Microtubules are polar cytoskeletal polymers composed of α/β-tubulin subunits that assemble in a head-to-tail fashion, forming protofilaments which then laterally associate to form tubes. Tubulin subunits are added to growing plus end in an expanded GTP-bound state whereas subunits within the microtubule lattice are primarily in a compacted GDP-bound state. Emerging data in the field indicate that microtubule-associated proteins (MAPs) alter the conformation of tubulin to locally expand or compact the lattice, which can regulate the formation of tubulin post-translational modifications (PTMs). End-binding (EB) proteins recognize and are sensitive to the nucleotide state at growing microtubule ends and promote lattice compaction. Although much has been done to understand the role of EBs at plus ends, their roles in regulating the tubulin code are unresolved. To address this, we utilized two EB knockout (KO) systems. We found that the levels of microtubule detyrosination are increased in cells with short-term loss of EBs but are unaffected in long-term, stable EB KOs. We hypothesized that cells compensate for EB loss by altering the levels of lattice-regulating MAPs. Using quantitative mass spectrometry, we identified jupiter microtubule associated homolog 2 (JPT2) as a protein that is upregulated in long-term EB KO cells. We show that JPT2 overexpression in short-term EB KO cells reduces detyrosination, suggesting that it regulates lattice conformation. In cells, JPT2 is evicted from the lattice upon Taxol-induced lattice expansion, suggesting that JPT2 prefers a compacted lattice. In in vitro reconstitution assays, JPT2 binds to growing plus ends and has a higher affinity for microtubules in a GDP-Pi state than the GTP-like or GDP states. These results reveal JPT2 as a MAP sensitive to lattice conformation and provide new insights into how plus end proteins regulate the tubulin code.
Pimm et al. (Sun,) studied this question.