Little is known about the molecular components and interactions of the planar cell polarity pathway that regulate neuronal polarity. This study uses a prkl-1 induced backwards locomotion defect as an array to perform a prkl-1 suppressor screen in C. elegans looking for new components of the planar cell polarity pathway involved in the neuronal polarization of VC4 and VC5. The screen discovered twelve new alleles of vang-1, one new allele of fntb-1 and five new mutations in unknown polarity genes. fntb-1 encodes for the worm ortholog of Farnesyltransferase β-subunit and is important for neuronal polarization. Acting cell and non-cell autonomously, fntb-1 regulates the function and localization of prkl-1 through the recognition of a CAAX motif. Therefore, fntb-1 modifies prkl-1 to regulate the neuronal polarity of VC4 and VC5.

In the field of human factors engineering the issue of how to present electronic text to people has been studied intensely for over 35 years. However, one major consideration that has largely been overlooked in these studies is how visual impairments affect reading of computer text. Specifically, the issue of how text can be modified to improve readability of CRTs for individuals with low vision. A 2x5x2x3 (visual capability, font size, polarity, and contrast) mixed-factor, repeated-measures experimental design was used to determine if changes in font size, contrast polarity, and/or contrast can improve reading speeds and reduce error rate for people with low vision. The results of this experiment show that alterations in text can be made that do not affect unimpaired vision readers while dramatically improving the reading capabilities of the impaired vision population. For character size, 12 and 14 point font sizes were found to be too small for the visually impaired population examined. In general, 18 and 30 point font sizes were equal to each other and to the 24 point font size, but for some interactions these two were found to produce longer response times and higher error rates. Thus, a 24 point font size is recommended. Unlike previous research with visually impaired participants, this experiment found that negative (white-on-black) polarity worsened reading performance. It is thought that this discrepancy is a result of polarity's interaction with small font sizes. For this reason, it is recommended that for font sizes of 18 points and below, positive polarity should be used. For 24 and 30 point sizes either polarity is satisfactory, though previous research (Legge, Pelli, Rubin, and Schleske, 1985b; NRC, 1995; Rubin and Legge, 1989) suggests negative polarity might be better for some visually impaired readers.. Contrasts of 3:1, 7:1, and 18:1 were used in this experiment and had no significant effect for either vision group. However, contrast did significantly interact with both font size and polarity. For font sizes of 18 points or below, it is recommended that contrasts of 18:1 be used for either polarity, but this is very important if negative polarity is used. The above recommendations are based on a small group of impaired vision readers. Visual impairments vary widely and the sample used in this experiment represented only a portion of them, with respect to both cause and severity. Wherever possible, computer text should be tailored to the unique needs of its users. / Master of Science

International Telemetering Conference Proceedings / October 18-21, 2004 / Town & Country Resort, San Diego, California / This paper presents an alternative approach to high-precision bearing estimation for navigation and guidance in homing and docking of underwater vehicles. This new technique is significantly simpler than the conventional methods in terms of computation complexity and yet produces results of superior precision and consistency.

A cell's composition is not uniform, but is comprised of many molecular gradients to compartmentalize functions into specialized subcellular domains. This organization is called polarity–the asymmetry of morphology and composition. Though it's a feature of nearly all prokaryotic and eukaryotic cells, polarity is plastic and highly dynamic, and is continuously instructed by the crosstalk between extracellular cues and internal effector pathways. One of the master regulators of polarity is the Par complex, canonically comprised of Cdc42, Par6, Par3 and atypical protein kinase C (aPKC). The Par complex defines the apical domain of epithelia and the neuronal axon, directs cell migration and the assembly of cell junctions, and restricts other polarity complexes to their respective domains. We have identified a novel polarity protein that counteracts the activities of the Par complex in cells. PKMζ, a truncated isoform of aPKC normally found in neurons, competes with full-length aPKC for substrate interactions. This competition results in the disruption of the canonical Par complex and its instruction of cell polarity, manifesting as a block in axon specification in developing neurons, or as a loss of the apical-basal axis of epithelial polarity. By eliminating PKMζ's ability to compete with aPKC for interaction with Par3, the effect on polarity is mitigated, while RNAi-mediated reduction of Par3 levels similarly rescues PKMζ-associated defects. We further report that PKMζ is aberrantly transcribed in certain epithelial cancers, and its expression correlates with grade. Malignant epithelial phenotypes are driven by PKMζ's Par3-dependent disruption of polarity, and its Par3- independent promotion of anoikis resistance. We demonstrate that PKMζ, as the catalytic fragment of aPKC, is surprisingly competent to influence polarity independently of its kinase activity, while other aPKC isoforms require their catalytic function to permit apical development. Together, this body of work presents PKMζ as an endogenous inhibitor of Par complex function, whose presence provides bistability to the dynamics of symmetry-breaking.

