and were significantly enhanced by coinjection with the precursor protein, proOmpA, or active LamB signal peptide. Channel activity was blockable with sodium azide or adenylyl 5’-(β, γ-methylene)-diphosphonate, a non-hydrolyzable ATP analog, both of which are known to inhibit SecA protein activity. Channel activity was also stimulated by oocyte endogenous precursor proteins, which could be inhibited by puromycin. In the presence of puromycin, exogenous proOmpA or LamB signal peptides, but not defective signal peptides, stimulated the ionic currents. We also measured SecA-dependent currents with membranes depleted of SecYEG. Wild-type LamB signal peptides, or precursor proteins stimulated ionic currents following a co-injection of SecYEG¯ membranes with puromycin. Excess exogenous SecA stimulated ionic currents through SecYEG¯ membranes. Similar activities of added SecA were observed with reconstituted membranes depleted of SecYEG. Currents through such SecYEG-depleted membranes were also stimulated by addition of defective LamB signal peptides and unfolded mature PhoA protein. In contrast, currents produced by the membranes containing wild-type SecYEG were not so stimulated, but ionic currents were stimulated through mutant strains, similar to PrlA (SecY) suppressors, e.g. PrlA4, or PrlA665 membranes, suggesting that the proofreading function of SecY was bypassed in these membranes. We have observed that azide can inhibit ionic currents when E. coli wild-type MC4100 membranes were injected with proOmpA or LamB signal peptides into Xenopus oocytes. However, such inhibition was lost when observed with oocyte-endogenous signal peptides in the absence of bacterial signal peptides. Moreover, azide did not show complete inhibition upon using SecYEG¯ membranes or SecYEG¯ reconstituted membranes plus excess SecA in the presence or absence of LamB signal peptides. Such conformational alterations reflect different sensitivity in response to azide during the opening of protein-conducting channels.

Embryonic epiboly has become an important developmental model for studying the mechanisms underlying collective movements of epithelial cells. In the last couple of decades, most studies of epiboly have utilized Xenopus or zebrafish as genetically tractable model organisms, while the avian epiboly model has received virtually no attention. Here, we re-visit epiboly in quail embryos and characterize several molecular markers of epithelial-to-mesenchymal transition (EMT) in the inner zone of the extraembryonic Area Opaca and at the blastoderm edge. Our results show that the intermediate filament vimentin, a widely-used marker of the mesenchymal phenotype, is strongly expressed in the edge cells compared to the cells in the inner zone, and that epiboly is inhibited when embryos are treated with Withaferin-A, a vimentin-targeting drug. Laminin, an extracellular matrix protein that is a major structural and adhesive component of the epiblast basement membrane, is notably absent from the blastoderm edge, and shows three distinct morphological regions approaching the leading edge. While these expression profiles are consistent with a mesenchymal phenotype, several other epithelial markers, including cytokeratin, β-catenin, and E-cadherin, were present in the blastoderm edge cells. Moreover, the results of a BrDU proliferation assay suggest that expansion of the edge cell population is primarily due to recruitment of cells from the inner zone, and not proliferation. Taken together, our data suggest that the edge cells of the avian blastoderm have characteristics of both epithelial and mesenchymal cells, and could serve as an in-vivo model for cancer and wound healing studies.

Thèse diffusée initialement dans le cadre d'un projet pilote des Presses de l'Université de Montréal/Centre d'édition numérique UdeM (1997-2008) avec l'autorisation de l'auteur.

In vertebrate oocytes, meiotic progression is driven by the sequential translational activation of maternal messenger RNAs stored in the cytoplasm. This activation is mainly induced by the cytoplasmic elongation of their poly(A) tails, which is mediated by the cytoplasmic polyadenylation element (CPE) present in their 3’ untranslated regions (3´ UTRs). Sequential, phase-specific translation of these maternal mRNAs is required to complete the two meiotic divisions. Although the earlier polyadenylation events in prophase I and metaphase I are driven by the CPE-binding protein 1 (CPEB1), 90% of this protein is degraded by the anaphase promoting complex in the first meiotic division. The low levels of CPEB1 during interkinesis and in metaphase II raise the question of how the cytoplasmic polyadenylation required for the second meiotic division is achieved. In this work, we demonstrate that CPEB1 activates the translation of the maternal mRNA encoding CPEB4, which, in turn, recruits the cytoplasmic poly(A) polymerase GLD2 to “late” CPE-regulated mRNAs driving the transition from metaphase I to metaphase II, and, therefore, replacing CPEB1 for “late” meiosis polyadenylation.

Thesis (Ph. D.)--University of Virginia, 2008. / Includes bibliographical references. Also available in electronic form as viewed 2/16/2009.

Thesis (Ph. D.)--University of Washington, 1998. / Vita. Includes bibliographical references (leaves [90]-109).

