Thesis (Ph. D.)--Cornell University, August, 2008. / Vita. Includes bibliographical references (leaves 126-141).

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The circadian system in mammals is arranged as a hierarchical network of oscillators, with the master pacemaker of circadian rhythms located in the suprachiasmatic nuclei (SCN) of the hypothalamus and peripheral oscillators in most other organ and tissue systems of the body. The molecular machinery responsible for generating circadian rhythms is composed of interlocked transcriptional-translational feedback loops with the gene Brain Muscle Arnt-like 1 (Bmal1) functioning as a core positive regulator. Using the mouse, Mus musculus as a model system, we studied the post-transcriptional mechanisms regulating Bmal1 expression in the SCN pacemaker and in peripheral oscillators. Target prediction algorithms were used to identify microRNAs (miRNAs) predicted to target Bmal1. We profiled the temporal expression of miR-142-3p in the mouse SCN in vivo and in an immortalized SCN cell line and observed robust circadian rhythms in its expression in the SCN. Following luciferase-reporter and site-directed mutagenesis analyses, we identified miR-142-3p as a bona-fide post-transcriptional repressor of Bmal1. The temporal expression of potential Bmal1-targeting miRNAs was also examined in the circulation in mouse serum. In mice housed in a light-dark cycle, diurnal oscillations were observed in serum levels of miR-152 and miR-494, but not miR-142-3p expression. Luciferase reporter studies indicated that miR-494, both independently and synergistically with miR-142-3p, repressed the Bmal1 3′ UTR. Overexpression of these miRNAs disrupted ensemble circadian rhythms of PER2::LUCIFERASE activity in cultured fibroblasts. Overexpression of the miRNAs also increased their extracellular levels and their intracellular accumulation in recipient cultures exposed to conditioned medium. Furthermore, inhibition of exocytosis and endocytosis affected ensemble circadian rhythms in cultured fibroblasts. The results thus implicate miR-142-3p and miR-494 in the regulation of Bmal1 expression in the SCN and peripheral oscillators and suggest that miRNAs may function as both, intracellular and extracellular (cis- and trans- acting) signals, modulating the core clock mechanism in the SCN and in fine-tuning the synchronization of circadian rhythmicity between cell-autonomous oscillators in the periphery.

Intercellular signalling is essential for multicellular organisms to coordinate growth and development, and is mediated by a huge variety of proteins. Some signalling pathways rely on the proteolytic cleavage of membrane proteins by a relatively newly discovered process of regulated intramembrane proteolysis (RIP), the cleavage of proteins within a transmembrane domain. There are four classes of intramembrane cleaving proteases (ICliPs) – Rhomboids, Site-2-proteases, Signal peptide peptidases and γ-secretase. Of all the ICliPs studied to date, γ-secretase is unique, as it is comprised of a four-protein complex, and is only found in multicellular organisms. A vast amount of research is carried out on the γ-secretase complex, not just because of its role in developmentally important pathways, such as NOTCH signalling, but also due to its role in Alzheimer’s disease. The β-amyloid precursor protein (APP) is cleaved by γ-secretase, and defects in this process result in the release of abnormal peptides that form the senile plaques in the brains of Alzheimer’s disease patients. Homologues of the four components of γ-secretase (PRESENILIN (PS), NICASTRIN (NCT), ANTERIOR PHARYNX DEFECTIVE-1 (APH-1) and PRESENILIN ENHANCER-2 (PEN-2)) are found in plants. The aim of this thesis was to characterise the potential γ-secretase components in Arabidopsis thaliana, to determine whether they form a complex, and to analyse what role, if any, they play in plant signalling. The members of the putative Arabidopsis γ-secretase complex (AtPS1 and 2, AtNCT, AtAPH1 and AtPEN2) were identified through BLAST searches, and found to be uniformly expressed. Analysis of T-DNA insertion mutants in each of these genes, and combinations there of, revealed no gross morphological differences to wild type under normal growth conditions and when subjected to a range of stresses. Protein fusions to GFP under the control of the 35S promoter were constructed and stably transformed into plants. AtPEN2:GFP is expressed throughout the plant, and accumulates in BFA sensitive Golgi bodies in roots. AtPS1:GFP, only accumulates strongly in developing seeds. Native blue PAGE was used to look for high molecular weight complexes (HMW) containing AtPEN2:GFP and AtPS1:GFP. Both fusion proteins were found in similar sized HMW complexes. A variety of methods were used to look for substrates of the iv putative γ-secretase complex in Arabidopsis, and although no specific substrates were identified, a potential role in seed development has been established.

