Biology subjects

Fabritius, A. S.

Publications and source records attributed to Fabritius, A. S..

3 recordsLinked to original sources

bioRxiv2024

The Structure of Cilium Inner Junctions Revealed by Electron Cryo-tomography

The cilium is a microtubule-based organelle critical for many cellular functions. Its assembly initiates at a basal body and continues as an axoneme that projects out of the cell to form a functional cilium. This assembly process is tightly regulated. However, our knowledge of the molecular architecture and the mechanism of assembly is limited. By applying electron cryo-tomography and subtomogram averaging, we obtained subnanometer resolution structures of the inner junction in three distinct regions of the cilium: the proximal region of the basal body, the central core of the basal body, and the flagellar axoneme. The structures allowed us to identify several basal body and axoneme components. While a few proteins are distributed throughout the entire length of the organelle, many are restricted to particular regions of the cilium, forming intricate local interaction networks and bolstering local structural stability. Finally, by knocking out a critical basal body inner junction component Poc1, we found the triplet MT was destabilized, resulting in a defective structure. Surprisingly, several axoneme-specific components were found to "infiltrate" into the mutant basal body. Our findings provide molecular insight into cilium assembly at its inner junctions, underscoring its precise spatial regulation.

cell biology↗

bioRxiv2021

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

Doublet microtubules (DMT) provide a scaffold for axoneme assembly in motile cilia. Aside from /{beta} 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 -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.

cell biology↗

bioRxiv2020

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

Motile cilia and flagella are built from stable populations of doublet microtubules that comprise their axonemes. Their unique stability is brought about, at least in part, by a network of Microtubule Inner Proteins (MIPs) found in the lumen of their doublet microtubules. Rib72A and Rib72B were identified as microtubule inner proteins (MIPs) in the motile cilia of Tetrahymena thermophila. Loss of these proteins leads to ciliary defects and loss of multiple MIPs. We performed mass spectrometry coupled with proteomic analysis and bioinformatics to identify the MIPs lost in RIB72A/B knockout (KO) Tetrahymena cells. From this analysis 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.

molecular biology↗