3D Nanoscale Architecture and Physiology of the Human Airway Epithelium

Understanding how transcriptional changes lead to cellular-level architectural changes that drive cellular function is a central challenge in single-cell biology. An example of cellular remodelling is observed in the differentiation of airway basal stem cells into multiciliated cells within the human respiratory epithelium – a tissue barrier crucial for protection against bacteria, viruses, and particulate matter. In this thesis, I present the first isotropic three-dimensional map of the human airway epithelium at the nanometre scale, utilising state-of-the-art Focused Ion Beam-Scanning Electron Microscopy (FIB-SEM), which reveals extensive remodelling of cellular topology, organelle distribution, cytoskeletal arrangement, and basal body maturation during multiciliogenesis. Furthermore, observations of organelle remodelling were accompanied by single-cell RNA sequencing (scRNA-seq) analysis, which revealed Gene Ontology (GO) enrichment of pathways related to oxidative phosphorylation (OXPHOS), alongside increased expression of genes involved in mitochondrial fatty acid oxidation (FAO) and glycogenesis. In parallel, a broad downregulation of genes associated with glycolysis was observed, suggestive of a metabolic shift. These transcriptional changes potentially support the intensive energy demands of sustained ciliary beating during multiciliogenesis. Building on this integrative analysis, I identified and focused on a novel organelle–cytoskeletal interaction between rootlets – a cytoskeletal structure emanating from the base of the motile cilium – and the mitochondrial network. Despite their conserved morphology, the function of rootlets and their interaction with the mitochondria in human airway epithelial cells remains poorly understood. Using a combination of advanced imaging techniques, this thesis determines the cellular and molecular architecture of rootlets across various scales, their formation during multiciliogenesis, and their intricate linkage to basal bodies. Additionally, super-resolution live-cell imaging demonstrates that rootlets oscillate during ciliary beating. Notably, I found that rootlets not only play a role in ciliary beating coordination, but also may regulate ATP generation, raising the possibility of a previously uncharacterised link between motile cilia and mitochondria. While the precise nature of this interaction remains to be fully elucidated, these findings suggest a spatial alignment that may reflect a functional association crucial for meeting the energy demands of cilia beating. By linking this mechanism to the broader physiological role of airway defence, my research sheds light on how cytoskeletal and organelle interactions contribute to essential cellular processes. Altogether, this thesis provides nanoscale insights into the structural, functional, and dynamic aspects of airway epithelial biology, advancing our understanding of how cytoskeletal and organelle interactions drive vital cellular processes.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2025

Author dc:creator

• Vijayakumaran, Aaran

Advisor dc:contributor.advisor

• Sawarkar, Ritwick

• Cellular architecture
• Focused Ion Beam-Scanning Electron Microscopy (FIB-SEM)
• Human airway epithelium
• Motile cilia
• Multiciliogenesis
• Organelle–cytoskeletal interactions
• Rootlets

Licence

• https://www.repository.cam.ac.uk/bitstreams/7eaf4a98-33f8-4cc1-8c40-efa90fe0279a/download
• http://purl.org/NET/rdflicense/allrightsreserved

Language dc:language

eng

DOI dc:identifier.doi

https://doi.org/10.17863/CAM.121654

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/389911