Periodic Reporting for period 4 - CentrioleBirthDeath (Mechanism of centriole inheritance and maintenance)
Periodo di rendicontazione: 2021-07-01 al 2022-12-31
Every organelle within a particular cell type has a characteristic positioning and copy number in the cell, reflecting its function. Organelle inheritance and its persistence upon cell division, fertilization and disease were already passionately discussed by pioneering cell biologists. Amongst those, a structure that always raised much interest is the centriole, which assembles centrosomes and cilia, critical structures for cell division, polarity, motility and signaling, often deregulated in human disease. Additionally, centrioles and cilia are highly conserved throughout the eukaryotic tree of life, but have been lost in a variety of species, being an excellent study case to analyze the evolution of eukaryotic cellular structures. This project aims at investigating, in an integrative and quantitative way, how centrioles are formed in the right numbers at the right time and place, and how they are maintained to ensure their function and inheritance. We first ask how centrioles guide their own assembly position (right place) and centriole copy number (right number), as well as how this process is coordinated with the cell cycle (right time). Our recent work highlighted several properties of the system, including positive and negative feedbacks and local cues. Guided by that conceptual framework we explore critical hypotheses through a combination of biochemistry, quantitative live cell microscopy and computational modeling. Finally, we ask how differentiating cells regulate centriole maintenance/elimination in the context of the organism. We focus this analysis on natural examples where centrioles are either removed (e.g. oogenesis and skeletal muscle) or maintained (e.g. ciliated neurons). By studying centriole disappearance in the female germline we uncovered that centrioles need to be actively maintained by their surrounding material, the PCM. We propose to investigate how the PCM provides stability to the centrioles, whether this is differently regulated in different cell types and the possible consequences of its misregulation for the organism (infertility and ciliopathy-like symptoms). Furthermore, we envision to identify novel ways by which cells communicate and regulate each other within an organism. Understanding how these different mechanisms are regulated in nature will provide novel insights to diagnostics and treatment of human disease originated by centriole and cilia abnormalities. Besides scientific research, our mission is also one of training scientists and communicating science to lay people.
Centrioles assemble centrosomes and cilia, critical structures for cell division, polarity, motility and signaling, which are often deregulated in human disease. We address, in an integrative and quantitative way, how centrioles are formed in the right numbers at the right time and place, and how they are maintained to ensure their function and inheritance. During the period covered by this report we have published new aspects of the mechanisms controlling centriole biogenesis and structure, the first Aim of this project (Gouveia and Zitouni et al, 2019; Ito et al, 2019; Jana et al, 2018). Moreover, we have produced the tools needed to address the second aim, regarding coordination of the centrosome cycle with the cell cycle. Finally, we have published data related to the third aim (Jana et al, 2018), focused on centriole stability and homeostasis in different tissues.
Besides being able to form close to a pre-existing structure (canonical duplication), centrosomes can form de novo, a process that is mostly unknown. We explored the initial steps of de novo centrosome biogenesis, using frog egg extracts. These extracts are made from eggs and are therefore devoid of centrioles. Adding PLK4, the major trigger of centriole biogenesis, in acentriolar cells is known to induce de novo centriole formation. We observed that adding PLK4 in frog extracts induces the formation of microtubule organizing centers. Using this system, we unveiled a new function for Plk4 (Gouveia and Zitouni et al, 2019), which can self assemble in vitro into condensates that are able to recruit α- and β-tubulins, as well as STIL, a substrate of PLK4, and the microtubule nucleator γ-tubulin, leading to the formation of acentriolar MTOCs, and reflecting the possible initial steps for de novo centriole biogenesis. We suggest a new mechanism of action for PLK4, where it forms a self-organizing catalytic scaffold that recruits centriole components, PCM factors and α- and β-tubulins, leading to MTOC formation (Gouveia and Zitouni et al, 2019). This work opens exciting new avenues regarding how PLK4 and the first steps of centriole biogenesis are regulated.
An important question is what determines where centrioles are formed, when no centriole is there to catalyze the process. We explored the evolutionary conserved interactions of centrosomal components and uncovered that pericentrin, a component of the pericentriolar material (PCM), and that is often overexpressed in cancer, can recruit one of the first components needed to make centrioles, SAS-6 (Ito et al, 2019). SAS-6 is a critical component of the cartwheel structure, which helps defining the ninefold centriole symmetry (see for review Nabais et al, 2019). Our work suggests that the PCM not only recruits and concentrates microtubule-nucleators, but also the centriole assembly machinery, promoting biogenesis close by, an hypothesis we are now further testing. These observations are a relevant contribution for the comprehension of cellular mechanisms controlling centriole number and a new step forward to identify and correct deregulated processes in human disease.
