Super-resolution microscopy reveals coupling between mammalian centriole subdistal appendages and distal appendages

Abstract

Subdistal appendages (sDAPs) are centriolar elements that are observed proximal to the distal appendages (DAPs) in vertebrates. Despite the obvious presence of sDAPs, structural and functional understanding of them remains elusive. Here, by combining super-resolved localization analysis and CRISPR-Cas9 genetic perturbation, we find that although DAPs and sDAPs are primarily responsible for distinct functions in ciliogenesis and microtubule anchoring, respectively, the presence of one element actually affects the positioning of the other. Specifically, we find dual layers of both ODF2 and CEP89, where their localizations are differentially regulated by DAP and sDAP integrity. DAP depletion relaxes longitudinal occupancy of sDAP protein ninein to cover the DAP region, implying a role of DAPs in sDAP positioning. Removing sDAPs alter the distal border of centrosomal γ-tubulins, illustrating a new role of sDAPs. Together, our results provide an architectural framework for sDAPs that sheds light on functional understanding, surprisingly revealing coupling between DAPs and sDAPs.

Modeling a Microtubule Filaments Mesh Structure from Confocal Microscopy Imaging

“…Recent super resolution imaging for mammalian centriole subdistal appendages and distal appendages [ 20 ] showed a clear filament assembly structure of microtubules, with intriguing biological findings. For detailed discussions of molecular interactions on the supramolecular level, complex 3D models made by Blender were effectively used.…”

Chemical Analysis of Single Cells and Organelles

“…Figure 4 provides examples of the super-resolved architecture of cellular compartments that can be revealed by super-resolution microscopy. These examples are discussed throughout this section and include images of a subset of microtubules around the mother centriole in human retinal pigment epithelial cells ( Figure 4 A), dSTORM images of an axial view of centrioles showing the radial distribution of various critical proteins that were not resolvable with wide-field (WF) imaging ( Figure 4 B), 53 time-lapse STED imaging of mitochondrial dynamics in cells ( Figure 4 C), 54 representative dSTORM images of chemically fixed HeLa cells expressing vimentinBC2T ( Figure 4 D, right) 55 and WF/dSTORM images with intracellular live-cell labeling and imaging of clickable microtubule-associated protein (EMTB) in COS-7 cells ( Figure 4 D, left), 56 rapid time-lapse imaging of peroxisomes with smart RESOLFT ( Figure 4 E), 47 dendritic and axonal profiles with MemBright (blue) to visualize glutamate AMPA receptor clusters (green) aggregated at the dendrite in front of the axonal profile with an interleaved 3D STORM stack ( Figure 4 F), 57 a DNA-PAINT image of NUP96-SNAP in U2OS cells ( Figure 4 G, left) and NUP107-GFP in HeLa cells ( Figure 4 G, right) revealing the nuclear pore complexes, 58 and images of the Golgi apparatus with panExM/STED ( Figure 4 H). 59 …”

“…Scale bar: 250 nm. Panels A and B are reproduced from Chong, W. M.; Wang, W. J.; Lo, C. H.; et al eLife 2020 , 9 , 1–21 (ref ( 53 )). Panel C is reproduced and adapted with permission from Proceedings of the National Academy of Sciences; Wang, C.; Taki, M.; Sato, Y.; et al J. Proc.…”

With Age Comes Maturity: Biochemical and Structural Transformation of a Human Centriole in the Making

“…Accumulation of SDA and DA components begins on younger mother centrioles in G2 2 and it involves a sequential incorporation of these components from the centriole’s MT wall toward the periphery [ 19 , 31 , 209 , 210 , 211 ]. The assembly of SDAs is initiated by the recruitment of Odf2 around the centriole’s MTs in G2 2 , followed by the sequential recruitment of SDA components Cep128, CCDC68, CCDC120, Nde1, Centriolin, Ninein, and Cep170 ( Figure 4 D) [ 209 , 211 , 212 ]. The assembly of DAs starts with the recruitment of C2CD3, followed by CCDC41/CEP83 in G2 2 .…”