title: Different mutations in the beta-tubulin TUBB4B result in a novel spectrum of dominant-negative ciliopathies
Creators: name: McPhie, Fraser
Publisher: name: The University of Edinburgh
Year: 2024
Subjects: subject: cilia
subject: Primary ciliary dyskinesia
subject: PCD
subject: tubulin
subject: TUBB4B
subject: tubulin mutation
subject: microtubules
subject: syndromic PCD mutation
subject: motile cilia

description: Microtubules are polymers of tubulins which provide the cell with shape and structure. They can form highly dynamic structures such as the mitotic spindle, or highly stable lattices, like those which form the ciliary axoneme. Each tubulin molecule that integrates into a microtubule is a heterodimer that consists of a single alpha and a single beta tubulin monomer. Given there are 9 genes (isotypes) for alpha tubulin and 10 for beta tubulin in humans, it remains unclear whether isotypes are interchangeable or whether each isotype conveys unique properties onto the microtubule in which it is integrated, resulting in a ‘tubulin code’ for different microtubule structures. Genetic studies of the tubulinopathies, a group of typically neurodevelopmental or neurodegenerative diseases, suggests that there are cell-type specific functions for different tubulin isotypes. This thesis examines the disease mechanism in a cohort of 11 primary ciliary dyskinesia (PCD) patients with 4 novel dominant mutations we identified in the beta tubulin isotype TUBB4B. This is the first recorded case of a tubulin mutation causing PCD as well as a spectrum of dominant negative ciliopathies. These patients exhibit a drastic reduction of the number of basal bodies and motile cilia axonemes in the airway cells. This suggests that TUBB4B fulfils a specific role in the generation of basal bodies and axonemes in multiciliated cells, whilst the cytoplasmic microtubules appear unaffected. To understand the function of TUBB4B and how these mutations are causing disease, my thesis work aims to: (1) examine how these different mutations in TUBB4B cause distinct types of ciliopathies, and (2) ask whether TUBB4B is a cilia-specific beta tubulin isotype. Based on patient features, the mutations have been split into two categories: those causing PCD-only, and those causing PCD as well as sensorineural disease (SND) (early onset vision and hearing loss). Overexpression constructs for each patient variant were used in vitro to study how each behaves in a cellular context. Data from the overexpression constructs shows that mutations located at the site of alpha-beta heterodimer formation cause PCD-only. They disrupt the ability of TUBB4B to form heterodimers and robustly integrate into microtubules. In contrast, the syndromic mutation is found in the intraluminal interface and does not impair heterodimer formation. It may however subsequently disrupt polymerisation. It can integrate into cytoskeletal microtubules, primary and motile cilia, but ultimately results in fewer basal bodies and shorter axonemes. To investigate the functional role of TUBB4B and which microtubules networks it integrates into in vivo, I generated a mouse line in which Tubb4b is tagged at the N-terminus with the small, rationally designed ALFA tag; Tubb4bALFA allele. When maintained as a heterozygote, this is the first viable, endogenously tagged tubulin mouse to date. Examination of TUBB4B expression across developmental stages in the mouse revealed TUBB4B is widely expressed, but highly enriched in all motile cilia studied and was particularly enriched in the cilia of the trachea and choroid plexus. The enrichment of TUBB4B in the motile cilia helps to explain why this cohort of patients have developed PCD. Combining in vivo work studying the normal spatiotemporal localisation across development of TUBB4B and in vitro work examining the effect of over-expressed patient variants shows the importance of TUBB4B in cilia biology. Additionally, the Tubb4bALFA mouse line will be a valuable tool to study a single tubulin isotype with unparalleled resolution in cytoskeletal dynamics in different cell types.