Systematic Identification of Microtubule Inner Proteins Reveals JPT2 as a Key Regulator of Lumen Microenvironment and Drug Sensitivity

Abstract Microtubules are primarily studied for the interactions of proteins that bind to their outer surfaces and ends, while the regulatory mechanisms within the microtubule lumen, particularly in singlet microtubules critical for essential cellular processes, remain largely unexplored. Our study provides the first systematic identification of key regulatory proteins within the single microtubule lumen. Using proximity-dependent biotin identification (Bio-ID) coupled with mass spectrometry, we identified candidate microtubule inner proteins (MIPs), including Jupiter microtubule-associated homolog 2 (JPT2). JPT2 binds directly to microtubules and specifically localizes within the lumen, where it modulates the luminal environment by inhibiting acetylase MEC17 and independently affects the binding and efficacy of Paclitaxel. Furthermore, our screening identified additional MIPs that influence cellular sensitivity to Paclitaxel, indicating a link between luminal regulation and drug responsiveness. These discoveries reveal JPT2’s critical role in singlet microtubule regulation and suggest new therapeutic targets for enhancing cancer drug sensitivity.