The correct polarity of a neuron underlies its ability to integrate precise circuitries in the nervous system. The goal of my thesis was to investigate the pathways that establish and maintain neuron polarity/orientation in vivo. To accomplish this, I used bipolar VC4/5 motor neurons, which innervate the C. elegans egg-laying musculature, as a model system. Vulval proximal VC4/5 neurons extend axons in the left-right (LR) orientation, around the vulva; whereas vulval distal VC1-3,6 neurons extend axons along the anterior-posterior (AP) axis. A previous study showed that vang-1, a core planar cell polarity (PCP) gene, suppresses AP axon growth in VC4/5 neurons. In order to identify new components of this pathway we performed genetic screens for mutants with abnormal VC4/5 polarity/morphology. We isolated and mapped alleles of farnesyl transferase b (fntb-1) and of core PCP genes, prickle- 1 (prkl-1) and dishevelled-1 (dsh-1); all of which display tripolar VC4/5 neurons, similar to vang-1 lof. In prkl-1 and dsh-1 mutants, primary LR and ectopic AP VC4/5 axons are born simultaneously, suggesting an early role in establishing polarity. In addition, prkl-1 and dsh-1 act persistently to maintain neuron morphology/orientation. Genetic analysis of double mutants suggests that prkl-1 interacts with vang-1 in a common PCP pathway to prevent AP axon growth, while dsh-1 also acts in a parallel pathway. Furthermore, prkl-1 functions cell autonomously in neurons, whereas dsh-1 acts both cell autonomously and cell nonautonomously in epithelial cells. Notably, prkl-1 overexpression results in unipolar VC4/5 neurons, in a dose-dependent manner. In contrast, dsh-1 overexpression in VC4/5 neurons results in a lof phenotype, similar to vang-1 lof and overexpression phenotype. Remarkably, prkl-1 overexpression restores normal VC4/5 polarity in dsh-1 and vang-1 mutants, which is suggestive of a downstream role for prkl-1. Both PRKL-1 and DSH-1 are expressed in iii uniformly distributed puncta at the plasma membrane of VC4/5, similar to VANG-1; suggesting that their asymmetric distribution is not critical for neuron polarity. Furthermore, we found that the vulva epithelium induces prkl-1 expression in VC4/5; indicating a functional relationship between the egg-laying organ and neuron morphology. Moreover, a structure-function analysis of PRKL-1 revealed that the conserved PET domain and the Cterminal region are crucial to prevent AP axon growth, whereas the three LIM domains are dispensable for this role. In addition, we showed that dsh-1 also regulates the morphology of AP-oriented PDE neurons. dsh-1 promotes the formation of PDE posterior axons, contrary to its function in VC5 neurons; which indicates a context-dependent role for dsh-1 in neuronal polarity. Altogether, this thesis implicates the PCP signalling pathway in a previously unknown role, in establishing and maintaining neuronal polarity, by controlling AP axon growth in response to organ-derived polarizing cues.

We have previously demonstrated that Llgl1 loss results in a gain of mesenchymal phenotypes and a loss of apicobasal and planar polarity. We now demonstrate that these changes represent a fundamental shift in cellular phenotype. Llgl1 regulates the expression of multiple cell identity markers, including CD44, CD49f, and CD24, and the nuclear translocation of TAZ and Slug. Cells lacking Llgl1 form mammospheres, where survival and transplantability is dependent upon the Epidermal Growth Factor Receptor (EGFR). Additionally, Llgl1 loss allows cells to grow in soft-agar and maintain prolonged survival as orthotopic transplants in NOD-SCID mice. Lineage tracing and wound healing experiments demonstrate that mammosphere survival is due to enhanced EGF-dependent migration. The loss of Llgl1 drives EGFR mislocalization and an EGFR mislocalization point mutation (P667A) drives these same phenotypes, including activation of AKT and TAZ nuclear translocation. Together, these data indicate that the loss of Llgl1 results in EGFR mislocalization, promoting pre-neoplastic changes.

