Periodic Reporting for period 1 - GTR (Dissecting gamma-TuRC composition and activity by Single Molecule Pull-down)
Période du rapport: 2017-09-01 au 2019-08-31
The main purpose of this project was to dissect the composition and activity of gamma-tubulin ring complexes (γTuRCs), the main nucleator of microtubule (MT) polymerization.
MTs are filamentous structures, composed of α- and β-tubulin dimers, that mediate essential processes such as intracellular transport, cell migration or chromosome segregation during cell division. MT polymerization in vivo requires specific MT nucleators which regulate MT assembly at MT organizing centers (MTOCs). The major MT nucleator is the γTuRC, a multi-protein complex assembled from γ-tubulin and gamma complex proteins (GCPs) 2-6. According to the current model, activated γTuRCs nucleate MTs by providing a template for MT assembly: the helical arrangement of ~13 γ-tubulin molecules in the γTuRC resembles the arrangement α-β-tubulin subunits in a MT, thereby serving as a platform for the addition of alpha-beta-tubulin dimers. Throughout the cell cycle, γTuRC localizes to the centrosome, the major MTOC in animal somatic cells. Apart from residing in the pericentriolar material (PCM), where γTuRC nucleates MTs that extend into the cytoplasm during interphase or are incorporated into the mitotic spindle during mitosis, γ-tubulin also localizes specifically to centrioles, the core structures of the centrosome. Here, γTuRC might acts as the nucleator of the MTs that constitute the centriolar cylinder. However, convincing data to support this hypothesis is still missing and it remains possible that γTuRC has functions beyond MT nucleation. It is conceivable that both γTuRC activity and its mode of action can be tuned to allow for the formation of distinct MT-based structures in a cell site- and/or cell cycle specific manner. This might be achieved through interactions with selective factors or through alterations in γTuRC subunit composition.
In this project, we set out to test these hypotheses. We established conditions to analyse γTuRC by SiMPull, an innovative in vitro approach that is based on the immobilization of single γTuRCs immunoprecipitated from crude cell extract on a PEG-passivated glass surface and imaging of these complexes by high-resolution microscopy. Complementary, we dissected γTuRC activity and composition in a relevant context, namely during centriole assembly. We were able to identify γTuRC interactors that target the complex to centrioles and, intriguingly, we could show that γTuRC might be required for stabilizing the centriolar cylinder when centrioles have already reached their final size. This novel function of γTuRC might be important to guarantee the stable inheritance of centrioles to the daughter cells during cell division and to stably maintain centrioles in non-cycling cells, to allow formation of cilia, cell protrusions that are templated by “mother” centrioles in resting or post-mitotic cells and that are required for cell signalling among other functions.
Overall, our findings provide novel insight into γTuRC activity, especially in the context of centrioles, which frequently show structural and numerical abnormalities in a broad range of diseases such as cancer, microcephaly and a range of complex disorders collectively termed ciliopathies.
MTs are filamentous structures, composed of α- and β-tubulin dimers, that mediate essential processes such as intracellular transport, cell migration or chromosome segregation during cell division. MT polymerization in vivo requires specific MT nucleators which regulate MT assembly at MT organizing centers (MTOCs). The major MT nucleator is the γTuRC, a multi-protein complex assembled from γ-tubulin and gamma complex proteins (GCPs) 2-6. According to the current model, activated γTuRCs nucleate MTs by providing a template for MT assembly: the helical arrangement of ~13 γ-tubulin molecules in the γTuRC resembles the arrangement α-β-tubulin subunits in a MT, thereby serving as a platform for the addition of alpha-beta-tubulin dimers. Throughout the cell cycle, γTuRC localizes to the centrosome, the major MTOC in animal somatic cells. Apart from residing in the pericentriolar material (PCM), where γTuRC nucleates MTs that extend into the cytoplasm during interphase or are incorporated into the mitotic spindle during mitosis, γ-tubulin also localizes specifically to centrioles, the core structures of the centrosome. Here, γTuRC might acts as the nucleator of the MTs that constitute the centriolar cylinder. However, convincing data to support this hypothesis is still missing and it remains possible that γTuRC has functions beyond MT nucleation. It is conceivable that both γTuRC activity and its mode of action can be tuned to allow for the formation of distinct MT-based structures in a cell site- and/or cell cycle specific manner. This might be achieved through interactions with selective factors or through alterations in γTuRC subunit composition.
In this project, we set out to test these hypotheses. We established conditions to analyse γTuRC by SiMPull, an innovative in vitro approach that is based on the immobilization of single γTuRCs immunoprecipitated from crude cell extract on a PEG-passivated glass surface and imaging of these complexes by high-resolution microscopy. Complementary, we dissected γTuRC activity and composition in a relevant context, namely during centriole assembly. We were able to identify γTuRC interactors that target the complex to centrioles and, intriguingly, we could show that γTuRC might be required for stabilizing the centriolar cylinder when centrioles have already reached their final size. This novel function of γTuRC might be important to guarantee the stable inheritance of centrioles to the daughter cells during cell division and to stably maintain centrioles in non-cycling cells, to allow formation of cilia, cell protrusions that are templated by “mother” centrioles in resting or post-mitotic cells and that are required for cell signalling among other functions.
