Formation of a tubular organ, such as the heart, requires cells to integrate positional and polarity signals in order to enclose a fluid or gas transporting lumen. Developing tubes must establish a site for lumen initiation, demarcate membrane domains, and modulate cell polarization and morphology. The Drosophila melanogaster embryonic heart is a mesodermal tube model displaying a unique polarity, reminiscent of vertebrate vasculature. We have characterized a role for the transmembrane adhesion receptor αPS3βPS1 integrin and its cytoplasmic adaptor Talin in heart tubulogenesis. βPS1 and Talin are early indicators of the luminal site and Talin-mediated integrin function is essential for cardioblast polarization and morphology prior to and during lumen development. Careful analysis of hearts in embryos homozygous for a null allele of rhea, the gene encoding Talin, reveals that Talin is required to correctly orient the heart cell polarity such that a continuous central open lumen is enclosed. Without proper integrin or Talin function, the luminal determinants Slit and its receptor Robo are not stabilized within the heart, a central lumen fails to form, and the midline is instead marked by continuous adhesion. Furthermore, although Talin is essential for proper βPS1 integrin localization within the heart, either of Talin’s two integrin binding sites are sufficient to stabilize βPS1 along the luminal domain and establish an open cardiac tube. Taken together, our findings reveal an instructive role for integrins and Talin in communicating polarization cues central to heart tubulogenesis. / Thesis / Doctor of Philosophy (PhD)

The retinal pigment epithelium (RPE) is a transporting epithelium with polarized membrane domains. A unique characteristic of these cells is that their apical surface does not face a lumenal space, but is directly apposed to a layer of neurons (photoreceptors) and their associated extracellular matrix. Because the interaction occurring at this site is important for retinal attachment and particle phagocytosis, an attempt was made to identify epithelial molecules which potentially could mediate cell-cell or cell-matrix adhesion. In the present report, the subcellular localization of β1-integrins, the main receptors for extracellular matrix ligands, has been examined within Xenopus RPE. Several previously characterized antibodies were used in this analysis including: two rabbit polyclonal antibodies directed against purified chick muscle fibronectin receptor (pAbs No. 3818 and No. 2999), and a monoclonal antibody specific for Xenopus β1-integrin subunit (mAb 8C8). In Western blots of whole epithelial cell extracts, each of the antibodies intensely labeled a 115 kDa band, consistent with β1-integrin reactivity. One of the reagents (pAb No. 3818) also weakly stained unidentified bands of 50 and 100 kDa. Pre-clearing experiments demonstrated that pAb No. 3818 and mAb 8C8 both recognize the same detergent-soluble integrin: when cell extracts were depleted of β1-integrin by immunoprecipitation with mAb 8C8, the 115 kDa antigen recognized by pAb No. 3818 was not observed. Consistent with their similar immunochemical reactivities, each of the antibodies produced equivalent immunocytochemical staining of many eyecup tissues, including extraocular skeletal muscle cells, scleral and choroidal fibroblasts and vascular endothelium of the choroid and neural retina. In the native RPE, and isolated sheets of epithelium, however, qualitative differences in labeling between these antibodies were evident. Analysis by confocal microscopy showed that, while all three antibodies stained the basal surface of the epithelium, pAb No. 3818 also strongly labeled the apical microvillar surface. As the adjacent photoreceptors did not cross-react with this antibody in control experiments, the apical RPE staining could not be accounted for as contamination with retinal tissues during isolation. Furthermore, when the apical cell surface was selectively biotinylated in situ, and biotinylated proteins precipitated by streptavidin-agarose, β1-integrin was detected by immunoblotting with both mAb 8C8 and pAb No. 3818. This domain-specific material, however, represented only a fraction of the whole cell surface integrin: substantially greater amounts of tagged molecules could be detected when isolated epithelial sheets were biotinylated, most likely representing the basal protein. Based on these results, it can be concluded that β1-integrin is present in both basal and apical RPE plasma membranes. Molecules present in the apical, membrane may represent components of adhesion receptors responsible for retina-epithelium interactions.

