Morphological Remodeling of the Endoplasmic Reticulum During Mitosis and Its Influence on the Integrity of the Spindle Apparatus

<p>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 <em>Drosophila</em> 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 <em>Rab1</em> 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 <em>Rab1</em> 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 <em>Rab1</em> 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 <em>Rab1</em> 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, <em>ReepB</em>, that display division failures during cortical divisions. Additionally, <em>ReepB</em> 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 <em>ReepB</em> 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 <em>Drosophila</em> 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 <em>ReepB</em> 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.</p>

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Year dc:date.available

2024

Author dc:creator

• Rollins, Katie R.

Contributors dc:contributor

• J. Todd Blankenship
• Dinah Loerke
• Cedric Asensio
• Joe Angleson

• Cleavage divisions
• Drosophila melanogaster
• Endoplasmic reticulum
• Mitosis
• Spindle apparatus
• Biochemistry, Biophysics, and Structural Biology
• Cell and Developmental Biology
• Cell Biology
• Developmental Biology
• Life Sciences
• Molecular Biology

Statement dc:rights

• <p>Copyright is held by the author. User is responsible for all copyright compliance.</p>

Language dc:language

English (eng)

Repository record dc:identifier

https://digitalcommons.du.edu/etd/2395

OAI identifier oai:identifier

oai:digitalcommons.du.edu:etd-3390