Periodic Reporting for period 1 - CENTROMD (Deciphering the molecular dynamics of centriole and centrosome biogenesis)
Période du rapport: 2020-12-01 au 2022-11-30
Centrioles are cellular structures that form two key organelles: the centrosome, composed of a centriole pair surrounded by the pericentriolar matrix (PCM), and the basal body, which forms the cilium, an intricate structure that is essential for cell motility and environment sensing. Centrosomes and cilia perform many important functions in animal cells and their dysfunction is associated with an extensive array of pathologies, including cancer, dwarfism, microcephaly and obesity. Centrosome amplification (CA) constitutes an abnormal numerical increase in centrosome numbers, and can arise due to centriole overduplication during the cell cycle. CA has been reported in nearly all solid or hematologic human cancers, and can by itself lead to tumorigenesis. Elucidating the mechanisms that regulate centriole and centrosome biogenesis is of paramount importance for cell biology and cancer research.
The aim of this action was to investigate the dynamic behaviour of key players involved in centrosome and centriole biogenesis, using cutting-edge super-resolution microscopy and the Drosophila embryo as an in vivo model. Centrioles organize the PCM, a complex protein machine that has many functions and that assembles/disassembles very quickly. The molecular principles that allow such a complex machine to undergo drastic variations in size are unclear but it is evident that proper centrosome size is important for mitotic fidelity. Although many hundreds of proteins localise to
centrosomes, only Cnn, Spd-2 and Polo kinase (the Drosophila homologues of human Cdk5Rap2,CEP192 and Plk1, respectively) are essential for the assembly of the mitotic PCM in flies. We developed and implemented methods to characterise the behavior of individual molecules of Spd2 and Cnn as they move through the PCM and found that these are incorporated at the centre close to the centriole and gradually flux outwards. In general terms, we found that molecules flux slower when the centrosome is growing in preparation for mitosis and faster when the target size has been reached and that flux is influenced by microtubule pulling on the PCM, phosphorylation of Cnn and Spd2 and from constant incorporation of new molecules at the surface of the centriole generating a pushing motion.
Our findings indicate that flux dynamics of individual key components underlie the growth of the centrosome to its correct size in preparation for mitosis.
The aim of this action was to investigate the dynamic behaviour of key players involved in centrosome and centriole biogenesis, using cutting-edge super-resolution microscopy and the Drosophila embryo as an in vivo model. Centrioles organize the PCM, a complex protein machine that has many functions and that assembles/disassembles very quickly. The molecular principles that allow such a complex machine to undergo drastic variations in size are unclear but it is evident that proper centrosome size is important for mitotic fidelity. Although many hundreds of proteins localise to
centrosomes, only Cnn, Spd-2 and Polo kinase (the Drosophila homologues of human Cdk5Rap2,CEP192 and Plk1, respectively) are essential for the assembly of the mitotic PCM in flies. We developed and implemented methods to characterise the behavior of individual molecules of Spd2 and Cnn as they move through the PCM and found that these are incorporated at the centre close to the centriole and gradually flux outwards. In general terms, we found that molecules flux slower when the centrosome is growing in preparation for mitosis and faster when the target size has been reached and that flux is influenced by microtubule pulling on the PCM, phosphorylation of Cnn and Spd2 and from constant incorporation of new molecules at the surface of the centriole generating a pushing motion.
Our findings indicate that flux dynamics of individual key components underlie the growth of the centrosome to its correct size in preparation for mitosis.
We established experimental and analytical methodologies to follow individual molecule behavior in Drosophila embryos and then proceeded to collect data for thousand of molecules. We built a catalogue of single molecule behaviors for Spd-2, Cnn and dTACC. For each molecule the following were measured: time of incorporation and exit from the PCM, total binding duration, timing in regards to the phases of the cell cycle, distance from the centriole when bound and when exiting, movement throughout the PCM. The main conclusion coming from these studies was that both Spd-2 and Cnn bind to the PCM at the centre, close to Asl and then move outwards to the edge of the PCM, after which they exit. We call this movement FLUXING.
The major conclusions from this study are 1) molecules of Spd-2 are incorporated to the PCM mostly during early s-phase, with few incorporating during mitosis. Molecules of Cnn, in reverse, are mostly incorporated during mitosis and late S-phase, with few incorporated during early S-phase; 2) Spd-2 molecules spend on average l.7 minutes in the PCM whereas Cnn molecules spend 2 minutes in the PCM; 3) Fluxing Spd-2 and Cnn molecules move at speeds between 3 and 30 nm/s through the PCM, with Spd-2 molecules on average moving faster than Cnn; 4) Flux speed is determined by several factors: molecules binding initially closer to the centriole tend to move faster through the PCM than those that bind further from it and flux speed is faster (for Spd-2) during earlier S-phase than during later S-phase. We found that dTACC bound and unbound from the centrosome at a fast rate and seemingly at random distance from the centre, in clear contrast to the directional outwards movement of Spd2 and Cnn.
