Semester

Summer

Date of Graduation

2024

Document Type

Dissertation

Degree Type

PhD

College

School of Medicine

Department

Not Listed

Committee Chair

Visvanathan Ramamurthy

Committee Co-Chair

Seonhee Kim

Committee Member

Seonhee Kim

Committee Member

Elena Pugacheva

Committee Member

Aaron Robart

Committee Member

Maxim Sokolov

Abstract

Microtubules are ubiquitous cytoskeletal filaments essential in the function of eukaryotic cells. These polymers, comprised of αβ-tubulin heterodimers, are critical for cell division, intracellular transport, and formation of the axonemes of cilia and flagella. This functional diversity of microtubules is attributed to the incorporation of various α- and β-tubulin isotypes encoded by different genes. Indeed, mutations in human tubulin genes have been linked to various tissue-specific diseases, suggesting a unique function for each tubulin isotype. Mutations in the β4B-tubulin gene (TUBB4B) are associated with sensorineural hearing loss, Leber Congenital Amaurosis (LCA), a severe childhood blindness, and primary ciliary dyskinesia (PCD), a motile ciliopathy. However, the functional role of this particular tubulin isotype and the pathophysiology of diseases caused by TUBB4B mutations are not fully understood. In this work, we investigate the functional role of β4B-tubulin using the Tubb4b mouse models.

We found that mice lacking β4B-tubulin (TUBB4B) are profoundly deaf. Furthermore, we discovered that the deafness observed in these animals is due to defects in both the middle and inner ear. In the middle ear, the Tubb4b−/− (knockout) exhibited motile cilia defects, leading to the development of otitis media. In the inner ear, the lack of TUBB4 leads to disorganized and reduced densities of microtubules in pillar cells, suggesting a critical role for TUBB4B in providing mechanical support for auditory transmission.

We also investigated the importance of murine β4B-tubulin in vision. The Tubb4b knockout and knock-in, carrying the mutations observed in TUBB4B patients, failed to recapitulate vision loss. Furthermore, β6-tubulin transcript and protein levels were upregulated in the retina lacking TUBB4B, suggesting a functional compensation mechanism. Moreover, we have demonstrated that β4B-tubulin is differentially expressed in murine and monkey retina, suggesting that the discrepancy in visual phenotype seen in TUBB4B patients and Tubb4b knock-in mice is probably due to the difference in expression pattern between humans and mice.

Our work also highlights the importance of β4B-tubulin in male fertility. Tubb4b−/− male mice failed to produce offspring. The lack of TUBB4B resulted in seminiferous tubule degeneration and failure to produce sperm cells. Our studies revealed that β4B-tubulin is essential for the expansion of differentiating spermatogonia, as the number of differentiating spermatogonia was significantly reduced in mice lacking TUBB4B.

Altogether, this work provides a strong groundwork for future investigations of the β4B-tubulin roles in motile cilia and in microtubules of cochlear supporting cells, as well as for developing a model system to investigate the role of β4B-tubulin in vision and mechanisms behind blindness caused by TUBB4B mutations.

Embargo Reason

Publication Pending

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Available for download on Wednesday, July 30, 2025

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