Les plasmodesmes sont des nanopores membranaires qui traversent la paroi des cellules végétales. Ces nanostructures jouent un rôle central en communication intercellulaire et agissent comme des centres de signalisation capables de générer et de relayer le signal de cellule à cellule via l’activité de récepteurs. En tant qu’éléments clés de la communication intercellulaire, les plasmodesmes coordonnent les processus liés à la croissance et au développement des plantes ainsi que les réponses aux stress environnementaux. Dans cette étude, nous avons identifié trois récepteurs de la famille des récepteurs riches en leucine (LRRRLK), capables de relocaliser de manière dynamique au niveau des plasmodesmes après un stress abiotique. L'association plasmodesmale est rapide et survient en moins de 2 minutes. Cette association dynamique n’est pas un comportement général des protéines associées à la membrane plasmique ou aux microdomaines membranaires. En focalisant notre étude sur SAK1 (Sucrose Activated Kinase) nous avons démontré que l'association dynamique aux plasmodesmes est indépendante de la composition en stérols ou en sphingolipides et est partiellement dépendante du statut de phosphorylation de cette protéine. Nous avons identifié un strech d'acide aminé polybasique dans le domaine Juxtamembrane (JMD) de SAK1, décrit chez l’homme comme interagissant avec les lipides anioniques, qui est indispensable à l'association conditionnelle aux plasmodesmes. Au total, nos données indiquent que les changements dans la membrane signature moléculaire des domaines spécialisés plasmodesmes accompagne les réponses aux stimuli externes. / Plasmodesmata pores sustain intercellular communication in plants. They act as specialized signalling hubs clustering receptor activities, and are capable of generating and relaying signalling from cell-to-cell. As key elements in intercellular communication, plasmodesmata coordinate processes related to plant growth, development and environmental stresses responses. In this study, we identified three PM-located Leucine Rich Repeat Receptor Like Kinases (LRR-RLKs) that are able to dynamically and conditionally relocate to plasmodesmata upon abiotic stress. Plasmodesmal association occurs within 2 minutes and is not a general behaviour of PM or microdomain-associated proteins. Focusing on SAK1 (SUCROSE ACTIVATED KINASE) we demonstrated that the plasmodesmal dynamic association is neither dependent of sterol or sphingolipid composition and nor driven by the protein phosphorylation status. Importantly, we identified a polybasic amino acid motif in the Juxtamembrane Domain (JMD) of SAK1 predicted to bind anionic lipids that is critical for conditional plasmodesmal association. Altogether our data indicate that changes in the membrane molecular signature of plasmodesmata specialized domains accompanies responses to external stimuli.

Individual cells in a genetically identical population, exposed to the same environment, can show great variation in their protein expression levels. This is due to noise, which is inherent in many biological processes, due in part to the low molecule numbers and probabilistic interactions which lead to stochasticity. Much of the work in the field of noise and its propagation in gene expression networks, whether it is experimental, modelling or theoretical, has been conducted on networks/systems that occur within a single cell. However, cells do not exist solely in isolation and understanding how cells are able to coordinate their behaviour despite this noise is an interesting area of expansion for the field. In this study, a synthetic intercellular communication system was designed that allows the investigation of how noise is propagated in intercellular communication. The communication system consists of separate sender and receiver cells incorporating components of the Lux quorum sensing system of Vibrio fischeri. The sender cell was designed so that the production of the signalling molecule, 3-oxohexanoyl homoserine lactone, is able to be controlled by addition of isopropyl-β-D-thio-galactoside (IPTG) and monitored via a reporter gene. The receiver cell was designed with a dual reporter system to enable the response of the cell to the signalling molecule to be monitored and the intrinsic and extrinsic noise contributions to the total noise to be calculated. Sender and the receiver cells were engineered in Escherichia coli. The functionality of the receiver cells was tested in the presence of known concentrations of the signalling molecule. The population response and the noise characteristics of the receiver cells in the homogeneous environment were determined from single cell measurements. The functionality of the sender cells was tested in the presence of a range of IPTG concentrations and the induction of expression from the LacI-repressible promoter was monitored. Mathematical models of the system were developed. Stochastic simulations of the models were used to investigate any unexplained behaviour seen in the characterisation of the cells. The full functionality of the intercellular communication system was then tested by growing the receiver in the collected media of the induced sender cells. The response of the receiver cells to the signalling molecule in the media was again characterised using single cell measurements of the reporter expression levels. The analysis of mixed populations of the sender and receiver cells was hampered by the technical limitations of the instruments used for the single cell measurements. Difficulties were encountered in simultaneous and specific measurement of the three reporter genes. Two methods for overcoming this issue were proposed using microscopy, and one of these methods was shown to have potential in overcoming the issue.

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Indiana University-Purdue University Indianapolis (IUPUI) / Glioblastoma is a lethal cancer that arises from support cells in the nervous system and kills around 20,000 people in the United States each year. While much is known about the highly malignant primary glioblastoma, the natural history of lower grade glioma (LGG) is less understood. While the majority of LGGs are initiated by a mutation in isocitrate dehydrogenase, the events leading to their malignant progression into a grade IV tumor are not known. Analysis of primary tumor sample data has revealed that low transcript levels of Angiomotin-like-1 (AmotL1) strongly associate with poor outcomes of patients with these cancers. Follow-up RNA-sequencing of human embryonic astrocytes with AmotL1 silencing revealed the downregulation of many transcripts that encode proteins mediating gap junctions (GJ) between astrocytes, especially connexin-43 (Cx43). Cx43 protein oligomerizes to form functional channels comprising the astrocytic GJ. AmotL1 knockdown through RNA interference decreases Cx43 transcript and protein levels while increasing its distribution to GJs. This suggests increased GJ formation and intercellular communication, as similar localization patterns are observed in differentiated astrocytes. Astrocytes with AmotL1 knockdown also display a pronounced pancake-like morphology, suggesting that the actin cytoskeleton is affected. Imaging reveals that cells with reduced AmotL1 have characteristic losses in both stress fibers and focal actin under the cell body but notable increases in cortical F-actin. Consistent with previous studies, AmotL1 may promote increases in the number and thickness of F-actin fibers. Because actin binding to related angiomotins is inhibited by phosphorylation from the LATs kinases, I define the effects of expressing wildtype AmotL1 versus mutants that mimic or prevent phosphorylation by LATs1/2. Interestingly, expression of AmotL1 S262D in combination with NEDD4-1, a ubiquitin ligase, results in a profound loss of actin stress fibers. Dependence on NEDD4-1 suggests that this phenotype is due to the induced degradation of proteins that promote F-actin, e.g. RhoA. These results directly support a model in which phosphorylated AmotL1 specifically inhibits F-actin formation as opposed to unphosphorylated AmotL1 which is known to promote stress fiber formation. Thus, in addition to regulating polarity and YAP/TAZ transcriptional co-activators, AmotL1 plays major functions in dictating cellular F-actin dynamics. / 2021-01-01

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