Single-molecule localization microscopy reveals the ultrastructural root constitution of distal appendages in expanded mammalian centrioles

Abstract

Distal appendages (DAPs) are vital in cilia formation, mediating vesicular and ciliary docking to the plasma membrane during early ciliogenesis. Although numerous DAP proteins arranging a nine-fold symmetry have been studied using superresolution microscopy analyses, the extensive ultrastructural understanding of the DAP root structure developing from the centriole wall remains elusive owing to insufficient resolution. Here, we proposed a pragmatic imaging strategy for two-color single-molecule localization microscopy of swellable mammalian DAP proteins. Importantly, our imaging workflow enables us to push the resolution limit of a light microscope well close to an electron microscopy level, thus achieving an unprecedented λ/200 mapping precision inside intact cells. Upon this workflow, we unravel the ultra-resolved higher-order protein complexes of the core DAP. Intriguingly, C2CD3, microtubule triplet, and ODF2 jointly constitute the spatial basis of DAP, suggesting a unique configuration of the DAP assembly. Moreover, our results show that the distal-layered ODF2 labeled at the N- and C-terminus construct a fastening unit encircling the microtubule triplets. Together, we develop an organelle-based drift correction protocol and a two-color imaging solution with minimum crosstalk, allowing a robust localization microscopy imaging of expanded cellular structures deep into the gel-specimen composites.

Expansion microscopy of apicomplexan parasites

“…Another obvious advantage of U‐ExM is that it is compatible with most super‐resolution imaging modalities, increasing the potential usefulness of the technique. To date, Airyscan (Calla et al, 2023; Liffner & Absalon, 2021; Liffner et al, 2023; Morano et al, 2023) is the only super‐resolution technique that has been used on expanded parasites to the best of our knowledge, but stimulated emission depletion (STED) (Gambarotto et al, 2019), 3D structured illumination microscopy (SIM) (Woglar et al, 2022) and single molecule localization microscopy (SMLM) (Chang et al, 2023) have been applied to U‐ExM on other sample types. Additionally, U‐ExM may increase antibody accessibility for certain proteins, with reduced antibody competition having been previously observed for expanded microtubules (Gambarotto et al, 2019).…”

Centriole and transition zone structures in photoreceptor cilia revealed by cryo-electron tomography

“…We have not included distal or subdistal appendages in our models, which have been built from subtomogram average maps of limited volumes and are based primarily on the MT. These structures, which have been extensively studied in other cell types (Bowler et al, 2019; Chang et al, 2023) are visible in our maps, but would require much larger volumes in the boxing step carried out prior to subtomogram averaging.…”

Towards understanding centriole elimination

“…Obviously, monitoring several centriolar proteins reduces the risk of being misled in such cases. Moreover, expansion microscopy methods now enable resolutions that are sufficient to monitor the signature 9-fold radially symmetrical distribution of microtubules [109,110], and are likely to become a standard means to ascertain with reasonable throughput whether foci bearing centriolar proteins also harbour centriolar microtubules.…”

“…The exact mechanism through which SAS-1, and potentially C2CD3, confers stability to the centriole is unclear. However, given the striking 9-fold radially symmetric distribution of the two proteins uncovered by expansion microscopy, just within the confines of the microtubule wall, as well as their organelle stabilizing function, it is tempting to speculate that these proteins form an inner brace that somehow hold together centriolar microtubules [109,110,192]. Removing this brace may destabilize centrioles, thus potentially offering a handle to modulate organelle elimination.…”