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

Mathew Bowler, Dong Kong, Shufeng Sun, Rashmi Nanjundappa, Lauren Evans, Veronica Farmer, Andrew Holland, Moe R. Mahjoub, Haixin Sui & Jadranka Loncarek

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.

How Amira-Avizo Software is used

Segmentation of the tomogram and visualization of the distal appendage were carried out using the 3D interactive tool in Amira software (Thermo Fisher). Before segmentation, the “slicer” function in IMOD software was used to rotate the tomogram and look for a suitable orientation for convenient visualization and model building. Based on the orientation parameters in “slicer”, the tomogram was rotated using programs in Bsoft package. The re-orientated tomogram was imported into Amira software for structural visualization, model building, and movies making.