Date of Award
Summer 8-23-2025
Document Type
Dissertation
Degree Name
Ph.D. in Biological Sciences
Organizational Unit
College of Natural Science and Mathematics, Biological Sciences
First Advisor
James Todd Blankenship
Second Advisor
Dinah Loerke
Third Advisor
Schuyler van Engelenburg
Fourth Advisor
Mira Pronobis
Copyright Statement / License for Reuse
All Rights Reserved.
Keywords
Centrosomal, Non-centrosoman, Nuclear dynamics, Epithelium, Drosophila embryos
Abstract
The morphogenesis of developing tissues relies on extensive cellular rearrangements in shape, position, and identity. A key process in reshaping tissues is cell intercalation-driven elongation, where epithelial cells align and intercalate along a common axis. Typically, analyses focus on how peripheral cortical forces influence cell shape changes. Less attention is given to how inhomogeneities in internal structures, particularly the nucleus, impact cell shaping. Here, we examine how pulsed contractile and extension dynamics interact with the nucleus in elongating Drosophila embryos. Our data show that tightly packed nuclei in apical layers hinder tissue remodeling/oscillatory behaviors. We identify two mechanisms for resolving internuclear tensions: nuclear deformation and dispersion. Embryos with non-deformable nuclei use nuclear dispersion to maintain near-normal extensile rates, while those with non-dispersible nuclei due to microtubule inhibition exhibit disruptions in contractile behaviors. Disrupting both mechanisms leads to severe tissue extension defects and cell extrusion. These findings highlight the critical role of nuclear shape and positioning in the topological remodeling of epithelia.
To further understand how nuclear dispersion along the apical-basal axis is regulated during the germband extension (GBE), we examined the structural reorganization of microtubule (MT) arrays over time. We find that the initial centrosome-derived MT network, which forms an inverted basket-like structure surrounding the nucleus, transitions into non-centrosomal MT (ncMT) arrays enriched at the apical cortex as GBE progresses. Disruption of Patronin, a minus-end tracking proteins (-TIP), impaired both perinuclear and apical MT pools, while inducing ectopic MT accumulation at centrosomes. This led to a failure in nuclear dispersion, with nuclei remaining densely packed in the apical domain. Patronin inhibition also reduced the proportion of nuclei undergoing active displacement and significantly slowed nuclear migration. Inhibiting CLASP, a plus-end tracking protein (+TIP), produced a similar disruption in MT organization, with even greater centrosomal enrichment. CLASP loss resulted in a striking reduction in the number of actively dispersing nuclei, disrupted apical-basal nuclear orientation and impaired nuclear anchoring to the apical cortex. Detailed examination of centrosomal MT enrichment in CLASP- and Patronin-depleted embryos revealed a competitive relationship between centrosomal and non-centrosomal MT populations, suggesting an antagonistic regulatory mechanism. We also observed compensatory interactions between γ-tubulin and patronin at centrosomes: γ-tubulin inhibition led to increased Patronin levels and enhanced total MT abundance, with only mild effects on nuclear dispersion. Finally, perturbation of EB1 disrupted the apical shift of the MT basket and impaired nuclear anchoring to the cortex, although apical-basal nuclear orientation was largely preserved. Together, these findings highlight the importance of properly organized and dynamically remodeled MT networks – particularly the transition from centrosomal to non-centrosomal arrays – for accurate nuclear positioning, orientation, and dispersion during epithelial tissue remodeling.
Copyright Date
8-2025
Publication Statement
Copyright is held by the author. User is responsible for all copyright compliance.
Rights Holder
Rashmi Budhathoki
Provenance
Received from ProQuest
File Format
application/pdf
Language
English (eng)
Extent
138 pgs
File Size
15 MB
Recommended Citation
Budhathoki, Rashmi, "Centrosomal and Non-centrosomal Microtubule Networks Coordinate to Drive Nuclear Dynamics in Developing Epithelium" (2025). Electronic Theses and Dissertations. 2618.
https://digitalcommons.du.edu/etd/2618