In a separate study which was focused on understanding the formation of diverse ciliary bases, a structure composed by the centrioles, we uncovered that the cartwheel components, SAS-6 and its partner STIL, also play an important role in basal body elongation, proposing thus a novel role for these centriole components. Moreover, by characterizing 15 different components of basal bodies, we have also observed that all basal bodies are surrounded by several components of the PCM, suggesting the PCM might be involved in their maintenance, as we had originally predicted in Aim3. This survey is an essential tool to comprehend the processes and molecules that ensure the maintenance of these long lived cellular structures, one of the main objectives of Aim3. Defects in cilia can often cause human disorders, including ciliopathies, tissue-degeneration and ageing-related phenotypes.
Besides being able to form close to a pre-existing structure (canonical duplication), centrosomes can form de novo, a process that is mostly unknown. We explored the initial steps of de novo centrosome biogenesis, using frog egg extracts. These extracts are made from eggs and are therefore devoid of centrioles. Adding PLK4, the major trigger of centriole biogenesis, in acentriolar cells is known to induce de novo centriole formation. We observed that adding PLK4 in frog extracts induces the formation of microtubule organizing centers. Using this system, we unveiled a new function for Plk4 (Gouveia and Zitouni et al, 2019), which can self assemble in vitro into condensates that are able to recruit α- and β-tubulins, as well as STIL, a substrate of PLK4, and the microtubule nucleator γ-tubulin, leading to the formation of acentriolar MTOCs, and reflecting the possible initial steps for de novo centriole biogenesis. We suggest a new mechanism of action for PLK4, where it forms a self-organizing catalytic scaffold that recruits centriole components, PCM factors and α- and β-tubulins, leading to MTOC formation (Gouveia and Zitouni et al, 2019). This work opens exciting new avenues regarding how PLK4 and the first steps of centriole biogenesis are regulated.
An important question is what determines where centrioles are formed, when no centriole is there to catalyze the process. We explored the evolutionary conserved interactions of centrosomal components and uncovered that pericentrin, a component of the pericentriolar material (PCM), and that is often overexpressed in cancer, can recruit one of the first components needed to make centrioles, SAS-6 (Ito et al, 2019). SAS-6 is a critical component of the cartwheel structure, which helps defining the ninefold centriole symmetry (see for review Nabais et al, 2019). Our work suggests that the PCM not only recruits and concentrates microtubule-nucleators, but also the centriole assembly machinery, promoting biogenesis close by, an hypothesis we are now further testing. These observations are a relevant contribution for the comprehension of cellular mechanisms controlling centriole number and a new step forward to identify and correct deregulated processes in human disease.
In a separate study which was focused on understanding the formation of diverse ciliary bases, a structure composed by the centrioles, we uncovered that the cartwheel components, SAS-6 and its partner STIL, also play an important role in basal body elongation, proposing thus a novel role for these centriole components. Moreover, by characterizing 15 different components of basal bodies, we have also observed that all basal bodies are surrounded by several components of the PCM, suggesting the PCM might be involved in their maintenance, as we had originally predicted in Aim3. This survey is an essential tool to comprehend the processes and molecules that ensure the maintenance of these long lived cellular structures, one of the main objectives of Aim3. Defects in cilia can often cause human disorders, including ciliopathies, tissue-degeneration and ageing-related phenotypes.
Our results elucidated different mechanisms regulating the birth of new centrioles. PLK4 forms condensates that promote recruitment of tubulins, centriolar and PCM components leading to MTOC formation. Moreover we have shown that the PCM is likely to define an important pathway for the initial steps of biogenesis, at least via Sas-6 recruitment. We are now focusing on how PLK4 is regulated in time and space using Drosophila egg extracts which are easy to manipulate. We are also particularly interested to understand whether and how the PCM might regulate this process. Our knowledge of the molecular composition of different bases of cilia in the organism will now be fundamental to understand how these structures are maintained both structurally and functionally along the life of the organism. Altogether this grant will produce new knowledge on the regulation of centriole birth, number and maintenance in time and space.