Date of Award
6-15-2024
Document Type
Dissertation
Degree Name
Ph.D.
Organizational Unit
College of Natural Science and Mathematics, Biological Sciences
First Advisor
J. Todd Blankenship
Second Advisor
Dinah Loerke
Third Advisor
Cedric Asensio
Fourth Advisor
Joe Angleson
Keywords
Cleavage divisions, Drosophila melanogaster, Endoplasmic reticulum, Mitosis, Spindle apparatus
Abstract
The Endoplasmic Reticulum (ER) is the largest organelle in the cell by surface area and forms contacts with other organelles throughout the cell. In recent work, the ER has been implicated in numerous processes within the cell beyond its canonical roles of protein synthesis and calcium storage. The ER also undergoes a remarkable transition in morphology during cell division, with mitotic divisions requiring a massive remodeling of various membranous organelles such as the nuclear envelope and Golgi. However, whether changes in ER behaviors modulate mitotic events is less clear. The rapid cleavage divisions in the early Drosophila embryo are a potent system to understand the dynamic changes that underlie successful mitotic events, as well as uncover the distinct challenges that occur during the rapid cleavage cycles that exist in many animal embryos. In a screen for Rab family GTPases that display dynamic function at these stages, we identified Rab1. Disrupting Rab1 function in the early embryo led to an enhanced buildup of ER at the spindle poles and, interestingly, produced highly penetrant defects in the division cycles. Clustering of ER around the mitotic space is negatively correlated with spindle length and microtubule intensity. Importantly, the recruitment of several key centrosomal proteins at mitotic onset to the spindle poles is weakened after Rab1 disruption leading to reduced γ-tubulin function, suggestive of a defect in centrosomal maturation. We implicate Dynein in maintaining some ER contact with the spindle poles in embryos and find that dual disruption of Rab1 and Dynein can rescue maturation defects. These results demonstrate that ER levels must be carefully tuned during mitotic processes to ensure proper assembly of the division machinery. However, the ER’s structural complexity raises the question of whether the accumulation of ER alone, or the structural disruption of the polar ER may be driving the phenotypes seen in Rab1 disrupted embryos and to what extent either factor may contribute to the weakened centrosomes. To this end, we explore the contributions of the ER shaping proteins in mitosis. Through screening ER shaping protein depletions for mitotic failures, we find one, ReepB, that display division failures during cortical divisions. Additionally, ReepB disruption affects the ER coated structure when depleted, resulting in a ‘frilled ER’ phenotype and a reduction of ER adherence to the spindle space. Additionally, division failures occur in the ReepB depleted background in conjunction with the ER aberrant morphologies. Interestingly, ReepA, a member of the same family of proteins, when overexpressed does not rescue ReepB phenotypes in Drosophila embryos and instead increases ER structural aberrance. To further understand the Reep subclasses we overexpressed each fly Reep in Cos7 cells, resulting in varied phenotypes with ReepB appearing to remodel ER tubules more readily. We then turn back to fly embryos to elucidate the mechanism behind ReepB spindle disruption. Together these data suggest that ReepA and ReepB balance in division cycles is pivotal for ER morphological maintenance and that ReepA and ReepB have some degree of antagonism when expressed in conjunction.
Copyright Date
6-2024
Copyright Statement / License for Reuse
All Rights Reserved.
Publication Statement
Copyright is held by the author. User is responsible for all copyright compliance.
Rights Holder
Katie R. Rollins
Provenance
Received from ProQuest
File Format
application/pdf
Language
English (eng)
Extent
124 pgs
File Size
4.8 MB
Recommended Citation
Rollins, Katie R., "Morphological Remodeling of the Endoplasmic Reticulum During Mitosis and Its Influence on the Integrity of the Spindle Apparatus" (2024). Electronic Theses and Dissertations. 2395.
https://digitalcommons.du.edu/etd/2395