High-resolution characterization of centriole distal appendage morphology and dynamics by correlative STORM and electron microscopy

Abstract

Centrioles are vital cellular structures that form centrosomes and cilia. The formation and function of cilia depends on a set of centriole’s distal appendages. In this study, we use correlative super resolution and electron microscopy to precisely determine where distal appendage proteins localize in relation to the centriole microtubules and appendage electron densities. Here we characterize a novel distal appendage protein ANKRD26 and detail, in high resolution, the initial steps of distal appendage assembly. We further show that distal appendages undergo a dramatic ultra-structural reorganization before mitosis, during which they temporarily lose outer components, while inner components maintain a nine-fold organization. Finally, using electron tomography we reveal that mammalian distal appendages associate with two centriole microtubule triplets via an elaborate filamentous base and that they appear as almost radial finger-like protrusions. Our findings challenge the traditional portrayal of mammalian distal appendage as a pinwheel-like structure that is maintained throughout mitosis.

Phosphorylation of multiple proteins involved in ciliogenesis by Tau Tubulin kinase 2

“…Our observation that TTBK2 is able to phosphorylate components of DA and proteins associated with centriole distal end does not come as a complete surprise, given its proposed role in the assembly of the appendages (26). Moreover, thanks to the recently revealed details on DA structure and organization (21,59) it is tempting to speculate that TTBK2 might be able to target additional basal body proteins beside those included in our screen. In our initial screening, CP110 and Rab8a did not shift upon TTBK2 co-expression, hence we did not include them in the subsequent detailed analysis.…”

“…Initially, five DA proteins, CEP164 (17), CEP83 (18), CEP89 (19), FBF1 (15,20) and SCLT1 (15) have been identified. Recent identification of new DA components suggests the list might not yet be complete (21,22).…”

Procentriole microtubules as drivers of centriole reduplication

“…In addition, stable expression of centriolar marker Centrin1-GFP allows a direct read out of centriole duplication and reduplication status. To analyze localization of proteins within centrosomes, we used Stochastic Optica Reconstruction Microscopy (STORM), which allows detection of fluorophores with ~22 nm resolution (Bates et al, 2007;Bowler et al, 2019).…”

“…Multiple STORM images were taken from two or more independent experiments. The resolution in our STORM experiments was estimated based on the analysis of cytoplasmic MTs, as described in (Bowler et al, 2019).…”

Correlative cryo-structured illumination fluorescence microscopy and soft X-ray tomography elucidates reovirus intracellular release pathway

“…Correlative light and electron microscopy (CLEM) has been used extensively to date, and recent advances include the addition of super resolution visible-light fluorescence [15][16][17] . FM data, at cryogenic and non-cryogenic temperatures, have also been correlated with SXT, but in all cases it has either been diffraction-limited [18][19][20][21][22] or involved a chemical fixation step 23 with the associated risk for artefact formation 24,25 .…”