No description available.

<p> This thesis describes a study of the photosolvolysis of N-benzoyl-5-bromo-7-nitroindoline. The investigation was instigated because N-acyl-5-bromo-7-nitroindolines have been considered as potential photoprotecting groups for the photolithographic formation of charge-mosaic membranes. However, no kinetic data, and only scant mechanistic information, has been reported for the photofragmentation process.</p> <p> Both absorption and emission spectra of N-benzoyl-5-bromo-7-nitroindoline were obtained. Based on the electronic spectra, it would seem that the two lowest energy singlet excited states are nearly isoenergetic and their relative energies are a function of the solvent medium.</p> <p> The distribution of photoproducts obtained from irradiation of N-benzoyl-5-bromo-7-nitroindoline in various solvent systems was investigated. It was shown that the photosolvolysis reaction is a function of the solvent properties. Thus 5-bromo-7-nitroindoline and benzoic acid were the only photoproducts formed in acetonitrile. In acetonitrile-water, 5-bromo-7-nitrosoindole as well as 5-bromo-7-nitroindoline and benzoic acid were identified. A correlation between solvent polarity, ET(30), and 5-bromo-7-nitrosoindole formation was observed.</p> <p> The mechanism of the fragmentation mode was examined. The presence of a free radical intermediate is suggested from the photoproduct distribution in tetrahydrofuran (THF). Observation of an electron spin resonance (e.s.r.) spectrum during irradiation of N-benzoyl-5-bromo-7-nitroindoline in an e.s.r. spectrometer cavity, 18_0 incorporation into benzoic acid, and a linear free energy relationship with sigma dot (σ•) corroborated the intermediacy of a free radical in the fragmentation process.</p> <p> Quantum yields of N-benzoyl-5-bromo-7-nitroindoline disappearance in different solvents and quenchers were measured in order to establish the reactive excited state and the effect of the medium on the efficiency of the transformation process. A solvent dependency on both the excited state lifetime and photosolvolysis reaction efficiency was noted. The results were indicative of a change in mechanism with a variation in solvent polarity and/or hydrogen bonding ability.</p> <p> A mechanism compatible with experimental results and applicable to previously reported N-acyl-2-nitroaniline photofragmentations is proposed.</p> / Thesis / Doctor of Philosophy (PhD)

No description available.

No description available.

