Date of Award
Fall 2023
Document Type
Dissertation
Degree Name
Doctor of Philosophy (PhD)
Department
Genetics
First Advisor
Liem Jr, Karel
Abstract
Microtubules (MTs) are a key component of the cytoskeleton and participate in multiple cellular functions and processes. MTs are made of α- and β-tubulin proteins that are encoded by multiple, individual isotype genes. Over the recent decades there has been mounting evidence that despite the high similarity among tubulin proteins, these various isotypes have unique functions rather than being functionally redundant. First, tubulins have different expression and localization patterns. Second, mutations in different tubulin genes result in a variety of disease phenotypes, some of which are exclusively tied to specific tubulins. Finally, multiple tubulin studies have shown that tubulin composition within MTs directly influences MT properties such as stability and flexibility. A few tubulin proteins have even been associated with specific cell types and structures. However, there are still many tubulins whose functions have yet to be defined. In this dissertation I focus on identifying ciliary tubulins. Cilia are stable MT-based cell surface organelles that are present on almost every cell in the body and are critical for development and homeostasis. Abnormalities in these structures can cause a wide variety of developmental defects. Identifying ciliary tubulins offers important insight into the structural requirements of cilia. Among mammalian tubulins, the β-tubulin class IV (Tubb4) isotype proteins, β-tubulin class Iva (Tubb4a) and β-tubulin class IVb (Tubb4b), standout as candidates for playing essential roles in ciliary structure. The Tubb4 isotypes have nearly identical amino acid sequences and both carry a predicted ciliary motif sequence. Tubb4 protein has also been detected via a Tubb4 specific antibody in ciliated cell types of various organisms. However, the potential function of each Tubb4 isotype in cilia has yet to be tested. Here, I use multiple knockout (KO) mouse lines to explore the individual requirement and function of the mammalian Tubb4 isotypes in multiple ciliated cell types. My in-depth analysis of Tubb4a and Tubb4b expression derived from single-cell RNA sequencing (scRNAseq) data and LacZ reporter staining showed that the expression patterns of the Tubb4 isotypes are distinctly different from one another and indicate the Tubb4b rather than Tubb4a has a ciliary function. Tubb4b’s ciliary function is further confirmed by ciliopathic phenotypes observed only in Tubb4b KO mice. I report that Tubb4a is nonessential, and that Tubb4b critically functions in the axonemes of motile cilia on multiciliated cells (MCCs) in multiple tissues. Work from this study enhances our understanding of tubulin isotype specificity in different MT contexts and cilia biology across different cell types.
Recommended Citation
Sewell, Mycah Theair, "Determining the Role of Beta-Tubulin 4b in Cilia and Development" (2023). Yale Graduate School of Arts and Sciences Dissertations. 1180.
https://elischolar.library.yale.edu/gsas_dissertations/1180
