Final Report Summary - CENTRIOLEELONGATION (Role of Microtubule Regulators in Centriole Elongation)
Centrioles are essential for the formation of several microtubule organizing structures including cilia, flagella and centrosomes. These structures are involved in a variety of functions, from cell motility to division. Despite the essential functions of centrioles and their role in human disease, their biogenesis is poorly understood. The goal of my project was to investigate centriole biogenesis, in particular centriole elongation, its kinetics and regulation. In addition, I was involved in another project from my laboratory where we found that Bld10 is a centrosomal protein that binds microtubules and it is involved in centriole elongation (Carvalho-Santos et al, 2012).
PLK4 is a tumor suppressor kinase and a member of the polo-like kinase family. It is a key regulator of centriole biogenesis since its kinase activity is required for centriole duplication. New centrioles arise next to existing ones, the canonical pathway, but they can also form de novo. PLK4 overexpression triggers centrioles overduplication in both cases and its absence induces progressive centriole loss. This project establishes an in vitro system to study centriole biogenesis using Xenopus egg extracts. In addition, it uncovers new roles for PLK4 in centriole biogenesis and the molecular mechanisms of the cytoskeleton, in particular its microtubule network. Taking advantage of an in vitro system we have obtained an extensive series of results. PLK4 induces microtubule organizing centers (MTOCs) formation in Xenopus egg extracts in specific phases of the cell cycle, which suggests a cell cycle dependent regulation of PLK4 activity. In addition, this MTOCs formation is dependent on PLK4 levels. We report that PLK4 binds specifically to microtubules which suggests a new role for PLK4, suggesting that PLK4 plays a critical role to mediate microtubule dynamics to enable acentrosomal asters assembly. We tested whether those asters might form on top of centrioles or whether the aster is the first thing that forms. Supporting the second hypothesis we observed that PLK4 induced aster-like structures have centriolar proteins colocalization, including gamma tubulin. Moreover, the formation of these acentrosomal aster-like structures is Dynein independent showing that their behavior is more similar to the one reported for centrosomal MTOC’s. Therefore centrosome protein recruitment might be the first event triggered by PLK4. Indeed, depletion of centrosomal proteins, such as SAS6, induces a striking delay in PLK4 aster formation, suggesting a role for centrosomal proteins in this process. However, when analyzed by electron microscopy, these asters do not show a 9 fold centriole structure, suggesting PLK4 asters grow on top of centriole-like amorphous structures. We are further investigating how different motor proteins, centrosome proteins and microtubule regulators are involved and interact with each other to form these MTOCs. Finally, the development of two independent screens regarding centriole biogenesis and elongation is an important step toward a better understanding of centriole biology and will contribute in the near future with novel players in centriole biogenesis and elongation that may uncover novel mechanisms.
Taken together, this project made several contributions to cell biology and to the understanding of the molecular mechanisms of cytoskeleton-based processes. It will hopefully provide in the near future a better understanding of centriole biology and open exciting new avenues to address long standing questions.
PLK4 is a tumor suppressor kinase and a member of the polo-like kinase family. It is a key regulator of centriole biogenesis since its kinase activity is required for centriole duplication. New centrioles arise next to existing ones, the canonical pathway, but they can also form de novo. PLK4 overexpression triggers centrioles overduplication in both cases and its absence induces progressive centriole loss. This project establishes an in vitro system to study centriole biogenesis using Xenopus egg extracts. In addition, it uncovers new roles for PLK4 in centriole biogenesis and the molecular mechanisms of the cytoskeleton, in particular its microtubule network. Taking advantage of an in vitro system we have obtained an extensive series of results. PLK4 induces microtubule organizing centers (MTOCs) formation in Xenopus egg extracts in specific phases of the cell cycle, which suggests a cell cycle dependent regulation of PLK4 activity. In addition, this MTOCs formation is dependent on PLK4 levels. We report that PLK4 binds specifically to microtubules which suggests a new role for PLK4, suggesting that PLK4 plays a critical role to mediate microtubule dynamics to enable acentrosomal asters assembly. We tested whether those asters might form on top of centrioles or whether the aster is the first thing that forms. Supporting the second hypothesis we observed that PLK4 induced aster-like structures have centriolar proteins colocalization, including gamma tubulin. Moreover, the formation of these acentrosomal aster-like structures is Dynein independent showing that their behavior is more similar to the one reported for centrosomal MTOC’s. Therefore centrosome protein recruitment might be the first event triggered by PLK4. Indeed, depletion of centrosomal proteins, such as SAS6, induces a striking delay in PLK4 aster formation, suggesting a role for centrosomal proteins in this process. However, when analyzed by electron microscopy, these asters do not show a 9 fold centriole structure, suggesting PLK4 asters grow on top of centriole-like amorphous structures. We are further investigating how different motor proteins, centrosome proteins and microtubule regulators are involved and interact with each other to form these MTOCs. Finally, the development of two independent screens regarding centriole biogenesis and elongation is an important step toward a better understanding of centriole biology and will contribute in the near future with novel players in centriole biogenesis and elongation that may uncover novel mechanisms.
Taken together, this project made several contributions to cell biology and to the understanding of the molecular mechanisms of cytoskeleton-based processes. It will hopefully provide in the near future a better understanding of centriole biology and open exciting new avenues to address long standing questions.