Proteomic analysis of microtubule inner proteins (MIPs) in Rib72 null<i>Tetrahymena</i>cells reveals functional MIPs

Fabritius, Amy S.; Bayless, Brian A.; Li, Sam; Stoddard, Daniel; Heydeck, Westley; Ebmeier, Christopher C.; Anderson, Lauren; Gunnels, Tess; Nachiappan, Chidambaram; Whittall, Justen B.; Old, William M.; Agard, David A.; Winey, Mark

doi:10.1101/2020.10.02.324467

Cited by 2 publications

(9 citation statements)

References 40 publications

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“…The average of the FAP115KO mutant reached 9.6 Å in resolution (Figure 4A, Figure S4A, Table 1). Compared to the wild-type, the bi-lobed structure of Fap115 is absent in the mutant (Figure 4B, Figure S4B), confirming the depletion of Fap115 in the mutant (Fabritius et al, 2020). However, Rib72A/B remain in the FAP115KO mutant structure (Figure 4B).…”

Section: Fap115 Knockout Results In Axoneme Defects Beyond Its Binding Sitementioning

confidence: 64%

“…The EF-hand protein Fap115 was identified as one of the MIPs lost in the Rib72A/B knockout mutants (Fabritius et al, 2020;Ma et al, 2019;Stoddard et al, 2018). In this study, we provide a detailed description of Fap115 binding to the DMT: The protein bridges pf A02 and A03 on the luminal wall of the A-tubule, making multiple interactions with the adjacent proteins; its NTD interacts with α/β tubulin in pf A02.…”

Section: Dmtmentioning

confidence: 95%

“…The conserved Rib72 protein is an elongated multidomain MIP essential for flagella function and for recruiting other MIPs to the organelle (Fabritius et al, 2020;Ma et al, 2019;Stoddard et al, 2018). A human homolog of Rib72, EFHC1, is linked to juvenile myoclonic epilepsy (Gonsales et al, 2020;Suzuki et al, 2020Suzuki et al, , 2004.…”

Section: Rib72a/b On the Luminal Wall Of A-tubulementioning

confidence: 99%

“…Remarkably, the axoneme of the motile cilium/flagellum is one of the most conserved structures during the evolution of the eukaryotic branches of the phylogenetic tree of life (Bornens, 2018;Carvalho-Santos et al, 2011;Cavalier-Smith, 2002;Mitchell, 2016;Satir et al, 2008;Satir and Christensen, 2007). In addition to comparative genomic and proteomic analyses in different organisms (Avidor-Reiss et al, 2004;Fabritius et al, 2020;Li et al, 2004;Ostrowski et al, 2002;Pazour et al, 2005;Smith et al, 2005), structural comparisons of cilia among different organisms have been carried out previously, showing both evolutionary conservation and diversity of this organelle (Carbajal-González et al, 2013;Khalifa et al, 2020;Lin et al, 2014;Nicastro et al, 2011;Pigino et al, 2012). However, most studies were limited to morphological descriptions, partly due to the limited resolution in the structures.…”

Section: Structural Conservation and Diversity Of Mips In Two Model Organismsmentioning

confidence: 99%

“…All strains were grown in the standard medium 2% SPP (2% protease peptone, 0.1% yeast extract, 0.2% glucose, 0.003% FeCl3) at 30°C. The FAP115 knockout strain was generated as described previously (Fabritius et al, 2020).…”

Section: Isolation Of Axoneme From the Tetrahymena Strainsmentioning

confidence: 99%

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Electron Cryo-Tomography Structure of Axonemal Doublet Microtubule from Tetrahymena thermophila

