Visualizing Specific Tubulin Isotypes and Pathogenic Variants in Cellular Microtubule Arrays.

Abstract

Eukaryotic cells depend on dynamic microtubule arrays to execute a wide range of functions vital for life. These microtubule filaments are formed through the polymerization of α/β-tubulin proteins, which can be generated from numerous tubulin genes, or isotypes. In the neuron, microtubule networks are responsible for a myriad of specialized functions, including neuronal migration, neurite extension, and long-range cargo transport. Pathogenic mutations in tubulin genes can result in a wide range of neurodevelopmental defects, collectively termed tubulinopathies. How distinct mutations within different tubulin genes lead to functional defects remains an area of active investigation. Neuronal microtubule networks are significantly challenging to visualize due to the dense, spatially confined microenvironments within the neuron. Further, the conserved nature of tubulin proteins makes the development of isotype-specific antibodies challenging. These factors pose a critical limitation to tubulinopathy research, making visualization of specific tubulin isotypes or mutant tubulin proteins inherently difficult. To address the current technological limitations, we describe a method for immunocytochemical labeling of a single ɑ-tubulin isotype harboring distinct pathogenic mutations. This approach utilizes a novel method for cellular tubulin visualization by inserting a hexahistidine (His6) epitope into a previously identified internal loop in the ɑ-tubulin protein. This modification acts as a visualization handle, providing direct evidence of the incorporation of ectopically expressed α-tubulin TUBA1A proteins into the cellular microtubule network using available 6×-His antibodies without disrupting tubulin function. We validate and describe this approach in human iNeurons and COS-7 cells-large, flat, fibroblast-like primate cells ideal for visualizing microtubule networks. This protocol highlights the efficacy of the His6-tag in wild-type TUBA1A, polymerization-defective mutant TUBA1A-N102D, and a highly polymer-stable mutant TUBA1A-E254A. This methodological pipeline can be harnessed to study the molecular and cellular phenotypes of numerous tubulin isotypes and pathogenic mutations.