Establishment and maintenacne of a polarized axis is essential for all organisms. Cells can either change their shape in response to extracellular cues or maintain a stable polarity axis via landmarks defined in relation to internal cues. In the fission yeast Schizosaccharomyces pombe,microtubules regulate cortical cell polarity together with the landmark protein Tea1. Tea1 is transported to cell tips on microtubule plus-­‐ends and deposited upon microtubule contact with the membrane. Although Tea1 has been shown to interact with several binding-­partners, Tea1 anchoring at the cell tip depends mostly on the membrane-­associated protein, Mod5. Tea1 and Mod5 accumulate in clusters at the cell tip in a mutually dependent manner. I used a combination of live-­‐cell imaging, FRAP (Fluorescence Recovery After Photobleaching) and computational modeling to dissect the dynamics of the Tea1-­‐Mod5 interaction. I have shown that although Tea1 is stably associated with the cell tip, Mod5 is mobile within the cell tip. I proposed a model in which Tea1 is stable at the cell tip due to self-­‐polymerization and association in the form of a cluster-­‐network. In the model, the role of Mod5 in the cluster-­‐network is to facilitate the formation of Tea1-­‐Tea1 interactions. Moreover, in the model, Mod5 is restricted to the cell tip due to iterative binding to and release from the Tea1 cluster-­‐network. The properties of the proposed Tea1 cluster-­‐ network might contribute to the behavior of Tea1 as a polarity landmark. I hypothesized that Tea1 transfer from the microtubules to the cell tip was regulated by phosphorylation. Tea1 phosphorylated residues were mapped using mass spectroscopy (MS), and identified to be mostly enriched within a central region of the protein. Using a combination of mutagenic analysis and live-­‐cell imaging I demonstrate that Tea1 phosphorylation might be required for its dissociation from the cluster-­‐network at the cell tip. This suggests that Tea1 interactions within the cluster network are phospho-­‐regulated by one of the several tip-­‐localized kinases. It has been shown in other organisms and in this thesis that comparison among MS samples requires quantitative MS methodologies. Thus, I developed a robust SILAC (Stable Isotope Labeling in Cell Culture) method to perform quantitative MS in S. pombe. As a proof-­‐of-­‐principle of the method I performed a proteome-­‐wide comparison between the late G2 and the G1/S transition of the cell cycle. The cell cycle proteome-­‐wide analysis not only quantified variation in expression levels of cell cycle regulated proteins but also identified novel cell cycle regulated proteins. It has been previously shown that Tea1, Tea3 and Mod5 can interact simultaneously, with each pair interacting independently of the third protein. I describe here a Mod5 mutagenic analysis screen designed to separate Tea1 and Tea3 binding site on Mod5. The Mod5-­‐mutants obtained from this analysis indicate that the Tea3-­‐Mod5 interaction may play a role in cell polarity establishment. Moreover, although Tea3 is non-­‐essential for the cluster-­‐network formation, Tea3 might be important for its compaction, which may be particularly important during de novo formation of cell polarity.

Planar cell polarity (PCP) is the organization of cellular characteristics within the plane of a tissue. PCP manifests both structurally, as in the directionality of insect bristles or mammalian skin hair, or dynamically, as in vertebrate neurulation, gastrulation, and oriented cell division in the kidney. Two well-conserved pathways are known to regulate PCP in invertebrates and in vertebrates: the Frizzled/PCP pathway and the Fat-Dachsous (Ft-Ds) pathway. The latter consists of the cadherins Ft and Ds, along with the Golgi kinase Four-jointed (Fj) and the transcriptional co-repressor Atrophin (Atro). Ft and Ds can bind each other, suggesting a mechanism for signal transduction. Fj phosphorylates Ft and Ds, modulating their binding affinities for each other. Atro is proposed to link Ft-Ds signaling with downstream events in the nucleus during eye development. The details of Ft-Ds binding, and the consequences of their interactions with other members of the pathway are poorly understood. In this work, I quantitatively analyzed Ft-Ds pathway mutant clones for their effects on ommatidial polarity in the Drosophila eye. My findings suggest that the Ft-Ds pathway regulates PCP independently of asymmetric cellular accumulation of Ft or Ds. I found that Atro has a position-specific role in regulating polarity in the eye, that Fj dampens clonal polarity signals, and that asymmetric accumulation of the atypical myosin Dachs is not essential for production and propagation of a long-range PCP signal. My observations suggest that Ft and Ds interact to modulate a secondary signal that regulates long-range polarity, that signaling by the Ds intracellular domain is dependent on Ft, and that ommatidial fate specification is genetically separable from long-range signaling.