Fernández

et al. 2021

Preprint

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Doublet microtubules (DMT) provide a scaffold for axoneme assembly in motile cilia. Aside from α/β tubulins, the DMT comprises a large number of non-tubulin proteins in the luminal wall of DMT, collectively named the microtubule inner proteins (MIPs). We used electron cryo-tomography to study axoneme DMT isolated from Tetrahymena thermophila. We present the structures of DMT at nanometer and sub-nanometer resolution. The structures confirm that MIP Rib72A/B binds to the luminal wall of the DMT by multiple DM10 domains, likely by recognizing the acetylated K40 residue of α-tubulin. We found Fap115, a MIP containing multiple EF-hand domains, located at the interface of four-tubulin dimers in the lumen of the A-tubule. It functions as a "molecular staple" stabilizing both lateral and longitudinal tubulin interfaces and playing a critical role in DMT stability. Defects caused by the depletion of Fap115 propagate along the axoneme due to extensive structural changes in the DMT at and beyond the Fap115 binding site. Finally, by comparing DMT structures from Tetrahymena and Chlamydomonas, we have identified a number of conserved MIPs as well as MIPs that are unique to each organism. This conservation and diversity of the DMT structures might be linked to their specific functions. Our work provides structural insights essential for understanding the roles of MIPs during motile cilium assembly and function, as well as their relationships to human ciliopathies.

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Section: Fap115 Knockout Results In Axoneme Defects Beyond Its Binding Sitementioning

confidence: 64%

Section: Dmtmentioning

confidence: 95%

Section: Rib72a/b On the Luminal Wall Of A-tubulementioning

confidence: 99%

Section: Structural Conservation and Diversity Of Mips In Two Model Organismsmentioning

confidence: 99%

Section: Isolation Of Axoneme From the Tetrahymena Strainsmentioning

confidence: 99%

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Electron Cryo-Tomography Structure of Axonemal Doublet Microtubule from Tetrahymena thermophila

Fernández

et al. 2021

Preprint

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FAP106 is an interaction hub required for assembly of conserved and lineage-specific microtubule inner proteins at the cilium inner junction

Shimogawa

Wijono

Wang

et al. 2022

Preprint

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Eukaryotic motile cilia/flagella are conserved organelles important for cell propulsion and fluid flow, typically built around a "9+2" axoneme of nine doublet microtubules (DMTs) encircling a central pair of singlet microtubules. The DMT lumen is lined with an interconnected network of microtubule inner proteins (MIPs), some conserved and others lineage-specific. MIPs augment the tubulin lattice of the DMT, directly impacting stability, fine structure, and motility, thus providing an important source of lineage-specific adaptations.Trypanosoma bruceiis a flagellated eukaryotic pathogen with distinctive motility that is critical for pathogen transmission and pathogenesis. Prior studies revealed lineage-specificT. bruceiMIPs, but their identities are unknown. To identifyT. bruceiMIPs, we examined flagellum structure and composition following knockdown of FAP106, a conserved MIP at the inner junction (IJ) connecting A- and B-microtubules of the DMT. FAP106 knockdown resulted in short flagella and defective parasite motility, supporting a role for MIPs inT. bruceiflagellum stability and motility. Cryogenic electron tomography (cryoET) and quantitative proteomics identified several conserved MIPs and lineage-specific MIP structures and MIP candidate proteins (MCs) that depend on FAP106 for stable assembly. We further demonstrate by knockdown and fitting AlphaFold models to cryoET maps that one of these, MC8, is a newly identified lineage-specific MIP required for normal parasite motility. This work provides an important advance toward elucidating the order of assembly of MIPs at the cilium inner junction and identifies trypanosome proteins specific to these deadly pathogens that represent targets to consider for therapeutic intervention.

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Proteomic analysis of microtubule inner proteins (MIPs) in Rib72 nullTetrahymenacells reveals functional MIPs

Cited by 2 publications

References 40 publications

Electron Cryo-Tomography Structure of Axonemal Doublet Microtubule from Tetrahymena thermophila

Electron Cryo-Tomography Structure of Axonemal Doublet Microtubule from Tetrahymena thermophila

FAP106 is an interaction hub required for assembly of conserved and lineage-specific microtubule inner proteins at the cilium inner junction

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