<p>Organelle transport in eukaryotic cells is regulated by a precisely coordinated activity of phosphoinositide lipids, small GTPases, and molecular motors. Despite the extensive study of functional activities of individual regulators, how these activities promote precise deliveries of particular membrane proteins to specific cellular locations remained unclear. Ankyrin-B, which is previously well recognized as a plasma membrane adaptor that assembles diverse specialized plasma membrane domains, exhibited an unexpected role in intracellular transport. This thesis establishes ankyrin-B as a master integrator of the polarized long range organelle transport via direct interactions with Rab GTPase Activating Protein 1 Like (RabGAP1L), phosphatidylinositol 3-phosphate (PI3P) and dynactin 4. In Chapter 2, I identified an ankyrin-B death domain binding partner, RabGAP1L, that specifically interacts with ankyrin-B on intracellular organelles and requires ankyrin-B for its proper localization. In Chapter 3, I demonstrated that ankyrin-B is a PI3P-effector in mouse embryonic fibroblasts (MEFs) and promotes the polarized transport of associated organelles in migrating cells in a RabGAP1L-dependent manner. I continued to investigate what membranes/membrane-associated proteins utilize the ankyrin-B/RabGAP1L pathway in Chapter 4 and identified α5β1-integrin as a cargo whose polarized transport and recycling are ankyrin-B-dependent. I further presented that ankyrin-B, through recruiting RabGAP1L to PI3P-positive/Rab22A-associated endosomes containing α5β1-integrin, promotes polarized recycling of α5β1-integrin in migrating mouse embryonic fibroblasts. In collaboration with James Bear (UNC Chapel Hill), we further demonstrated that this ankyrin-B/RabGAP1L-mediated pathway is required for haptotaxis along fibronectin gradients. In Chapter 5, I elucidated the in vivo interaction between ankyrin-B and RabGAP1L. I demonstrated that ankyrin-B specifically interacts with RabGAP1L at long axon tracts in the brain and at costameres in the skeletal muscle. I also show the phenotypic analysis of ankyrin-B floxDD mice as an initial attempt to determine the physiological function of the ankyrin-B death domain in vivo. Together, this thesis dissects an ankyrin-B-mediated molecular mechanism for polarized endosomal trafficking and α5β1-integrin recycling during directional cell migration, and provides new insights into how phosphoinositide lipids, Rab GTPases, and molecular motor activities are coordinated to control the directional transport of specialized membrane cargos.</p> / Dissertation

La polarité cellulaire, organisation asymétrique du matériel cellulaire dans l'espace et le temps, est fréquemment observée en biologie. Elle est nécessaire pour de nombreux mécanismes cellulaires essentiels allant de la division cellulaire et la migration au développement et la croissance polarisée. Comprendre comment la cellule génère et maintient cette polarité est crucial, les défauts de polarité étant liés à des maladies graves comme le cancer ou les maladies neurodégénératives. Chez la levure Saccharomyces cerevisiae, la polarité cellulaire est établie lorsque le module de la Rho GTPase Cdc42, qui comprend le facteur d'échange de nucléotide guanine (GEF) Cdc24 et la protéine scaffold Bem1, localise à un unique site à la membrane plasmique pour activer Cdc42 et ainsi, établir un axe de polarité utilisé pour la croissance et la division cellulaire. Les mécanismes responsables de l'activation de Cdc42 à un site unique au cortex pendant l'établissement de la polarité sont essentiels mais largement inconnus. En utilisant des expériences complémentaires d'imagerie in vivo et des expériences in vitro, je mis en évidence que le ciblage avide du module de Cdc42 à la membrane plasmique implique des interactions multivalentes entre des lipides anioniques et le module de Cdc42. En détail, j'ai démontré que la combinaison de plusieurs phospholipides anioniques, comprenant PS, PI4P et PI(4,5)P2, est nécessaire à la localisation de Bem1 et Cdc24 in vivo. J'ai identifié des groupements cationiques interagissant avec des lipides (CLICs) dans l'extrémité N-terminale de Bem1 qui étaient nécessaires et suffisants pour interagir avec des phospholipides anioniques. Réduire l’interaction de Bem1 avec les lipides en mutant la séquence CLICs a fortement diminué la localisation de Bem1 au niveau du cortex ainsi que la signalisation de Cdc42. En plus des CLICs de Bem1, le domaine PX de Bem1 et le domaine PH de Cdc24 augmentent davantage l'avidité du module GTPase pour les lipides anioniques et la combinaison des trois domaines est essentielle pour l'établissement de la polarité cellulaire. Ces résultats définissent pour la première fois le mécanisme de ciblage avide des activateurs de Cdc42 à la membrane plasmique pendant l'établissement de l'axe de polarité. / Cell polarity, the asymmetric organization of cell material in space and time, is frequently observed in biology. It is required for numerous essential cellular processes ranging from cell division and migration to development and polarized growth. Addressing how cells generate and maintain polarity is crucial, since defects in polarity are linked to severe diseases including cancer and neurodegeneration. In the budding yeast Saccharomyces cerevisiae, cell polarity is established when the Cdc42 Rho GTPase module, which includes the Guanine nucleotide Exchange Factor (GEF) Cdc24 and the scaffold protein Bem1, accumulate at a unique site on the plasma membrane to activate Cdc42 and establish the polarity axis used for cell growth and division. The mechanisms responsible for the site-specific activation of Cdc42 at the cortex during polarity establishment are essential but are largely unknown. Using complementary in vivo imaging and in vitro experiments, I found that the avid targeting of the Cdc42 GTPase module to the plasma membrane involves multivalent anionic lipid-Cdc42 module interactions. I found that a combination of anionic phospholipids, including PS, PI4P and PI(4,5)P2, are necessary for Bem1 and Cdc24 localization in vivo. I identified Cationic-enriched Lipid Interacting Clusters (CLICs) in the N-terminus of Bem1 that were necessary and sufficient for anionic phospholipid interactions. Reducing Bem1 lipid binding by mutating the CLICs strongly diminished the localization of Bem1 at the cortex and Cdc42 signaling. In addition to the Bem1 CLICs, the Bem1 PX domain and the Cdc24 PH domain increased the avidity of the GTPase module for anionic lipids, and a combination of all three domains was essential for the establishment of cell polarity. The results of my thesis define a mechanism of avid targeting of Cdc42 activators to the cortex during polarity axis establishment.

