The contents of nuclei are highly organized. Nuclear organization is facilitated by a dense protein network, called the nuclear lamina, which underlies the nuclear envelope. The nuclear lamina is composed of filamentous lamins and more than eighty lamin-associated proteins (LAPs). Among the first LAPs identified are LEM Domain (LEM-D) proteins, named after LAP2, emerin and MAN1. LEM-D proteins have many shared and unique functions that include providing structural support to the nucleus, regulating signal transduction pathways and gene expression, facilitating proper progression through the cell cycle and maintaining chromatin attachments at the nuclear periphery. Despite requirements for these processes in all cell types, loss of globally expressed LEM-D proteins causes tissue-restricted defects. Identification of the primary function in tissues susceptible to LEM-D protein loss is a persistent challenge in the nuclear lamina field. Research described here uses Drosophila as a model to understand LEM-D protein function. Loss of the Drosophila emerin homologue Otefin (Ote) causes a complex phenotype in the ovary wherein both somatic and germline cells are compromised. Using tissue-restricted expression experiments, it was determined that Ote function is only required in germline stem cells (GSCs) to maintain all cells in the ovary. Developmental, molecular and genetic analyses revealed that the primary defect in ote mutant ovaries is an early block in germline differentiation, followed by GSC death. Genetic rescue experiments revealed that both of these GSC defects are due to the activation of the DNA Damage Response (DDR) proteins ATR and Chk2. Interestingly, activation of ATR and Chk2 occurs independent of detectable canonical DDR triggers, including DNA damage. Immunohistochemical analyses suggest that Ote might be regulating chromatin condensation and/or heterochromatin organization in GSCs. Through studies of Ote, a rescue method was discovered that involves culturing animals at elevated temperatures. This novel rescue strategy, termed hyperthermia, acts independent of ATR or Chk2 inhibition. Interestingly, elevated temperatures leads to chromatin decondensation in Drosophila, suggesting that hyperthermia may rescue oogenesis by alleviating chromatin defects observed in ote mutant germ cells. Together, results from experiments discussed herein dissect a complex ovary phenotype to reveal the critical requirement for a nuclear lamina LEM-D protein. Investigations into Ote function have revealed several aspects of GSC biology. The ATR/Chk2 checkpoint activated in the absence of Ote uncovered a previously unidentified cause of female GSC death. Further, findings that ATR and Chk2 are activated in the absence of canonical triggers suggest that GSCs possess a system to monitor defects or changes in the nucleus that do not involve DNA damage. Therefore, studies of Ote function and ote mutant phenotypes have uncovered valuable insights into LEM-D protein function and revealed the existence of novel conditions required for GSC maintenance.

L'entrée en mitose est initiée par le complexe cycline B – Cdk1. La phosphorylation de ses substrats déclenche des transformations incluant la condensation des chromosomes, le bris de l'enveloppe nucléaire et la formation d'un fuseau mitotique. Ces transformations permettent à la cellule de se diviser. La protéine phosphatase 2A (PP2A) en complexe avec sa sous-unité B55/Twins (Tws) reconnaît et déphosphoryle les substrats de cycline B – Cdk1. Pour éviter la déphosphorylation précoce des phosphoprotéines mitotiques, PP2A-B55/Tws est inhibée en entrée de mitose. Cette inhibition de la phosphatase est attribuable au module Greatwall (Gwl) – endosulfines. Activée en entrée de mitose, la kinase Gwl phosphoryle les endosulfines, qui inhibent alors de manière spécifique PP2A-B55/Tws. Gwl est exportée du noyau vers le cytoplasme en prophase, avant le bris de l’enveloppe nucléaire. Les mécanismes de régulation spatiotemporelle du module Gwl – endosulfines – PP2AB55/Tws ne sont pas entièrement élucidés. De plus, les substrats ciblés par PP2AB55/Tws en sortie mitose ne sont pas tous identifiés à