Periodic Reporting for period 4 - ACCENT (Unravelling the architecture and the cartography of the human centriole)
Periodo di rendicontazione: 2021-07-01 al 2021-12-31
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 and expansion microscopy. 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.
During the funding period, we initiated and pushed forward the four aims as planned in the proposal. We notably developed an optimized expansion microscopy method named U-ExM allowing a nanometric protein mapping of the centriole. This method, which relies on the physical expansion of a sample, provides an unprecedented means to visualize preserved macromolecular structures and was published in Nature methods in 2019. U-ExM proved very powerful not only for studying centrioles but also to analyze the cytoskeleton structures of parasites such as Plasmodium or Toxoplasma (work published in Plos Biology 2021 and in eLife in 2020).
While U-ExM represents a stepping-stone to image centriolar structures, the field of super-resolution expansion microscopy still suffers from the lack of general fixations that would be optimal to image any kind of cellular structure. Therefore, in the line of Aim3, we developed a pipeline of cryo-fixation coupled to U-ExM amenable for the full preservation of cellular structures and organelles in cells (Laporte et al. Nature methods 2022)
In parallel to this method development, we conducted cryo-tomography of isolated cycling human centrioles as well as of three other evolutionary distinct species -Paramecium, Chlamydomonas, and Naegleria- and undertook the native reconstruction of the central core region of the centrioles. We revealed the presence of a helical inner scaffold spanning the central core region of centrioles in Paramecium and Chlamydomonas and found that parts of this structural entity are present in the human isolated centrioles, highlighting its evolutionary conservation. Furthermore, we mapped centriolar proteins within this novel architectural element using U-ExM and discovered a set of 4 proteins forming a complex that localizes where the inner scaffold structure is. This work has been published in Science Advances in 2020.
In addition to the central core region of the 4 species, we analyzed their proximal regions and carefully mapped the boundaries between the different structural elements. This work made also an important discovery on the organization of the cartwheel, a structure important for centriole biogenesis. This work has been published in EMBO Journal in 2021.
During this funding period, we also developed in vitro assays to dissect centriole formation, enabling notably the formation of microtubule doublets, which represent essential building blocks of centrioles and cilia, a work that was published in Science in 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. 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 been published in eLife in 2020.
Going beyond the original plan which focused solely on the central core region of centrioles, we conducted a mini screen of around 20 centriolar proteins using expansion microscopy to refine their localization at a nanometric scale and provide a molecular mapping of centrioles. This work led to an unprecedented understanding of centriolar molecular architecture, as well as centriole assembly. We completed this project during the last funding period.
Finally, prompted by our discovery of the inner scaffold and of its molecular components, we undertook a protein mapping using U-ExM of mouse photoreceptors cells. Indeed, these specialized ciliated cells contained a connecting cilium, a microtubule-based structure made of microtubule doublets that bridges the centriole to the axonemal outer segment. We discovered that the connecting cilium contains an inner scaffold-like structure, containing most of the identified centriolar inner scaffold components. Moreover, we discovered, using a mouse model of the human disorder retinitis pigmentosa, the molecular and structural mechanisms behind this degenerative disease of the retina.
While U-ExM represents a stepping-stone to image centriolar structures, the field of super-resolution expansion microscopy still suffers from the lack of general fixations that would be optimal to image any kind of cellular structure. Therefore, in the line of Aim3, we developed a pipeline of cryo-fixation coupled to U-ExM amenable for the full preservation of cellular structures and organelles in cells (Laporte et al. Nature methods 2022)
In parallel to this method development, we conducted cryo-tomography of isolated cycling human centrioles as well as of three other evolutionary distinct species -Paramecium, Chlamydomonas, and Naegleria- and undertook the native reconstruction of the central core region of the centrioles. We revealed the presence of a helical inner scaffold spanning the central core region of centrioles in Paramecium and Chlamydomonas and found that parts of this structural entity are present in the human isolated centrioles, highlighting its evolutionary conservation. Furthermore, we mapped centriolar proteins within this novel architectural element using U-ExM and discovered a set of 4 proteins forming a complex that localizes where the inner scaffold structure is. This work has been published in Science Advances in 2020.
In addition to the central core region of the 4 species, we analyzed their proximal regions and carefully mapped the boundaries between the different structural elements. This work made also an important discovery on the organization of the cartwheel, a structure important for centriole biogenesis. This work has been published in EMBO Journal in 2021.
During this funding period, we also developed in vitro assays to dissect centriole formation, enabling notably the formation of microtubule doublets, which represent essential building blocks of centrioles and cilia, a work that was published in Science in 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. 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 been published in eLife in 2020.
Going beyond the original plan which focused solely on the central core region of centrioles, we conducted a mini screen of around 20 centriolar proteins using expansion microscopy to refine their localization at a nanometric scale and provide a molecular mapping of centrioles. This work led to an unprecedented understanding of centriolar molecular architecture, as well as centriole assembly. We completed this project during the last funding period.
Finally, prompted by our discovery of the inner scaffold and of its molecular components, we undertook a protein mapping using U-ExM of mouse photoreceptors cells. Indeed, these specialized ciliated cells contained a connecting cilium, a microtubule-based structure made of microtubule doublets that bridges the centriole to the axonemal outer segment. We discovered that the connecting cilium contains an inner scaffold-like structure, containing most of the identified centriolar inner scaffold components. Moreover, we discovered, using a mouse model of the human disorder retinitis pigmentosa, the molecular and structural mechanisms behind this degenerative disease of the retina.
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 led to exciting results that have been translated into several publications in international journals as stated above. We developed several innovative techniques that will contribute significantly to the study of centriole structure, organization, and function as well as to other cellular organelles or structures in the cell behind centrioles.
By the end of the project, we have achieved a near-complete molecular mapping of centrioles from human cells. This work allows correlating structural elements to centriolar proteins and is an invaluable tool to understand the functional significance of particular structural elements of centrioles based on these novel and combinatorial approaches.
Finally, the discovery made on the centriole’s structure was used to better understand a human disease condition called retinitis pigmentosa, a degenerative disease of the retina.
By the end of the project, we have achieved a near-complete molecular mapping of centrioles from human cells. This work allows correlating structural elements to centriolar proteins and is an invaluable tool to understand the functional significance of particular structural elements of centrioles based on these novel and combinatorial approaches.
Finally, the discovery made on the centriole’s structure was used to better understand a human disease condition called retinitis pigmentosa, a degenerative disease of the retina.