Dissertation
Understanding the Role of Nuclear Architecture in Drosophila Germline Stem Cell Maintenance
University of Iowa
Doctor of Philosophy (PhD), University of Iowa
Spring 2020
DOI: 10.17077/etd.005436
Abstract
Tissue homeostasis relies upon a stable population of stem cells. Upon each division, stem cells give rise to one daughter that maintains a stem cell identity and a second daughter that enters a differentiation pathway. Regulation of this process requires the integrity of the nuclear lamina (NL), an extensive protein network that lies beneath the nuclear envelope, providing structural support for nuclear functions. The Drosophila germline stem cells (GSCs) possess a NL checkpoint, whose activation is associated with a thickened and lobulated NL structure. Activation of the NL checkpoint involves the ATM- and Rad3-related (ATR) and Checkpoint kinase 2 (Chk2) kinases, leading to a block in germ cell differentiation and death of GSCs. Loss of D-emerin/Otefin causes NL checkpoint activation. D-emerin/Otefin is a NL-associated LAP2-emerin-MAN1-domain (LEM-D) protein that directly associates with the histone and DNA binding protein Barrier-to-autointegration factor (BAF). Through this interaction, D-emerin/Otefin builds nuclear architecture in interphase cells and contributes to the nuclear envelope reformation at the end of mitosis. Data presented in this thesis expand the understanding of contributions of D-emerin/Otefin to both interphase and mitotic cells. First, I studied the partnership between D-emerin/Otefin and BAF during development. I found that even though Drosophila expresses three LEM-D proteins, only D-emerin/Otefin alone is required for global enrichment of BAF at the NL, revealing an unexpected dependence on a single LEM-D protein. Prompted by these observations, I studied BAF contributions to GSC maintenance, testing whether BAF contributes to D-emerin/Otefin function in these cells. I show that germ cell-specific BAF knockdown causes phenotypes that mirror d-emerin/otefin mutants. Loss of BAF disrupts NL structure, blocks differentiation and promotes germ cell loss, phenotypes that are partially rescued by inactivation of the ATR and Chk2 kinases. These data suggest that similar to D-emerin/Otefin, BAF loss activates the NL checkpoint in GSCs. Second, motivated by the shared functions of D-emerin and BAF in GSCs, I investigated whether NL reformation was affected in the NL mutant backgrounds. Surprisingly, analyses of wild type controls revealed the unexpected discovery that Drosophila ovarian GSCs undergo an atypical mitosis, characterized by retention of the NL. Further studies established that this type of mitosis is most similar to the “semi-open” mitosis found in the filamentous fungus Aspergillus nidulans. Strikingly, progenitor germ cells display greater disruption of the mitotic NL, suggesting a developmental switch in mitosis that correlates with adoption of an asymmetric division. Lastly, I tested whether NL components are required to maintain the semi-open GSC mitosis. I show that loss of the B-type Lamin or D-emerin/Otefin affects centrosome positioning and spindle structure. Strikingly, the NL deformation found d-emerin/otefin GSCs correlates with increased centrosome embedding. Furthermore, the interphase centrosomes in d-emerin/otefin mutants retain pericentriolar material (PCM), including key microtubule binding components. PCM retention remains in chk2-/-, d-emerin/otefin-/- double mutants, suggesting PCM retention is upstream of the NL checkpoint activation. Based on these findings, I propose that D-emerin/Otefin is required for centrosome disassembly at the end of mitosis, with failed disassembly resulting in activation of the NL checkpoint. My studies represent the first report of a semi-open mitosis in a stem cell population and suggest centrosome dysfunction as a potential trigger of the NL checkpoint in GSCs. Because basic principles and mechanisms of stem cell homeostasis are shared between cell types and organisms, these findings have implications for mechanisms associated with NL dysfunction in other systems, such as laminopathies.
Details
- Title: Subtitle
- Understanding the Role of Nuclear Architecture in Drosophila Germline Stem Cell Maintenance
- Creators
- Tingting Duan
- Contributors
- Pamela K Geyer (Advisor)Miles A Pufall (Advisor)Brandon S Davies (Committee Member)Albert J Erives (Committee Member)Daniel L Weeks (Committee Member)Marc S Wold (Committee Member)
- Resource Type
- Dissertation
- Degree Awarded
- Doctor of Philosophy (PhD), University of Iowa
- Degree in
- Biochemistry
- Date degree season
- Spring 2020
- DOI
- 10.17077/etd.005436
- Publisher
- University of Iowa
- Number of pages
- xvi, 117 pages
- Copyright
- Copyright 2019 Tingting Duan
- Language
- English
- Description illustrations
- color illustrations
- Description bibliographic
- Includes bibliographical references (pages 102-117).
- Public Abstract (ETD)
Underneath the inner nuclear envelope lies an extensive protein network, the nuclear lamina (NL). Main components of the NL are the lamins, which interact with LAP2-emerin-MAN1-domain (LEM-D) proteins and the chromatin and DNA binding protein Barrier-to-autointegration factor (BAF). Mutations in genes encoding lamin, LEM-D proteins and BAF underlie a group of fetal rare genetic disorders called progeria. These disorders are characterized by clinical features and phenotypes that resemble accelerated physiological ageing. Many studies suggest that progeria is a result of defective stem cell-driven tissue regeneration caused by adult stem cell defects. Drosophila melanogaster is a good model to study contributions of the NL to adult stem cells, as it allows examinations of stem cell functions in their natural environments. I studied ovarian germline stem cells (GSCs), a well-defined adult stem cell population in a non-essential tissue, the adult ovary. In the Drosophila ovary, loss of LEM-D protein D-emerin/Otefin and BAF causes NL distortion, similar phenotypes observed in progeria patient-derived cells. The distorted NL in mutant GSCs activates a NL checkpoint that involves two kinases ATM- and Rad3-related (ATR) and Checkpoint kinase 2 (Chk2). The activation of this checkpoint blocks germ cell differentiation and promotes GSC death. In most eukaryotic cells, the NL breaks down at the onset of mitosis to allow the reformation of mitotic spindles and reforms during mitotic exit. As both D-emerin/Otefin and BAF are involved in NL reformation during mitosis, we investigated whether NL checkpoint activation is associated with failed NL reformation. To begin these analyses, we examined the sequential events during GSC mitosis in wild type ovaries, including NL breakdown, mitotic spindle formation and NL reformation. Surprisingly, we found that lamin and D-emerin/Otefin do not disseminate but instead form a mitotic NL that surrounds the mitotic spindle, suggesting that GSCs undergo an atypical “semi-open” mitosis similar to yeast. Strikingly, loss of either lamin or D-emerin/Otefin causes mitotic spindle defects, indicating that the mitotic NL is required in GSC mitosis. In addition to mitotic spindle defects, d-emerin/otefin mutant GSCs display thickened lamin structure around the microtubule organization center, centrosome. As loss of D-emerin/Otefin activates the NL checkpoint, we investigated the connection between centrosomes and the NL checkpoint. We found that centrosome compositions are altered in d-emerin/otefin mutant GSCs and is upstream of Chk2 activation. Taken together, our data represent the first report of a semi-open mitosis in a stem population and suggest that centrosome dysfunction may be a trigger of the NL checkpoint.
- Academic Unit
- Biochemistry and Molecular Biology
- Record Identifier
- 9983966295402771
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