Overall, our findings provide novel insight into γTuRC activity, especially in the context of centrioles, which frequently show structural and numerical abnormalities in a broad range of diseases such as cancer, microcephaly and a range of complex disorders collectively termed ciliopathies.
For SiMPull, I generated human cell lines stably expressing GFP-tagged γTuRC subunits with the objective to pull-down γTuRCs using a biotinylated anti-GFP antibody (AB). Immuno fluorescence analysis revealed that GFP-tagged γTuRC subunits localize like endogenous γTuRC, suggesting that tagged proteins are incorporated into γTuRC. I also tried to tag endogenous γTuRC subunits with FLAG or Myc-His using CRISPR/Cas9, with the aim to pull-down γTuRCs using biotinylated anti-FLAG or -Myc ABs. Both endogenous GCP2 and GCP6 could be tagged successfully, at least one of the corresponding alleles. However, I started SiMPull with high-molecular weight fractions of a sucrose gradient containing GFP-tagged γTuRC. The glass surface to which the biotinylated AB is coupled has to be efficiently passivated with PEG to prevent unspecific binding of proteins. I modified the construction of flow chambers in respect to published protocols, allowing more stringent washing steps and easier handling. While I could detect only a few GFP “dots” in the negative control, I could detect GFP-tagged complexes in variable amounts when using biotinylated anti-GFP AB in different concentrations. I also adjusted the neutravidin concentration (allows the coupling of biotinylated ABs to the glass) and the concentration of cell extract in order to detect a suitable amount of γTuRCs, using both TIRF and conventional wide-field microscopy. I systematically tested all in the lab available primary ABs against γTuRC components/interactors in my experimental set-up and tried to establish conditions under which γTuRCs are able to nucleate microtubules.
I also dissected the composition and activity of γTuRC in the context of centrioles. Although γTuRC is implicated in centriole assembly in different systems, the underlying mechanism is unknown and the hypothesis that it acts as the nucleator of the MTs that constitute the centriolar cylinder, remains to be tested. To determine the role of γTuRC at centrioles, I analyzed γTuRC centriole localization by expansion microscopy in human cell lines. I found that γTuRC localizes to the outer surface of the centriolar cylinder, to the centriole lumen and potentially also to the very proximal part of centrioles. Using RNAi, I found that γTuRC is recruited to the centriole lumen by the augmin complex and that augmin depletion results in the destabilization of centrioles. This finding suggests a novel, non-canonical role of γTuRC at centrioles. To determine if the distinct centriolar pools of γTuRC differ in respect to theit subunit composition, I depleted several GCPs and analyzed the effect on γTuRC centriole localization. These experiments did not provide clear result yet.
Together, I established an innovative in vitro assay in the lab that not only allows the dissection of γTuRC activity and composition, but that can also easily be exploited to address other relevant questions in the future. Moreover, my findings provide novel insight into γTuRC and augmin activity, ultimately contributing to a better understanding of the structure and assembly pathway of centrioles, which are key to a variety of essential cellular processes by forming the core of the centrosome.
I discussed my findings with several colleagues in the field, either during their visits to IRB Barcelona or at international conferences and some of my data were presented by my supervisor at the CSH Asia Conference “Cilia & Centrosomes” (China, 2019) in form of an invited talk. I am currently preparing a manuscript to publish our findings in a high-profile scientific journal.
I also dissected the composition and activity of γTuRC in the context of centrioles. Although γTuRC is implicated in centriole assembly in different systems, the underlying mechanism is unknown and the hypothesis that it acts as the nucleator of the MTs that constitute the centriolar cylinder, remains to be tested. To determine the role of γTuRC at centrioles, I analyzed γTuRC centriole localization by expansion microscopy in human cell lines. I found that γTuRC localizes to the outer surface of the centriolar cylinder, to the centriole lumen and potentially also to the very proximal part of centrioles. Using RNAi, I found that γTuRC is recruited to the centriole lumen by the augmin complex and that augmin depletion results in the destabilization of centrioles. This finding suggests a novel, non-canonical role of γTuRC at centrioles. To determine if the distinct centriolar pools of γTuRC differ in respect to theit subunit composition, I depleted several GCPs and analyzed the effect on γTuRC centriole localization. These experiments did not provide clear result yet.
Together, I established an innovative in vitro assay in the lab that not only allows the dissection of γTuRC activity and composition, but that can also easily be exploited to address other relevant questions in the future. Moreover, my findings provide novel insight into γTuRC and augmin activity, ultimately contributing to a better understanding of the structure and assembly pathway of centrioles, which are key to a variety of essential cellular processes by forming the core of the centrosome.
I discussed my findings with several colleagues in the field, either during their visits to IRB Barcelona or at international conferences and some of my data were presented by my supervisor at the CSH Asia Conference “Cilia & Centrosomes” (China, 2019) in form of an invited talk. I am currently preparing a manuscript to publish our findings in a high-profile scientific journal.
This project does not only provide novel insight into γTuRC composition and activity, ultimately contributing to a better understanding of the structure and assembly pathway of centrioles, but also promises to make an important impact on human health by discovering and unraveling novel and previously unappreciated centriolar mechanisms as cause of different human diseases and conditions as both structural and numerical abnormalities of centrioles have been linked to a wide range of diseases such as cancer, microcephaly and a number of complex disorders collectively termed ciliopathies.