Facing a camera, Manoël Prouteau (Biochemist and Cell Biologist, postdoc in the Loewith lab, UNIGE) presents his recent work and gives emphasis on the structural problem behind it. Christian Sieben (Biophysicist, postdoc in the Manley lab, EPFL) comments with a few lines to explain the biophysical approach used to solve the problem. They are from different disciplines but meet through chemical biology.

 

Perspective by Dr. Christian Sieben

Target of rapamycin TOR is a well-conserved protein kinase in eukaryotes. TOR has been shown to nucleate into two large multiprotein complexes, TORC1 and TORC2. By integrating extracellular signals, such as available nutrients, both complexes are involved in cell growth and stress response. In budding yeast cells, TORC1 is either distributed in a uniform granular arrangement around the vacuole or forms distinct single foci. Although these foci have been observed before, their structural organisation remained largely unknown.

In our paper, we investigated the organisation of these TORC1 foci (which we termed TOROIDs) inside intact yeast cells. Due to their small size, foci appear as single spots using conventional confocal microscopy. However, when we calibrated the GFP signal per focus to a single GFP protein, we estimated that each focus contains several hundred TORC1 dimers. To further elucidate TOROID’s organisation at the nanometer scale in vivo, we used super-resolution microscopy. In reconstructed STORM images, we found particles of different size and shape. After aligning and averaging several hundred TOROID particles, we found that the observed shapes arise from different orientations of the same object, which we concluded is organised as a hollow cylinder.

This project shows how different microscopy techniques can lead to a deep structural understanding of protein complexes across multiple length scales. Super-resolution microscopy helped to decipher the TORC1 structure in vivo and well complemented a large body of biochemistry, live-cell imaging and cryon-electron microscopy data to unravel how TORC1 function is regulated through the assembly of higher order structures.

Go Further!

Prouteau M., Desfosses A., Mozaffari N.L., Sieben C., Bourgoint C., Demurtas D., Mitra A.K., Guichard P., Manley S., Loewith R., “TORC1 Organised in Inhibited Domains (TOROIDs) regulate TORC1 activity”, Nature, 2017. Read the publication