Purification of Ciliary Tubulin from Chlamydomonas reinhardtii

摘要

© 2020 Wiley Periodicals LLCCilia and flagella play essential roles in environmental sensing, cell locomotion, and development. These organelles possess a central microtubule–based structure known as the axoneme, which serves as a scaffold and is crucial for the function of cilia. Despite their key roles, the biochemical and biophysical properties of the ciliary proteins are poorly understood. To address this issue, we have developed a novel method to purify functional tubulins from different parts of the axoneme, namely the central pair and B-tubule. We use the biflagellate green alga Chlamydomonas reinhardtii, a model organism for studying cilia due to the conserved structure of this organelle, availability of genetic tools and a large collection of mutant strains. Our method yields highly purified functional axonemal tubulins in sufficient quantities to be used for in vitro biochemical and biophysical studies, such as microtubule dynamic assays. It takes 7 to 8 days to grow enough cells; the isolation of the flagella and the purification of the axonemal tubulins require an additional two full days.© 2020 Wiley Periodicals LLC. Basic Protocol 1: Growth and harvest of large volume of cell culture. Support Protocol: Assembly of homemade concentration apparatus. Basic Protocol 2: Isolation of flagella. Basic Protocol 3: Tubulin extraction and purification.