STED Microscopy with Optimized Labeling Density Reveals 9-Fold Arrangement of a Centriole Protein

Abstract

Super-resolution fluorescence microscopy can achieve resolution beyond the optical diffraction limit, partially closing the gap between conventional optical imaging and electron microscopy for elucidation of subcellular architecture. The centriole, a key component of the cellular control and division machinery, is 250 nm in diameter, a spatial scale where super-resolution methods such as stimulated emission depletion (STED) microscopy can provide previously unobtainable detail. We use STED with a resolution of 60 nm to demonstrate that the centriole distal appendage protein Cep164 localizes in nine clusters spaced around a ring of ∼300 nm in diameter, and quantify the influence of the labeling density in STED immunofluorescence microscopy. We find that the labeling density dramatically influences the observed number, size, and brightness of labeled Cep164 clusters, and estimate the average number of secondary antibody labels per cluster. The arrangements are morphologically similar in centrioles of both proliferating cells and differentiated multiciliated cells, suggesting a relationship of this structure to function. Our STED measurements in single centrioles are consistent with results obtained by electron microscopy, which involve ensemble averaging or very different sample preparation conditions, suggesting that we have arrived at a direct measurement of a centriole protein by careful optimization of the labeling density.

The Rac1 regulator ELMO controls basal body migration and docking in multiciliated cells through interaction with Ezrin

“…Cep164, as a marker of the mother basal body in primary cilia, was reported to localise to all basal bodies in MCCs (Lau et al, 2012). Our findings show a colocalisation of ELMO1 with Cep164 in both cell types ( Fig.…”

The Centrosome and Its Duplication Cycle

“…Superresolution techniques overcome the limits imposed on conventional microscopy by diffraction of light and resolve what was previously seen by conventional immunostaining as an unstructured blob into a tiny cylinder (Sillibourne et al 2011;Fu and Glover 2012;Lau et al 2012;Lawo et al 2012;Mennella et al 2012;Sonnen et al 2012). In this way, the mature Drosophila centrosome, for example, has been resolved into five major regions and three for its engaged daughter (see Fig.…”

CEP164-null cells generated by genome editing show a ciliation defect with intact DNA repair capacity

“…Immunoelectron microscopy demonstrated the localisation of CEP164 to the distal end of the mother centriole . Dual photoactivated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM) imaging has localised CEP164 in a ring around the centriole barrel with a periodic enrichment of the signal within the ring (Sillibourne et al, 2011), and stimulated emission depletion microscopy has found that the enriched CEP164 signal corresponds to nine symmetricallyarranged clusters around the centriole, indicative of its association with each of the nine distal appendages (Lau et al, 2012).…”