In order to find the determinants behind the observed fluxing behavior of Spd2 and Cnn, we repeated the single molecule analysis with either a) colchicine to depolymerise microtubules, where we found that Cnn stopped fluxing and would accumulate at the centrosome, whereas Spd2 was seemingly unaffected; constructed and used mutants of Spd-2 that lack the canonical phosphorylation sites – Spd2ALL and Spd2CONS, or mutants of Polo kinase and found that these were still able to flux, particularly at a faster rate indicating that phosphorylation of Spd2/Cnn contributes to keeping these proteins at the centrosome during expansion. We’re currently developing models to describe how fluxing of these proteins contributes to centrosome growth.
In conclusion, the implementation of this action provided a detailed model of how centrosomal key components flux through the centrosome during the cell cycle, the determinants of said flux and how this flux contributes to centrosome size growth and maintenance of size, which are crucial aspects that ensure mitotic fidelity. These findings are being used to prepare a manuscript suitable for publication.
Over the course of this action these findings were regularly disseminated to the community at conferences and symposiums, both internally in the department and at international conferences.
The major conclusions from this study are 1) molecules of Spd-2 are incorporated to the PCM mostly during early s-phase, with few incorporating during mitosis. Molecules of Cnn, in reverse, are mostly incorporated during mitosis and late S-phase, with few incorporated during early S-phase; 2) Spd-2 molecules spend on average l.7 minutes in the PCM whereas Cnn molecules spend 2 minutes in the PCM; 3) Fluxing Spd-2 and Cnn molecules move at speeds between 3 and 30 nm/s through the PCM, with Spd-2 molecules on average moving faster than Cnn; 4) Flux speed is determined by several factors: molecules binding initially closer to the centriole tend to move faster through the PCM than those that bind further from it and flux speed is faster (for Spd-2) during earlier S-phase than during later S-phase. We found that dTACC bound and unbound from the centrosome at a fast rate and seemingly at random distance from the centre, in clear contrast to the directional outwards movement of Spd2 and Cnn.
In order to find the determinants behind the observed fluxing behavior of Spd2 and Cnn, we repeated the single molecule analysis with either a) colchicine to depolymerise microtubules, where we found that Cnn stopped fluxing and would accumulate at the centrosome, whereas Spd2 was seemingly unaffected; constructed and used mutants of Spd-2 that lack the canonical phosphorylation sites – Spd2ALL and Spd2CONS, or mutants of Polo kinase and found that these were still able to flux, particularly at a faster rate indicating that phosphorylation of Spd2/Cnn contributes to keeping these proteins at the centrosome during expansion. We’re currently developing models to describe how fluxing of these proteins contributes to centrosome growth.
In conclusion, the implementation of this action provided a detailed model of how centrosomal key components flux through the centrosome during the cell cycle, the determinants of said flux and how this flux contributes to centrosome size growth and maintenance of size, which are crucial aspects that ensure mitotic fidelity. These findings are being used to prepare a manuscript suitable for publication.
Over the course of this action these findings were regularly disseminated to the community at conferences and symposiums, both internally in the department and at international conferences.
The biophysical nature of the centrosome constitutes a major unsolved mystery in cell biology. Of particular note is the dramatic and rapid increase in mass during PCM expansion, prior to mitosis, when this complex machine made of several hundreds of proteins assembles (and later disassembles) into a structure of precisely defined size. There is currently an intense debate about the role of phase separation in assembling the PCM scaffold, where constituent molecules would separate into biomolecular condensates that behave with liquid-like properties, similarly to P-bodies and lipid droplets.
In this action we studied the dynamics of single molecules of key centrosomal players as they move throughout the PCM and determined the molecular interactions responsible for this movement. In the context of elucidating the biophysical properties of the centrosome, it is crucial to understand if proteins can randomly diffuse throughout the PCM (supporting a biomolecular condensate hypothesis) or if they follow a vectoral outwards flux, indicating that the PCM is more structured. Our findings support the latter hypothesis and we're currently applying these techniques to other PCM proteins to understand how they interact with the underlying PCM scaffold and developing novel mathematical models to describe centrosome biogenesis.
The research described in this action contributes to elucidate aspects related to long-standing questions in cell biology, focusing on several key processes that are known to be involved in tumour development. As such, given the high-profile of the subject under study, the new knowledge generated by this action haa the potential to be impactful not only to the field of Cell Biology but also of broad interest to the scientific community. A manuscript detailing these findings is currently under preparation and will be submitted for peer review in the near future.
In this action we studied the dynamics of single molecules of key centrosomal players as they move throughout the PCM and determined the molecular interactions responsible for this movement. In the context of elucidating the biophysical properties of the centrosome, it is crucial to understand if proteins can randomly diffuse throughout the PCM (supporting a biomolecular condensate hypothesis) or if they follow a vectoral outwards flux, indicating that the PCM is more structured. Our findings support the latter hypothesis and we're currently applying these techniques to other PCM proteins to understand how they interact with the underlying PCM scaffold and developing novel mathematical models to describe centrosome biogenesis.
The research described in this action contributes to elucidate aspects related to long-standing questions in cell biology, focusing on several key processes that are known to be involved in tumour development. As such, given the high-profile of the subject under study, the new knowledge generated by this action haa the potential to be impactful not only to the field of Cell Biology but also of broad interest to the scientific community. A manuscript detailing these findings is currently under preparation and will be submitted for peer review in the near future.