Microtubules are dynamic polymers of α/β-tubulin heterodimers central to cellular processes, such as cell division, cell migration, and organelle transportation. Higher eukaryotes not only have microtubule architectures with diverse structures and functions but also encode substantially expanded α- and β-tubulin gene families (i.e., isotypes). Genetics and cell biology studies suggest that each tubulin gene has unique cellular functions. In particular, point mutations in specific human α- or β-tubulin isotypes have been associated with human diseases. The discoveries of disease-related mutations suggest that, even with the high degree of identity on the primary sequence basis, tubulin isotypes do not complement each other in functions.
With our strategy to generate isotypically pure recombinant human tubulin (Ti. et al. 2018 Dev Cell; Ti. et al. 2020 STAR Protocols), we employ chemical biology tools, high-resolution fluorescence/light microscopy-based in vitro reconstitution assays, cryogenic electron microscopy (cryo-EM) structural analysis of microtubules and in vivo cell biology to dissect the underlying molecular mechanisms for tubulin isotypes to regulate microtubule networks. Currently, we are pursuing the following goals:
1. Dissecting the interplay between tubulin isotypes and post-translational modifications
α- and β-tubulins are subject to the dynamic regulation of various post-translational modifications (PTMs). Both tubulin isotypes and PTMs have been shown to control the intrinsic properties of microtubules (e.g., lattice organization, polymerization dynamics, and mechanical resilience). We are dissecting the interplay between isotypes and PTMs that controls microtubule properties, manifesting in the cellular structures and functions.
2. Revealing the pathological mechanisms of disease-associated tubulin mutations
Mutations in tubulin isotypes have been associated with many human diseases, including neurological disorders, impaired oocyte maturation, and defective platelet formation. We are establishing an interdisciplinary platform that will correlate the effects of mutant tubulins on microtubule organization and stability with diseases' phenotypes.
3. Developing tools to investigate the functions of tubulin isotypes in cells
As tubulin mutagenesis in mammals causes complex phenotypes and sometimes embryonic lethality, it has been challenging to dissect the cellular functions of tubulin isotypes using classic approaches. By employing protein engineering and chemical biology strategies, we are developing tools that will open a new avenue to dissecting the critical roles of tubulin isotypes and PTMs in diverse cellular processes.