Thesis advisor: David R. Burgess / Polarity established by the first cleavages in sea urchin embryos was investigated in this thesis revealing precocious embryonic polarity. Studies of embryonic polarity have focused on protostomes such as <italics>C. elegans</italics>, and those on deuterostomes have focused on later developmental stages. I find asymmetries in the sea urchin membrane cell cortex as early as the first division after fertilization as a result of new membrane addition in the cleavage furrow. Membrane domains and the polarity determinants Par6, aPKC, and Cdc42 are polarized to the apical, or free, cell surface, while the cell-cell contact site remains distinct. Using immunofluorescence, fluorescence recovery after photobleaching (FRAP), and specific inhibitor treatments, myosin filaments were identified as the major regulator of membrane cortex polarity. However, membrane domains and cortical polarity determinants are differentially regulated with respect to blastomere dissociation. These asymmetries are required for proper spindle alignment and cleavage plane determination and are responsible for polarized fluid phase endocytosis. The work in this thesis and future studies addressing the connection between the membrane cortex and myosin filaments has and will lead to a greater understanding of the maintenance of embryonic polarity in cleavage stage sea urchin embryos. / Thesis (PhD) — Boston College, 2009. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

Understanding the establishment of polarity and the cell fate specification of epithelial cells is important for developmental biology, regenerative medicine and the study of cancer. In this thesis, models of pre-implantation epithelial development are used to investigate the relationship between these two processes. The trophoblast is an extraembryonic epithelial tissue which contributes to the placenta. Addition of BMP4 to mouse and human embryonic stem (mES) cells grown in culture has been suggested to induce differentiation of cells to the trophoblast lineage. The use of this differentiation method was investigated as a possible model of trophoblast polarisation and cell fate specification. Unfortunately, with the protocol and reagents available this model did not appear to physiologically recapitulate trophoblast development and was not reliable. The primitive endoderm is an epithelium which arises from the inner cell mass during mammalian pre-implantation development. It faces the blastocoel cavity and later gives rise to the extraembryonic parietal and visceral endoderm. When mES cells are grown in suspension they form aggregates of differentiating cells known as embryoid bodies. The outermost cell layer of an embryoid body is an epithelial cell type comparable to the primitive endoderm. Embryoid bodies were used here to study the polarisation and cell fate specification of the primitive endoderm. The outer cells of these embryoid bodies were found to gradually acquire the hallmarks of polarised epithelial cells and express markers of primitive endoderm cell fate. The acquisition of epithelial polarity occurred prior to the maximal expression of cell fate markers. Fgfr/Erk signalling is known to be required for specification of the primitive endoderm, but its role in polarisation of this tissue is less well understood. To investigate the function of this pathway in the primitive endoderm, embryoid bodies were cultured in the presence of a small molecule inhibitor of Mek. This inhibitor caused a loss of expression of markers of primitive endoderm cell fate and maintenance of the pluripotency marker Nanog. In addition, a mislocalisation of apico-basolateral markers and disruption of the epithelial barrier which normally blocks free diffusion across the epithelial cell layer occurred. Two inhibitors of the Fgf receptor elicited similar phenotypes, suggesting that Fgf receptor signalling promotes Erkmediated polarisation. This data shows that the formation of a polarised primitive endoderm layer in embryoid bodies requires the Fgfr/Erk signalling pathway.