Facing a camera, Dr. Christian Sieben (Manley lab, EPFL) presents his recent work and gives emphasis on the biochemical problem behind it. Niccolo Banterle (Gönczy lab, EPFL) comments with a few lines to explain the biological approach used to solve the problem. They are from different disciplines but meet through chemical biology.

 

 

 

Perspective by Dr. Niccolo Banterle

Cells need to perform diverse functions such as intracellular transport, sensing of the environment and division. To achieve these goals, thousands of proteins are organised at multiple levels within the cell. For intracellular transport and cell division, for example, the microtubule network is organised from microtubule organising centres such as the centrosome. At the core of centrosomes, electron microscopy uncovered beautiful microtubule-based nine-fold radially symmetric structures: the centrioles. Centrioles are ~350 nm wide and ~500 nm long organelles, comprising an estimated >150 different proteins. Centrioles need to duplicate once and only once per cell cycle. This duplication initiates on a torus located around one end of resident centrioles, to which key proteins are recruited to form the cartwheel, the first structure of newly formed centrioles. To understand how centrioles are built, a method is needed to locate individual proteins with utmost precision. Super-resolution fluorescence microscopy can achieve this goal in part, but the resolution in the axial direction is diffraction-limited. Conversely, electron microscopy provides the required resolution but lacks protein specificity. To overcome these limitations, we developed a technique that combines the best of both worlds. The high throughput STORM microscope developed by the Manley lab enabled us to generate datasets of purified centrioles comprising more than 1000 particles for each protein of interest. We then used algorithms that reconstruct the three-dimensional map of each protein. In this way, we could reconstruct the exact dimensions of the proteins Cep57, Cep63 and Cep152, which form the torus surrounding the end of resident centriole. Moreover, we adapted the reconstruction technique to map two proteins at the same time. In this way, we were able to reconstruct the orientation of the nascent cartwheel, revealing that it is loosely oriented and not-perfectly orthogonal to the resident centriole. The ability of mapping individual proteins with high isotropic resolution within protein complexes opens the perspective to soon achieve complete molecular maps of centrioles as well as other macromolecular complexes.

 

Go Further!

Sieben C., Banterle N., Douglass K.M., Gönczy P., Manley S., “Multicolor single-particle reconstruction of protein complexes”, Nat. Methods, 15(10):777-780, 2018. Read the publication