The role of Drosophila CPEB protein, Orb2, during asymmetric neural stem cell division Open Access

Robinson, Beverly (Summer 2022)

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Centrosomes serve as the primary microtubule-organizing centers of most cells, and centrosome dysregulation affects a range of cellular processes including cell division, cell-cell communication, cell motility, and intracellular transport of molecular cargo. Centrosomes attain diversity of function by altering their microtubule-nucleating activities through the cyclic shedding, recruitment, and rearrangement of a protein-rich lattice, called the pericentriolar material. Nevertheless, the mechanisms that centrosomes employ to modulate rapid and dynamic changes to pericentriolar material are still poorly understood. Identifying such mechanisms of centrosome regulation would not only expand our understanding of this versatile organelle, but also inform new therapeutic options for diverse, devastating, centrosome-related diseases. Developmental diseases such as microcephaly, congenital heart disease, and polycystic kidney disease are all associated with centrosome genes and manifest symptoms in the brain, heart, lungs, skin, eyes, kidneys, etc. Similarly, cancers associated with aberrant centrosome function have a poorer prognosis than those that do not. Thus, identifying mechanisms regulating centrosome activity may help inform the pathogenesis of human diseases. To understand disease pathogenesis associated with aberrant centrosome function, a basic understanding of the molecular mechanisms instructing pericentriolar material recruitment is required.

Toward this end, this thesis investigates a previously undefined mechanism of centrosome regulation: post-transcriptional regulation. Using neural stem cells from the Drosophila larval brain, we identified the conserved RNA-binding protein Orb2 as an important regulator of centrosome activity. Drosophila larval neural stem cells are a powerful model for studying centrosome biology, as the two centrosomes are differentially regulated both spatially and temporally. Differential recruitment of the pericentriolar material to the two centrosomes within a single interphase neural stem cell results in drastically different levels of microtubule-nucleating activity. This functional asymmetry ensures the segregation of differential fate determinants through proper alignment of the spindle pole along an invariant apical/basal axis, ultimately leading to asymmetric stem cell division. In support of Orb2 directing centrosome activity, orb2 loss generates a population of interphase neural stem cells with symmetrized, active centrosomes. Neural stem cells lacking orb2 are susceptible to mitotic spindle defects, despite maintaining cellular polarity. We show Orb2 is required cell autonomously to support centrosome asymmetry and maintain neural stem cell homeostasis. In addition, orb2 null brains are microcephalic, and Orb2 functions outside of neural stem cells to regulate brain size. We propose Orb2 plays opposing roles in centrosome regulation, possibly through the translational regulation of multiple mRNA substrates. These data support a novel paradigm of centrosome control in which activity is modulated by RNA-binding proteins. Using the insights gleaned from this work, we can build a more comprehensive understanding of the cellular mechanisms that regulate centrosome activity, which may inform the pathogenesis of human disorders arising from aberrant centrosome activity.

Table of Contents

Chapter 1. Understanding microcephaly through the study of centrosome regulation in Drosophila neural stem cells

1.1. Abstract

1.2. Introduction

1.3. Drosophila as a model to uncover cellular mechanism of NSC divisions

1.4. Centrosomes and polarity

1.5. Is centrosome asymmetry dispensable in Drosophila NSCs?

1.6. The SAC as a microcephaly fail-safe

1.7. Emerging roles of post-transcriptional control in preventing microcephaly

1.8. Summary

1.9. Acknowledgements

1.10. Supplemental Information

Chapter 2. Introduction to centrosome regulation and thesis rationale

2.1. Introduction

2.1.1. Centrosome structure

2.1.2. The centrosome cycle and inherent asymmetries

2.1.3. RNA localization to centrosomes

2.2. Evidence of post-transcriptional regulatory elements at the centrosome

2.2.1. CPE sites, known cis-elements and how they relate to centrosomal RNAs

2.2.2. CPEB RNA-binding proteins, the trans-element associated with CPE sites

2.3. Summary of thesis rationale

2.3.1. Hypothesis

2.3.2. Overview of experimental design

2.3.3. Summary of Chapter Two

2.4. Supplemental Information

Chapter 3. RNA-binding protein Orb2 causes microcephaly and supports centrosome asymmetry in neural stem cells

3.1. Abstract

3.2. Introduction

3.3. Results and Discussion

3.3.1. Orb2 localizes to centrosomes within cycling NSCs

3.3.2. Orb2 disrupts centrosome activity in interphase NSCs

3.3.3. Orb2-dependent centrosome regulation is cell autonomous

3.3.4. Loss of orb2 is associated with supernumerary centrosomes

3.3.5. Orb2 is required for mitotic spindle morphogenesis

3.3.6. Loss of orb2 results in microcephaly

3.3.7. Orb2 regulates PLP protein levels in larval brains

3.3.8. Model Summary

3.4. Materials and Methods

3.4.1. Fly Stocks

3.4.2. Immunofluorescence

3.4.3. Microscopy

3.4.4. Image analysis

3.4.5. Immunoblotting

3.4.6. Bioinformatics

3.4.7. Statistical analysis

3.5. Acknowledgements

3.6. Supplemental Information

Chapter 4. General Discussion

4.1. Summary of Results

4.2. Implications of Findings

4.3. Limitations and Future Directions

4.4. Conclusion

Chapter 5. References

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