The role for tubulin isotype diversity

Microtubules are long dynamic filaments present in the cytoplasm of cells that perform essential tasks. They separate chromosomes during cell division, act as tracks for motors to transport cargos across the cell and contribute to cell shape and architecture. Microtubules are assembled from alpha/beta-tubulin heterodimers. There are 9 alpha-tubulin and beta-tubulin genes in humans, that code for protein variants called isotypes.
In this grant, we seek to understand whether tubulin heterodimer assembly is random (hypothesis: any of the 9 alpha-tubulin isotypes can assemble with any of the 9 beta-tubulin isotypes), or whether it is determined (hypothesis: only certain combinations of alpha-tubulin isotypes can assemble with specific beta-tubulin isotypes).
Second we will determine how the tubulin variants affect the assembly of microtubules and the proteins that interact with microtubules to build tracks and cellular structures, and examine if tubulin variants can generate a variety of cellular structures.
Third, a growing number of patients are reported to have diseases caused by point mutations to specific tubulin genes. Very recently for the first time worldwide, we have identified patients with Primary Ciliary Dyskinesia, which have mutations in their tubulin genes. This disease causes mucus build-up in the lungs and inflammation of airways, with infections in the lungs, ear and nose. In these patients, the microtubules in the cilia are absent. Our last aim is to understand how these tubulin mutations cause the PCD disease, so that we can one day help and relieve patients' symptoms.

Alpha-tubulin and beta-tubulin heterodimers self-assemble into dynamic cytoskeletal polymers called microtubules. Microtubules are essential for cell architecture and polarity, transport and cell division. In humans there are 9 alpha-tubulin and 9 beta-tubulin isotypes, expressed at different levels in different cells and tissue types. While this diversity creates some redundancy, certain tubulin isotypes have unique properties giving rise to the tubulin code.
In this proposal, we hypothesize tubulin heterodimer composition is deterministic, giving rise to different types of microtubules with distinct properties. Missense mutations in tubulin isotypes cause distinct diseases, supporting the idea that tubulin isotypes are not all equivalent. These mutations are often dominant negative, making the disease mechanism hard to understand. Thus understanding the role of isotype diversity in microtubule biology is key to understanding the mechanisms that give rise to human tubulinopathies.
The objectives of this proposal are to define whether tubulin heterodimer assembly from isotypes is deterministic and identify the molecular determinants for specificity; define how tubulin isotype diversity control microtubule properties and modulate motors and microtubule-associated proteins; and explain how dominant negative mutations in tubulin cause pathogenesis.The study will focus on beta-tubulin isotypes, more divergent than alpha-tubulin isotypes in humans and targeted by most tubulin drugs, as a paradigm for isotype diversity. To understand molecular mechanisms that cause tubulinopathies, we will study how beta4B-tubulin mutations newly-discovered in patients with Primary Ciliary Dyskinesia cause disease at the cellular level.
Overall, the proposed work will impact both the research community by contributing to fundamental principles of tubulin biology, and the growing number of patients identified with tubulin-related disorders, for which little is currently known.