Proteomic analysis of microtubule inner proteins (MIPs) in Rib72 null Tetrahymena cells reveals functional MIPs.

The core structure of motile cilia and flagella, the axoneme, is built from a stable population of doublet microtubules. This unique stability is brought about, at least in part, by a network of microtubule inner proteins (MIPs) that are bound to the luminal side of the microtubule walls. Rib72A and Rib72B were identified as MIPs in the motile cilia of the protist Tetrahymena thermophila. Loss of these proteins leads to ciliary defects and loss of additional MIPs. We performed mass spectrometry coupled with proteomic analysis and bioinformatics to identify the MIPs lost in RIB72A/B knockout Tetrahymena axonemes. We identified a number of candidate MIPs and pursued one, Fap115, for functional characterization. We find that loss of Fap115 results in disrupted cell swimming and aberrant ciliary beating. Cryo-electron tomography reveals that Fap115 localizes to MIP6a in the A-tubule of the doublet microtubules. Overall, our results highlight the complex relationship between MIPs, ciliary structure, and ciliary function.

Functional refolding of the penetration protein on a non-enveloped virus.

A non-enveloped virus requires a membrane lesion to deliver its genome into a target cell1. For rotaviruses, membrane perforation is a principal function of the viral outer-layer protein, VP42,3. Here we describe the use of electron cryomicroscopy to determine how VP4 performs this function and show that when activated by cleavage to VP8* and VP5*, VP4 can rearrange on the virion surface from an 'upright' to a 'reversed' conformation. The reversed structure projects a previously buried 'foot' domain outwards into the membrane of the host cell to which the virion has attached. Electron cryotomograms of virus particles entering cells are consistent with this picture. Using a disulfide mutant of VP4, we have also stabilized a probable intermediate in the transition between the two conformations. Our results define molecular mechanisms for the first steps of the penetration of rotaviruses into the membranes of target cells and suggest similarities with mechanisms postulated for other viruses.

Tetrahymena RIB72A and RIB72B are microtubule inner proteins in the ciliary doublet microtubules.

Doublet and triplet microtubules are essential and highly stable core structures of centrioles, basal bodies, cilia, and flagella. In contrast to dynamic cytoplasmic micro-tubules, their luminal surface is coated with regularly arranged microtubule inner proteins (MIPs). However, the protein composition and biological function(s) of MIPs remain poorly understood. Using genetic, biochemical, and imaging techniques, we identified Tetrahymena RIB72A and RIB72B proteins as ciliary MIPs. Fluorescence imaging of tagged RIB72A and RIB72B showed that both proteins colocalize to Tetrahymena cilia and basal bodies but assemble independently. Cryoelectron tomography of RIB72A and/or RIB72B knockout strains revealed major structural defects in the ciliary A-tubule involving MIP1, MIP4, and MIP6 structures. The defects of individual mutants were complementary in the double mutant. All mutants had reduced swimming speed and ciliary beat frequencies, and high-speed video imaging revealed abnormal highly curved cilia during power stroke. Our results show that RIB72A and RIB72B are crucial for the structural assembly of ciliary A-tubule MIPs and are important for proper ciliary motility.

The nexin link and B-tubule glutamylation maintain the alignment of outer doublets in the ciliary axoneme.

We developed quantitative assays to test the hypothesis that the N-DRC is required for integrity of the ciliary axoneme. We examined reactivated motility of demembranated drc cells, commonly termed "reactivated cell models." ATP-induced reactivation of wild-type cells resulted in the forward swimming of ∼90% of cell models. ATP-induced reactivation failed in a subset of drc cell models, despite forward motility in live drc cells. Dark-field light microscopic observations of drc cell models revealed various degrees of axonemal splaying. In contrast, >98% of axonemes from wild-type reactivated cell models remained intact. The sup-pf4 and drc3 mutants, unlike other drc mutants, retain most of the N-DRC linker that interconnects outer doublet microtubules. Reactivated sup-pf4 and drc3 cell models displayed nearly wild-type levels of forward motility. Thus, the N-DRC linker is required for axonemal integrity. We also examined reactivated motility and axoneme integrity in mutants defective in tubulin polyglutamylation. ATP-induced reactivation resulted in forward swimming of >75% of tpg cell models. Analysis of double mutants defective in tubulin polyglutamylation and different regions of the N-DRC indicate B-tubule polyglutamylation and the distal lobe of the linker region are both important for axonemal integrity and normal N-DRC function. © 2016 Wiley Periodicals, Inc.