Biology subjects

Duarte, P.

Publications and source records attributed to Duarte, P..

5 recordsLinked to original sources

bioRxiv2022

Regulatory changes associated with the head to trunk developmental transition

Development of vertebrate embryos is characterized by early formation of the anterior tissues followed by the sequential extension of the axis at their posterior end to build the trunk and tail structures, first by the activity of the primitive streak and then of the tail bud. Embryological, molecular and genetic data demonstrate that head and trunk development are significantly different, indicating that the transition into the trunk formation stage involves changes in regulatory gene networks, that might include the acquisition of cell competence to respond to key regulatory factors. Herein, we explored the regulatory changes involved in this developmental transition by assessing the transcriptome and chromatin accessibility profiles from the posterior epiblast region of mouse embryos at embryonic day (E)7.5 and E8.5. We observed changes in various cell processes, including signaling pathways, ubiquitination, ion dynamics and metabolic processes involving lipids that could contribute to the functional switch in the progenitor region of the embryo. Our data also led to the identification of novel mechanisms controlling the differential Wnt functional requirements during head and trunk development. Moreover, we found substantial changes in chromatin accessibility mostly mapping to intergenic regions, indicating a significant switch in the regulatory elements controlling either head or trunk development. In addition, we tested the functional relevance of potential enhancers of Wnt5a and Nr2f2, identified in the accessibility assays, that reproduced the expression profiles of the target genes. Deletion of these regions by genome editing had limited effect on the expression of those genes, suggesting the existence of redundant enhancers that guarantee robust expression patterns. Overall, this study provides new insights on the regulatory mechanisms that change during the transition from head to trunk development. Author SummaryVertebrate main body axis is generated sequentially from head to tail. The developmental processes building head and trunk structures are significantly different, and the transition between these two stages requires substantial changes in functional gene regulatory networks. Herein, we explored such changes through genome wide analyses in developing mouse embryos. We observed significant differences in several signaling pathways and in the basic cell machinery, which may interact promoting a functional switch in the differentiating progenitor cells. We also found substantial changes in the accessibility of regulatory elements controlling either head or trunk formation, which conditioned the binding activity of key developmental transcription factors. Overall, our study gives relevant insights into the mechanisms regulating the head to trunk transition that, if disrupted, can lead to embryonic developmental arrest.

developmental biology↗

bioRxiv2022

Ana1/CEP295 is an essential player in the centrosome maintenance program regulated by Polo kinase

Centrioles play critical roles in our cells, being part of centrosomes and cilia, which are important microtubule organising centers (MTOC) with a variety of roles. While centrioles are very stable structures, they disappear in certain cell types upon differentiation, such as in oocytes. Little is known about the regulation of centriole structural integrity. We previously uncovered that the pericentriolar material (PCM), and its recruiter Polo kinase, are required for both the maintenance of centriole structural integrity and centrosome MTOC activity. Using an hypothesis driven RNAi screen, we show that both the cartwheel and the centriole wall play an important role in centrosome integrity. In particular, we uncovered that the centriole wall protein ANA1 is critical for the integrity of both new and mature centrioles, in Drosophila oogenesis as well as in cultured cells. Moreover, our results show that the activity of both Polo and the PCM in centriole integrity depends on ANA1. Our work suggests that the structural integrity of centrioles, once thought to be very stable organelles, depends on the turnover of key components, suggesting new perspectives for understanding the dysfunction of those structures in disease.

cell biology↗

bioRxiv2020

Plk4 triggers autonomous de novo centriole biogenesis and maturation

Centrioles form centrosomes and cilia. In most proliferating cells, centrioles assemble through canonical duplication, which is spatially, temporally and numerically regulated by the cell cycle and the presence of mature centrioles. However, in certain cell-types, centrioles assemble de novo, yet by poorly understood mechanisms. Here, we established a controlled system to investigate de novo centriole biogenesis, using Drosophila melanogaster egg explants overexpressing Polo-like kinase 4 (Plk4), a trigger for centriole biogenesis. We show that at high Plk4 concentration, centrioles form de novo, mature and duplicate, independently of cell cycle progression and of the presence of other centrioles. Plk4 concentration determines the kinetics of centriole assembly. Moreover, our results suggest Plk4 operates in a switch-like manner to control the onset of de novo centriole formation, and that distinct biochemical kinetics regulate de novo and canonical biogenesis. Finally, we investigated which other factors modulate de novo centriole assembly and reveal that proteins of the pericentriolar matrix (PCM) promote biogenesis, likely by locally concentrating critical components.

cell biology↗

bioRxiv2018

An ancestral role of pericentrin in centriole formation through SAS-6 recruitment

The centrosome is composed of two centrioles surrounded by a microtubule-nucleating pericentriolar matrix (PCM). Centrioles regulate matrix assembly. Here we ask whether the matrix also regulates centriole assembly. To define the interaction between the matrix and individual centriole components, we take advantage of a heterologous expression system using fission yeast. Importantly, its centrosome, the spindle pole body (SPB), has matrix but no centrioles. Surprisingly, we observed that the SPB can recruit several animal centriole components. Pcp1/pericentrin, a conserved matrix component that is often upregulated in cancer, recruits a critical centriole constituent, SAS-6. We further show that this novel interaction is conserved and important for centriole biogenesis and elongation in animals. We speculate that the Pcp1/pericentrin-SAS-6 interaction surface was conserved for one billion years of evolution after centriole loss in yeasts, due to its conserved binding to calmodulin. This study reveals an ancestral relationship between pericentrin and the centriole, where both regulate each other assembly, ensuring mutual localisation.\n\nShort summaryThe pericentriolar matrix (PCM) is not only important for microtubule-nucleation but also can regulate centriole biogenesis. Ito et al. reveal an ancestral interaction between the centriole protein SAS-6 and the PCM component pericentrin, which regulates centriole biogenesis and elongation.

cell biology↗

bioRxiv2018

PLK4 is a microtubule-associated protein that self assembles promoting de novo MTOC formation

Summary statementPLK4 binds to microtubules and self assembles into supramolecular assemblies that recruit tubulin and trigger de novo MTOC formation in Xenopus laevis extracts.\n\nAbstractThe centrosome is an important microtubule-organizing center (MTOCs) in animal cells and it consists of two barrel-shaped structures (centrioles), surrounded by the pericentriolar material (PCM), which nucleates microtubules. PCM components form condensates, supramolecular assemblies that concentrate microtubule nucleators. Centrosomes can form close to an existing structure (canonical duplication) or de novo. How centrosomes form de novo is not known. PLK4 is a master driver of centrosome biogenesis, which is critical to recruit several centriole components. Here, we investigate the beginning of centrosome biogenesis, taking advantage of Xenopus egg extracts, where we and others have shown that PLK4 can induce de novo MTOC formation (Eckerdt et al., 2011; Zitouni et al., 2016). Surprisingly, we observe that in vitro, PLK4 can self-assemble into supramolecular assemblies that recruit /{beta}-tubulin. In Xenopus extracts, PLK4 supramolecular assemblies additionally recruit the PLK4 substrate STIL and the microtubule nucleator, {gamma}-tubulin, and form acentriolar MTOCs de novo. The assembly of these robust microtubule asters is independent of dynein, similarly to centrosomes. We suggest a new mechanism of action for PLK4, where it forms a self-organizing catalytic scaffold that recruits centriole components, PCM factors and /{beta}-tubulin, leading to MTOC formation.

cell biology↗