Tubulin Lattice in Cilia is in a Stressed Form Regulated by Microtubule Inner Proteins

Abstract

17Cilia, the hair-like protrusions that beat at high frequencies to propel a cell or move fluid around 18 the cell, are composed of radially bundled doublet microtubules. The doublet microtubule is 19 composed of a 13-protofilament A-tubule, a partial 10-protofilament B-tubule and microtubule 20 inner proteins (MIPs) inside the tubulin lattice. In this study, we present the near-atomic 21 resolution map of the Tetrahymena doublet microtubules. The map demonstrates that the 22 network of microtubule inner proteins is weaving into the tubulin lattice, forming an inner sheath 23 of proteins. In addition, we also obtain the tubulin lattice structure with missing MIPs by Sarkosyl 24 treatment. In this structure, the tubulin lattice showed significant longitudinal compaction and 25 lateral angle changes between protofilaments. These results are evidence that the binding of 26MIPs directly affects and stabilizes the tubulin lattice. It is also suggested that the doublet 27 microtubule is an intrinsically stressed filament and this stress could be exploited in the 28 regulation of ciliary waveforms. 29 30 rigidity, damage resistance and stability similar to what microtubule-associated proteins affects 52 the properties of singlets (10). 53Recently, Rib72a/b were characterized as components of MIPs inside the A-tubule of 54Tetrahymena (11) while FAP45 and FAP52 were identified as MIPs in the B-tubule in 55Chlamydomonas (10). The Rib72a/b knockout caused reduced Tetrahymena swimming speed. 56The B-tubule of Chlamydomonas FAP45 and FAP52 double knockout mutant were more 57 vulnerable to depolymerization. These studies highlight the global effects of MIPs on motility and 58 stability of cilia. The resolution of the doublet structures presented in these studies, however, 59 was insufficient to uncover how these proteins affect the tubulin lattice. In this study, we 60 obtained near-atomic resolution maps of the doublet and the fractionated A-tubule from 61Tetrahymena cilia to understand the influence of MIPs to the tubulin lattice. 62

The inner junction complex of the cilia is an interaction hub that involves tubulin post-translational modifications

“…1B) (5). When comparing to the cryo-EM structure of the 48-nm repeating unit of Tetrahymena (7, 8), the IJ region bridging PF B10 and A1 remained intact (Figure 1A-D). Based on previous studies (15, 16, 23), we were able to localize FAP52, FAP45 in both Tetrahymena and Chlamydomonas (FAP52, light green and FAP45, yellow-green in Fig.…”

“…First, the B-tubule is held up by tether density 1 (red, Fig. 1H), referred to as MIP3b previously (7, 8). Tether density 1 connects the PF B9/B10 and A13.…”

“…Ciliary components, important for motility such as the outer and inner dynein arms, radial spokes and the dynein regulatory complex (DRC) are assembled onto the surface of the doublet (3-6). Inside the doublets, is a weaving network of proteins, termed microtubule-inner-proteins (MIPs), that bind to the inner lumen surface of the doublet (7, 8). These MIPs act to stabilize the microtubule and very likely regulate the ciliary waveform through interactions with the tubulin lattice (8).…”

“…Inside the doublets, is a weaving network of proteins, termed microtubule-inner-proteins (MIPs), that bind to the inner lumen surface of the doublet (7, 8). These MIPs act to stabilize the microtubule and very likely regulate the ciliary waveform through interactions with the tubulin lattice (8).…”

“…Cryo-EM structures of isolated doublets from Tetrahymena show that there are different tethering densities that connect the B-tubule to the A-tubule aside from the IJ (7, 8). Taken together, these data suggest that there is a complex interaction at the IJ region involving multiple proteins in addition to PACRG, FAP20, FAP45 and FAP52.…”