A cryo-ET study of ciliary rootlet organization

Reviewer #1 (Public Review):

Summary:
Ciliary rootlet is a structure associated with the ciliary basal body (centriole) with beautiful striation observed by electron microscopy. It has been known for more than a century, but its function and protein arrangement are still unknown. This work reconstructed the near-atomic resolution 3D structure of the rootlet using cryo-electron tomography, discovered a number of interesting filamentous structures inside, and built a molecular model of the rootlet.

Strengths:
The authors exploited the currently possible ability of cryo-ET and used it appropriately to describe the 3D structure of the rootlet. They carefully conducted subtomogram averaging and classification, which enabled an unprecedented detailed view of this structure. The dual use of (nearly) intact rootlets from cilia and extracted (demembraned) rootlets enabled them to describe with confidence how D1/D2/A bands form periodic structures and cross with longitudinal filaments, which are likely coiled-coil.

Weaknesses:
Some more clarifications are needed. This reviewer believes that the authors can address them.

Reviewer #2 (Public Review):

Summary:
This work performs structural analysis on isolated or purified rootlets.

Strengths:
To date, most studies of this cellular assembly have been from fluorescence microscopy, conventional TEM methods, or through biochemical analysis of constituents. It is clearly a challenging target for structural analysis due to its complexity and heterogeneity. The authors combine observations from cryo-electron tomograms, automated segmentations, subtomogram averaging, and previous data from the literature to present an overall model of how the rootlet is organised.

Their model will serve as a jumping-off point for future studies, and as such it is something of considerable value and interest.

Weaknesses:
It is speculative but is presented as such, and is well-reasoned, plausible, and thorough.

Reviewer #3 (Public Review):

Summary:
The study offers a compelling molecular model for the organization of rootlets, a critical organelle that links cilia to the basal body. Striations have been observed in rootlets, but their assembly, composition, and function remain unknown. While previous research has explored rootlet structure and organization, this study delivers an unprecedented level of resolution, valuable to the centrosome and cilia field. The authors isolated rootlets from mice's eyes. They apply EM to partially purified rootlets (first negative stain, then cryoET). From these micrographs, they observed striations along the membranes along the rootlet but no regular spacing was observed.

The thickness of the sample and membranes prevented good contrast in the tomograms. Thus they further purified the rootlets using detergent, which allowed them to obtain cryoET micrographs of the rootlets with greater details. The tomograms were segmented and further processed to improve the features of the rootlet structures. From their analysis, they described 3 regular cross-striations and amorphous densities, which are connected perpendicularly to filaments along the length of the rootlets. They propose that various proteins provide the striations and rootletin forms parallel coiled coils that run along the rootlet. Overall their data provide a detailed model for the molecular organization of the rootlet.

The major strength is that this high-quality study uses state-of-the-art cryo-electron tomography, sub-tomogram averaging, and image analysis to provide a model of the molecular organization of rootlets. The micrographs are exceptional, with excellent contrast and details, which also implies the sample preparation was well optimized to provide excellent samples for cryo-ET. The manuscript is also clear and accessible.

To further validate their model, it would have been useful to identify some components in the EM maps through complementary approaches (mass spectrometry, mutants disrupting certain features, CLEM). Some potential candidates are mentioned in the discussion.

This research marks a significant step forward in our understanding of rootlets' molecular organization.

Author Response

We appreciate the feedback from all the reviewers. We will incorporate their comments into the revised manuscript.

In response to reviewer three's suggestion regarding complementary approaches for identifying rootlet components, we'd like to provide further insight into the strategies we explored.

We performed mass spectrometry on our purified rootlets. This identified the rootlet components rootletin and CCDC102B and various axonemal components, due to the association between the rootlet and axoneme. However, due to the limitations in quantifying components using mass spectrometry, we were unable to confidently identify novel rootlet constituents present in quantities comparable to rootletin.

We further attempted cross-linking mass spectrometry on the rootlets to gain deeper insights to the interactions between rootletin molecules. Unfortunately, this effort resulted in a completely insoluble sample despite extended digestion times, leading to issues with mass spectrometry column clogging and rendering our results inconclusive.

We attempted to express rootlet components recombinantly and were able to purify fibres, but they did not contain the characteristic repeat pattern seen in native rootlets. We also considered purifying native rootlets from cultured cells, but realized the yield would be too low for cryo-ET studies.

We therefore regret that other approaches to validate our model are outside the scope of this current work.