Assessing the localization of centrosomal proteins by PALM/STORM nanoscopy
- 4 October 2011
- journal article
- research article
- Published by Wiley in Cytoskeleton
- Vol. 68 (11), 619-627
- https://doi.org/10.1002/cm.20536
Abstract
The structure of the centrosome was resolved by EM many years ago to reveal a pair of centrioles embedded in a dense network of proteins. More recently, the molecular composition of the centrosome was catalogued by mass spectroscopy and many novel components were identified. Determining precisely where a novel component localizes to within the centrosome remains a challenge, and until now it has required the use of immuno-EM. This technique is both time-consuming and unreliable, as it often fails due to problems with antigen accessibility. We have investigated the use of two nanoscopic techniques, photoactivated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM), as alternative techniques for localizing centrosomal proteins. The localization of a known centrosomal component, the distal appendage protein Cep164 was investigated by direct STORM (dSTORM) and resolved with a high spatial resolution. We further validated the use of nanoscopic PALM imaging by showing that the previously uncharacterized centrosomal protein CCDC123 (Cep123) localizes to the distal appendages, forming ring-like structures with a diameter of 500 nm. Our results demonstrate that both PALM and STORM imaging have great potential as alternatives to immuno-EM.Keywords
This publication has 92 references indexed in Scilit:
- Novel asymmetrically localizing components of human centrosomes identified by complementary proteomics methodsThe EMBO Journal, 2011
- Structural Basis of the 9-Fold Symmetry of CentriolesCell, 2011
- Nanoscale architecture of integrin-based cell adhesionsNature, 2010
- Photoactivatable fluorescent proteins for diffraction-limited and super-resolution imagingTrends in Cell Biology, 2009
- Plk4-Induced Centriole Biogenesis in Human CellsDevelopmental Cell, 2007
- SAS-6 defines a protein family required for centrosome duplication in C. elegans and in human cellsNature, 2005
- Proteomic characterization of the human centrosome by protein correlation profilingNature, 2003
- SAS-4 Is a C. elegans Centriolar Protein that Controls Centrosome SizeCell, 2003
- A general method for the covalent labeling of fusion proteins with small molecules in vivoNature Biotechnology, 2002
- Quantifying heterogeneity in a meta-analysisStatistics in Medicine, 2002