Periodic Reporting for period 2 - ACCENT (Unravelling the architecture and the cartography of the human centriole)
Reporting period: 2018-07-01 to 2019-12-31
Centrioles are evolutionary conserved macromolecular structures important for building centrosomes and templating cilia in many eukaryotes. As such, centrioles drive the proper execution of fundamental cell biological processes. Importantly defects in centrioles or cilia function have been associated to human diseases including ciliopathies and cancer. This research project aims at unraveling the architecture and the cartography of the human centriole, a deep-rooted question with a current limited knowledge. To do so, we undertook the development of innovative methods to tackle these challenging questions and to allow the nanometric protein mapping of centriolar proteins within architectural elements of the centriole. To this end, we are combining the use of state-of-the-art techniques such as cryo-electron microscopy, super-resolution microscopy to in vitro assays. Resolving such fundamental questions is of particular importance and will undoubtedly lead to a better understanding of centriolar and cilia function and might pave the way to further comprehension of human pathologies.
Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far
In order to achieve a nanometric protein mapping of the centriole, we developed a novel expansion microscopy method named UltraExM (Aim 3). This method relies on the physical expansion of a sample and provides an unprecedented mean to visualize preserved macromolecular structures from either purified samples or in cellulo specimens. Importantly, we could reveal for the first time the chirality and the 9-fold symmetry of centrioles using either conventional or super-resolution microscopes. This result published in Nature methods in February 2019 represents a major advance not only in the field of centriole biology but we also proved its general applicability on other systems. In parallel to that work, we performed cryo-tomography of isolated centrioles or in situ (Aim 2). We undertook the native reconstruction of the central core region of the centrioles not only from the isolated human sample but also from three other evolutionary distinct species. We revealed the presence of a helical inner scaffold spanning the central core region of centrioles in Paramecium and Chlamydomonas. Importantly, we found that parts of this structural entity are present in the human isolated centrioles, highlighting its evolutionary conservation. The reconstruction of the native architecture of the human central core region will be continued during the next funding period focusing on uncompressed samples. Furthermore, we mapped centriolar proteins within this novel architectural element and discovered a set of 4 proteins forming a complex that localizes where the inner scaffold structure is (Aim 1). This work has been accepted in Science Advances in December 2019. In addition, we developed in vitro assays to dissect centriole formation (Aim 4), enabling notably the formation of microtubule doublets, which represents essential building blocks of centrioles and cilia, a work that was published in Science in Jan 2019. Finally, we studied the first described human central core protein WDR90, which we hypothesize to be the protein forming the inner junction in the centriolar microtubule triplet by notably analyzing its ability to bind microtubules (Aims 1&4). Importantly, we discovered that WDR90 is a microtubule binding protein that localizes along the microtubule wall in the central core region and that its depletion impairs the inner scaffold structure, leading to centriole fracture. This work has just been submitted in an international journal and will be continued in the third funding period.
Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)
This research project aims at performing the cartography of centriolar proteins, which represents a challenge owing to the size and the number of this organelle in a cell. The experimental plan proposed in the ERC grant has been initiated during the two first funding periods and led to exciting results that have been translated in several publications in international journals. We developed innovative techniques that will contribute significantly to the study of centriole structure, organization and function. We expect to achieve a near-complete molecular mapping of centrioles from human cells and Chlamydomonas cells until the end of the project. In parallel, we aim at elucidating the native 3D architecture of human centrioles and correlating the structural elements to centriolar proteins. We also expect to understand the functional significance of particular structural elements of centrioles based on these novel and combinatorial approaches.