ce jour. Dans mon travail de thèse, j’ai trouvé que Tws peut transiter par le noyau via un signal de localisation nucléaire (NLS), mais que ses fonctions essentielles sont au cytoplasme. De plus, j’ai trouvé que l’unique endosulfine présente chez Drosophila melanogaster, Endos, a une localisation cytoplasmique. Cette localisation est requise pour qu’Endos soit efficacement phosphorylée par la forme active et cytoplasmique de Gwl. Endos phosphorylée lie ensuite PP2A-Tws pour l’inhiber. Empêcher la localisation cytoplasmique d’Endos avant le bris de l’enveloppe nucléaire entraîne des défauts mitotiques dépendants de l’activité de PP2A-Tws. Les substrats mitotiques de PP2A-Tws ne sont pas tous connus. Par des cribles de phosphoprotéomique, j’ai identifié des substrats mitotiques potentiels de PP2A-Tws. L’un des candidats hyperphosphorylés suite à la déplétion de Tws, Otefin (Ote), est une protéine de l’enveloppe nucléaire. Les sites de phosphorylation d’Otefin identifiés dans mes cribles sont adjacents à son domaine d’interaction avec BAF, une protéine liant l’ADN et certaines protéines de l’enveloppe nucléaire. L’introduction de mutations phosphomimétiques à ces sites abolit l’association d’Otefin avec BAF, en plus de retarder le recrutement d’Otefin à l’enveloppe nucléaire en sortie de mitose. Par ailleurs, l’association Otefin – BAF dépend de l’activité de PP2A-Tws. Enfin, la perte d’Otefin dans l’embryon syncytial de mouche affecte le développement. En somme, mes travaux ont permis d’approfondir notre compréhension mécanistique de la régulation spatiotemporelle du module Gwl – endosulfines – PP2A et d’identifier de nouveaux substrats potentiels de PP2A-Tws. / Mitosis is triggered by the cyclin B – Cdk1 complex that phosphorylates multiple substrates to promote transformations such as chromosome condensation, nuclear envelope breakdown and mitotic spindle formation. These transformations are required for cell division. The protein phosphatase 2A (PP2A) in complex with its B55/Twins (Tws) subunit dephosphorylates cyclin B – Cdk1 substrates. To prevent premature dephosphorylation of the mitotic phosphoproteins, PP2A-B55/Tws is inhibited upon mitotic entry. The Greatwall (Gwl) – endosulfines pathway is responsible for PP2AB55/Tws inhibition. Activated upon mitotic entry, the Gwl kinase phosphorylates small proteins called endosulfines to turn them into specific inhibitors of PP2A-B55/Tws. Gwl is exported from the nucleus to the cytoplasm before nuclear envelope breakdown. However, the mechanisms of spatiotemporal regulation of the Gwl – endosulfines – PP2A module are not entirely elucidated. Moreover, the identity of the proteins targeted by PP2A-Tws during mitotic exit is still unclear. During my PhD training, I found that Tws can transit through the nucleus via a nuclear localization signal (NLS), but its essential functions are cytoplasmic. Moreover, the sole endosulfine present in Drosophila melanogaster, Endos, has a cytoplasmic localization. Such localization is required for efficient phosphorylation of Endos by active and cytoplasmic Gwl. Once phosphorylated, Endos binds PP2A-Tws to inhibit its activity. Preventing the cytoplasmic localization of Endos prior to nuclear envelope breakdown causes mitotic defects that are PP2A-Tws-dependent. The mitotic substrates of PP2A-Tws are not all identified. By phosphoproteomic screening, I identified potential novel PP2A-Tws substrates. Among the hits that are hyperphosphorylated following Tws depletion, there is the nuclear envelope protein Otefin (Ote). The identified phosphosites on Otefin are adjacent to its domain of interaction with BAF, a protein binding DNA and nuclear envelope proteins. Introducing phosphomimetic mutations at these sites abolishes the Otefin – BAF association and delays Otefin recruitment at the reforming nuclear envelope during mitotic exit. Moreover, the Otefin – BAF association is PP2A-Tws-dependent. Finally, loss of Otefin in the syncytial embryo of the fly impairs development. Altogether, my results deepen our understanding of the spatiotemporal coordination of the Gwl – endosulfines – PP2A module and provide potential novel PP2A-Tws substrates.