SPEF1 mediates assembly of central pair microtubule complexes in cilia of Tetrahymena

SPEF1 is a conserved calponin-homology domain protein that decorates the seam of central pair microtubules in the distal segment of motile cilia. Here, we show that in the ciliate Tetrahymena, loss of SPEF1 disrupts the central pair throughout the middle segment, which comprises most of the cilium, producing gaps in the microtubule lattice, truncations of proximal microtubule regions, and loss of lateral projections. Although visible structural defects were restricted to the central microtubules, SPEF1 also localized to the ciliary outer doublets and to all classes of non-ciliary cortical microtubules. Within cilia, SPEF1 was strongly enriched near the distal tip but was also present throughout the middle segment, where it was more abundant on the central than on the outer doublet microtubules. In live cilia, most SPEF1 particles were stationary and turned over slowly; some underwent diffusion, but none moved by intraflagellar transport. Central pair defects arose early during ciliary assembly. We propose that SPEF1 functions as a general microtubule stabilizer whose activity is particularly important for the assembly of central pair microtubules within the middle ciliary segment.