Planar cell polarity (PCP) is the organization of cellular characteristics within the plane of a tissue. PCP manifests both structurally, as in the directionality of insect bristles or mammalian skin hair, or dynamically, as in vertebrate neurulation, gastrulation, and oriented cell division in the kidney. Two well-conserved pathways are known to regulate PCP in invertebrates and in vertebrates: the Frizzled/PCP pathway and the Fat-Dachsous (Ft-Ds) pathway. The latter consists of the cadherins Ft and Ds, along with the Golgi kinase Four-jointed (Fj) and the transcriptional co-repressor Atrophin (Atro). Ft and Ds can bind each other, suggesting a mechanism for signal transduction. Fj phosphorylates Ft and Ds, modulating their binding affinities for each other. Atro is proposed to link Ft-Ds signaling with downstream events in the nucleus during eye development. The details of Ft-Ds binding, and the consequences of their interactions with other members of the pathway are poorly understood. In this work, I quantitatively analyzed Ft-Ds pathway mutant clones for their effects on ommatidial polarity in the Drosophila eye. My findings suggest that the Ft-Ds pathway regulates PCP independently of asymmetric cellular accumulation of Ft or Ds. I found that Atro has a position-specific role in regulating polarity in the eye, that Fj dampens clonal polarity signals, and that asymmetric accumulation of the atypical myosin Dachs is not essential for production and propagation of a long-range PCP signal. My observations suggest that Ft and Ds interact to modulate a secondary signal that regulates long-range polarity, that signaling by the Ds intracellular domain is dependent on Ft, and that ommatidial fate specification is genetically separable from long-range signaling.

Les tissus épithéliaux sont des couches de cellules adhérentes qui servent de barrières entre différents compartiments morphologiques et procurent un transport directionnel de molécules. L’action coopérative de plusieurs déterminants de la polarité gouverne l’identité et la morphogenèse spécifiques de ces domaines : 1) le cytosquelette d’actomyosine, 2) les jonctions adhérentes (AJs) basées sur la E-cadhérine et 3) les complexes de polarité conservés au cours de l’évolution. Une perte de l’adhérence via la DE-cadhérine (DE-Cad) conduit à des défauts de polarité apico-basale, tandis que la localisation apicale de DE-Cad nécessite les protéines de polarité Crumbs (Crb) et Bazooka (Baz) (L’homologue de Par3 chez la mouche). Notablement, DE-Cad forme des amas qui co-localisent partiellement avec les amas de Baz, génèrent l’adhésion intercellulaire et transmettent la tension. Les mécanismes impliqués dans le contrôle de la taille, le nombre, la répartition et la dynamique des amas de DE-Cad restent peu connus.J’ai étudié le rôle de Crumbs et Baz dans la régulation de la distribution fine de DE-Cad. J’ai montré que Crb contrôle la distribution macroscopique de DE-Cad, au moins, partiellement via Baz. En générant des mutations de Baz sur des sites régulateurs variés grâce à de la transgenèse spécifique de site et en utilisant de la microscopie en temps réel quantitative, j’ai montré que Crb agit via le domaine d’oligomérisation CR1 et le site Ser980 de Baz afin d’ajuster les niveaux de DE-Cad. Remarquablement, j’ai aussi révélé que le domaine d’oligomérisation de Baz est inutile à la formation d’amas Baz-DE-Cad et j’ai caractérisé la réciprocité de l’interaction DE-Cad-Baz. / Epithelia are sheets of adherent cells that serve as barriers between distinct morphological compartments and provide directed transport of molecules.. The cooperative action of several polarity determinants governs the proper identity and morphogenesis of these domains: 1) actomyosin cytoskeleton; 2) E-Cadherin-based adherens junctions (AJs) and 3) evolutionarily conserved polarity complexes.A loss of DE-cadherin (DE-Cad) adhesion leads to apico-basal polarity defects, while the apical localization of DE-Cad requires the polarity proteins Crumbs (Crb) and Bazooka (Baz) (Par3 homolog in fly). Notably, DE-Cad builds clusters that display a certain degree of colocalization with the clusters of Baz, provide intercellular adhesion and transmit tension.I have addressed the role of Crumbs and Baz in the regulation of DE-Cad fine distribution. I demonstrated that Crb controls DE-cad macroscopic distribution, at least, partially via Baz. By generating Baz mutants on various regulatory sites using site-specific transgenesis and quantitative live-imaging microscopy, I showed that Crb acts via CR1 oligomerization domain and Ser980 site of Baz to adjust DE-Cad levels. I also revealed that Baz oligomerization domain is dispensable for Baz-DE-Cad clusters formation and characterized the reciprocity of DE-Cad-Baz crosstalk.