Single-molecule localization microscopy reveals the ultrastructural 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 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 expanded mammalian DAP. Importantly, our imaging workflow enables us to push the resolution limit of a light microscope well close to a molecular level, thus achieving an unprecedented mapping resolution inside intact cells. Upon this workflow, we unravel the ultra-resolved higher-order protein complexes of the DAP and its associated proteins. Intriguingly, our images show that C2CD3, microtubule triplet, MNR, CEP90, OFD1, and ODF2 jointly constitute a unique molecular configuration at the DAP base. Moreover, our finding suggests that ODF2 plays an auxiliary role in coordinating and maintaining DAP nine-fold symmetry. Together, we develop an organelle-based drift correction protocol and a two-color solution with minimum crosstalk, allowing a robust localization microscopy imaging of expanded DAP structures deep into the gel-specimen composites.

Stuck Photoswitching Event Analysis and Correction for Superresolution Single-Molecule Localization Microscopy

“…Take SCLT1, another member of the distal appendage protein; for example, our method removed stuck localizations recorded at two blades of the SCLT1 pattern (arrowheads in raw, Figure c) and enabled us to specify the protein orientation at individual blades with optimized processing (arrowheads in optimized, Figure c; Figure S8). The unique molecular chirality of the SCLT1 protein arrangement reappeared, consistent with the findings from the previous works. , Second example is given by ARL13B, a ciliary membrane marker (Figure d). The raw image displays a dot-like, dispersed distribution of ARL13B, as well as the morphology of the cilium with three bright spots of stuck localizations (arrowheads, Figure d).…”

“…The unique molecular chirality of the SCLT1 protein arrangement reappeared, consistent with the findings from the previous works. 25,52 Second example is given by ARL13B, a ciliary membrane marker (Figure 5d). The raw image displays a dot-like, dispersed distribution of ARL13B, as well as the morphology of the cilium with three bright spots of stuck localizations (arrowheads, Figure 5d).…”

Expanding boundaries – a cell biologist's guide to expansion microscopy

“…As discussed above, ExM increases resolution by physically expanding a sample. Of course, these expanded samples may then be imaged using super-resolution microscopy techniques to further increase resolution, and indeed, the major super-resolution techniques have been successfully combined with ExM, including structured illumination microscopy (SIM) ( Cahoon et al, 2017 ; Halpern et al, 2017 ), STED ( Gambarotto et al, 2019 ; Gao et al, 2018 ; Kim et al, 2019 ; Li et al, 2018 ; Pesce et al, 2019 ; Unnersjö-Jess et al, 2017 ) and, more recently, SMLM ( Chang et al, 2023 ; Shi et al, 2021 ; Xu et al, 2019 ; Zwettler et al, 2020 ). These approaches face specific challenges resulting from the nature of expanded samples.…”

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.…”