Microtubules are cytoskeletal structures involved in stability, transport and organization in the cell. The building blocks, the α- and β-tubulin heterodimers, form protofilaments that associate laterally into the hollow microtubule. Microtubule also exists as highly stable doublet microtubules in the cilia where stability is needed for ciliary beating and function. The doublet microtubule maintains its stability through interactions at its inner and outer junctions where its A- and B-tubules meet. Here, using cryo-electron microscopy, bioinformatics and mass spectrometry of the doublets of Chlamydomonas reinhardtii and Tetrahymena thermophila, we identified two new inner junction proteins, FAP276 and FAP106, and an inner junction-associated protein, FAP126, thus presenting the complete answer to the inner junction identity and localization. Our structural study of the doublets shows that the inner junction serves as an interaction hub that involves tubulin post-translational modifications. These interactions contribute to the stability of the doublet and hence, normal ciliary motility.

Mechanoreceptors are sensory cells that transduce mechanical stimuli into electrical signals and mediate the perception of sound, touch and acceleration. Ciliated mechanoreceptors possess an elaborate microtubule cytoskeleton that facilitates the coupling of external forces to the transduction apparatus. In a screen for genes preferentially expressed in Drosophila campaniform mechanoreceptors, we identified DCX-EMAP, a unique member of the EMAP family (echinoderm-microtubule-associated proteins) that contains two doublecortin domains. DCX-EMAP localizes to the tubular body in campaniform receptors and to the ciliary dilation in chordotonal mechanoreceptors in Johnston's organ, the fly's auditory organ. Adult flies carrying a piggyBac insertion in the DCX-EMAP gene are uncoordinated and deaf and display loss of mechanosensory transduction and amplification. Electron microscopy of mutant sensilla reveals loss of electron-dense materials within the microtubule cytoskeleton in the tubular body and ciliary dilation. Our results establish a catalogue of candidate genes for Drosophila mechanosensation and show that one candidate, DCX-EMAP, is likely to be required for mechanosensory transduction and amplification.

Cells increase microtubule dynamics to make large rearrangements to their microtubule cytoskeleton during cell division. Changes in microtubule dynamics are essential for the formation and function of the mitotic spindle, and misregulation can lead to aneuploidy and cancer. Using in vitro reconstitution assays we show that the mitotic spindle protein Cytoskeleton-Associated Protein 2 (CKAP2) has a strong effect on nucleation of microtubules by lowering the critical tubulin concentration 100-fold. CKAP2 increases the apparent rate constant k_a of microtubule growth by 50-fold and increases microtubule growth rates. In addition, CKAP2 strongly suppresses catastrophes. Our results identify CKAP2 as the most potent microtubule growth factor to date. These finding help explain CKAP2’s role as an important spindle protein, proliferation marker, and oncogene.

Regulation of microtubule dynamics by microtubule-associated proteins (MAPs) is essential for mitotic spindle assembly and chromosome segregation. Altered microtubule dynamics, particularly increased microtubule growth rates, were found to be a contributing factor for the development of chromosomal instability, which potentiates tumorigenesis. The MAP XMAP215/CKAP5 is the only known microtubule growth factor, and whether other MAPs regulate microtubule growth in cells is unclear. Our recent in vitro reconstitution experiments have demonstrated that Cytoskeleton-Associated Protein 2 (CKAP2) increases microtubule nucleation and growth rates, and here, we find that CKAP2 is also an essential microtubule growth factor in cells. By applying CRISPR-Cas9 knock-in and knock-out (KO) as well as microtubule plus-end tracking live cell imaging, we show that CKAP2 is a mitotic spindle protein that ensures faithful chromosome segregation by regulating microtubule growth. Live cell imaging of endogenously labeled CKAP2 showed that it localizes to the spindle during mitosis and rapidly shifts its localization to the chromatin upon mitotic exit before being degraded. Cells lacking CKAP2 display reduced microtubule growth rates and an increased proportion of chromosome segregation errors and aneuploidy that may be a result of an accumulation of kinetochore–microtubule misattachments. Microtubule growth rates and chromosome segregation fidelity can be rescued upon ectopic CKAP2 expression in KO cells, revealing a direct link between CKAP2 expression and microtubule dynamics. Our results unveil a role of CKAP2 in regulating microtubule growth in cells and provide a mechanistic explanation for the oncogenic potential of CKAP2 misregulation.