Thesis (Ph. D.)--University of Washington, 2005. / Vita. Includes bibliographical references (leaves 82-96).

No description available.

Historically, sperm has been considered merely as a carrier of genetic material at fertilisation. However, it is known that sperm supports embryonic development better than other cell types, suggesting that it might also have additional important, non-genetic contributions to embryonic development. The work described in this dissertation focuses on identifying the molecular determinants of developmental programming of sperm. First, the development of embryos derived from sperm and spermatids, immature precursors of sperm was compared. Sperm-derived embryos developed significantly better than spermatid-derived embryos. Further research aiming to identify the reasons for the developmental advantage of sperm led to the identification of proteins that are present specifically in sperm and not in spermatids. Moreover, egg factors which are preferentially incorporated into the sperm, but not into the spermatid chromatin were identified with the use of egg extracts, suggesting that the chromatin of sperm could be programmed to interact with the components of the egg. Subsequently, the reasons for developmental failure of spermatid-derived embryos were investigated. By comparing the sperm with spermatids it was shown that the programming of sperm to support efficient development is linked to its special ability to regulate expression of developmentally-important embryonic genes, and not to its ability to support DNA replication or rRNA production. Further characterisation of the sperm and spermatid chromatin with the use of genome-wide sequencing allowed me to link the correct regulation of gene expression in the embryo with a certain combination of epigenetic marks in the sperm, but not in the spermatid chromatin. Finally, it is shown that enzymatic removal of epigenetic modifications at fertilisation leads to misregulation of gene expression. This therefore suggests that epigenetic information contained in parental genomes at fertilisation is required for a proper regulation of embryonic transcription. My results support the hypothesis that the sperm is not only a carrier of genetic material, but also provides the embryo with epigenetic information for regulation of transcription after fertilisation. I believe that these findings advance our current understanding of the nature and mechanisms of sperm programming for embryonic development, and are important contributions to the emerging field of transgenerational inheritance of epigenetic traits in general.

Imaging Time-of-Flight secondary ion mass spectrometry (ToF-SIMS) has been developed to perform 2D imaging and depth profiling of biological systems with micron or submicron scale lateral resolution, which can be attributed to the advent of polyatomic ion beam particularly C60+ and new concept of ToF-SIMS instrument, the J105 3D Chemical Imager (J105). These recent advances in ToF-SIMS have opened a new dimension for biological analysis. In this study, 2D and 3D imaging have been performed on two biological systems, Xenopus laevis (X. laevis) zygote/embryo and murine embryonic fibroblasts NIH 3T3 BXB-ER cells to explore the capability of ToF-SIMS to handle the biological samples with extreme topography and high resolution depth profiling of microdomains, which still represent major challenges for the ToF-SIMS. The study on X. laevis embryo explored the capability of ToF-SIMS to handle spherical samples (approx. 1-1.2 mm in diameter), identify lipid species in mixtures of lipid extraction from the zygotes and image of an intact embryo in 2D/3D during dynamic biological events, e.g., fertilisation and early embryo development. For the first time the J105 and conventional BioToF-SIMS instrument were employed for the study of developmental biology. The major classes of lipid were identified through multiple lipid assay in a single analytical run using ToF-SIMS. Topography effects of the embryo were assessed through imaging a single intact zygote/embryo that revealed secondary ions loss at the edge of the single cell. However, the topography effects on the mass resolution could be minimised using the J105. Moreover, in situ lipid profiling of the zygote revealed different lipid compositions and intensities on the membrane of the animal and vegetal hemispheres. Furthermore, high resolution imaging and depth profiling that performed on a single intact cell in a time course study visualised the egg-sperm fusion sites on the membrane of the zygote 10 min post-insemination and lipids arrangement on the membrane of the embryo through the early development stages. Subcellular signalling upon the fertilisation was also spatially located on the serial cryosections of a single zygote. With the NIH 3T3 BXB-ER cells, the study firstly adopted a finely focused C60+ beam to track morphological changes and rearrangement of subcellular organelle mitochondria (0.5-2 µm) in response to the activation of Raf/ERK (extracellular signal regulated kinase) pathway using the J105. The SIMS images of the unlabelled cells showed the shifting of membrane distribution and nuclei shrinking following Raf/ERK activation. The mitochondria fluorescence probe within the cells were located 3-dimensionally using confocal microscopy and ToF-SIMS, which revealed the distribution pattern of condensing in the two sides of the nuclei following the Raf/ERK activation. Coupled with scanning electron microscopy (SEM), the three imaging modes showed good agreement in cellular morphological changes and subcellular mitochondrial rearrangement without or following Raf/ERK activation, demonstrating an integrated approaching to study the biological processes at subcellular dimension.