Biology subjects

de-Carvalho, J.

Publications and source records attributed to de-Carvalho, J..

6 recordsLinked to original sources

bioRxiv2023

The positioning mechanics of microtubule asters in Drosophila embryo explants

Microtubule asters are essential in localizing the action of microtubules in processes including mitosis and organelle positioning. In large cells, such as the one-cell sea urchin embryo, aster dynamics are dominated by hydrodynamic pulling forces. However, in systems with more densely positioned nuclei such as the early Drosophila embryo, which packs around 6000 nuclei within the syncytium in a crystalline-like order, it is unclear what processes dominate aster dynamics. Here, we take advantage of a cell cycle regulation Drosophila mutant to generate embryos with multiple asters, independent from nuclei. We use an ex vivo assay to further simplify this biological system to explore the forces generated by and between asters. Through live imaging, drug and optical perturbations, and theoretical modelling, we demonstrate that these asters likely generate an effective pushing force over short distances. Significance StatementUsing cytosolic explants from Drosophila syncytial embryos combined with quantitative microscopy and perturbations, de-Carvalho et al., reveal the mechanical forces separating Drosophila microtubule asters. Aster separation drives precise nuclear positioning in multinucleated embryo cells, a vital process for tissue formation and gene expression during subsequent embryo development.

biophysics↗

bioRxiv2022

Pulse-chase experiments reveal dynamics of RNA binding protein Exuperantia in Drosophila melanogaster egg chambers

In cells, mRNA can be associated with various proteins, forming ribonucleoprotein complexes (RNPs) which take part in spatiotemporal control of translation. In the Drosophila melanogaster developing egg chamber, a set of RNPs is transported from the nurse cells to the oocyte and targeted selectively to specific cellular locations. This mRNA sorting process leads to the final oocyte polarization pre-defining the body axes of the future embryo. However, how mRNA is encoded for selection and directed transport is mechanistically not well understood. A master mRNA involved in body axes formation is bicoid, which localizes anterolaterally and is essential for head and thorax definition of the embryo. A protein that was identified essential for bicoid anterior localization is Exuperantia (Exu). Here, we use a live imaging-based pulse-chase approach, which reveals selective transport dynamics of Exu from nurse cells to the oocyte during mid to late-stage oogenesis.

cell biology↗

bioRxiv2022

Follicle cell contact maintains main body axis polarity in the Drosophila melanogaster oocyte

In Drosophila melanogaster the anterior-posterior body axis is maternally established and governed by differential localization of partitioning defective (Par) proteins within the oocyte. At mid-oogenesis, Par-1 accumulates at the posterior end of the oocyte while Par-3/Bazooka is excluded there but maintains its localization along the remaining oocyte cortex. This mutual exclusion leads to a polarized microtubule network and accumulation of posterior determinant oskar later in oogenesis. Reciprocal biochemical interactions between Par proteins can explain their cortical exclusion and domain formation - for example, Par-1 excludes Par-3 by phosphorylation. However, past studies have proposed the need for somatic cells at the posterior end to initiate oocyte polarization by providing a trigger signal. To date, despite modern screening approaches and genetic manipulation, neither the molecular identity nor the nature of the signal is known. Here, we provide the first evidence that mechanical contact of posterior follicle cells (PFCs) with the oocyte cortex causes the posterior exclusion of Bazooka and maintains oocyte polarity. We show that Bazooka prematurely accumulates exclusively where posterior follicle cells have been mechanically detached or ablated. This occurs before Par-1 is removed suggesting that phosphorylation of Bazooka by Par-1 is not sufficient to maintain Bazooka exclusion in the absence of PFC contact. Furthermore, we provide evidence that PFC contact maintains Par-1 and oskar localization and microtubule cytoskeleton polarity in the oocyte. Our observations suggest that cell-cell contact mechanics modulates Par protein binding sites at the oocyte cortex.

developmental biology↗

bioRxiv2020

Aster repulsion drives local ordering in an active system

Biological systems are a form of active matter, which often undergo rapid changes in their material state, e.g. liquid to solid transitions. Yet, such systems often also display remarkably ordered structures. It remains an open question as to how local ordering occurs within active systems. Here, we utilise the rapid early development of Drosophila melanogaster embryos to uncover the mechanisms driving short-ranged order. During syncytial stage, nuclei synchronously divide (within a single cell defined by the ellipsoidal eggshell) for nine cycles after which most of the nuclei reach the cell cortex. Despite the rapid nuclear division and repositioning, the spatial pattern of nuclei at the cortex is highly regular. Such precision is important for subsequent cellularisation and morphological transformations. We utilise ex vivo explants and mutant embryos to reveal that microtubule asters ensure the regular distribution and maintenance of nuclear positions in the embryo. For large networks of nuclei, such as in the embryo, we predict - and experimentally verify - the formation of force chains. The ex vivo extracts enabled us to deduce the force potential between single asters. We use this to predict how the nuclear division axis orientation in small ex vivo systems depend on aster number. Finally, we demonstrate that, upon nucleus removal from the cortex, microtubule force potentials can reorient subsequent nuclear divisions to minimise the size of pattern defects. Overall, we show that short-ranged microtubule-mediated repulsive interactions between asters can drive ordering within an active system.

biophysics↗

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↗

bioRxiv2019

Astral microtubule crosslinking by Feo safeguards uniform nuclear distribution in the Drosophila syncytium

The early insect embryo develops as a multinucleated cell distributing the genome uniformly to the cell cortex. Mechanistic insight for nuclear positioning beyond cytoskeletal requirements is missing. Contemporary hypotheses propose actomyosin driven cytoplasmic movement transporting nuclei, or repulsion of neighbor nuclei driven by microtubule motors. Here, we show that microtubule crosslinking by Feo and Klp3A is essential for nuclear distribution and internuclear distance maintenance in Drosophila. Germline knockdown causes irregular, less dense nuclear delivery to the cell cortex and smaller distribution in ex vivo embryo explants. A minimal internuclear distance is maintained in explants from control embryos but not from Feo inhibited embryos, following micromanipulation assisted repositioning. A dimerization deficient Feo abolishes nuclear separation in embryo explants while the full-length protein rescues the genetic knockdown. We conclude that Feo and Klp3A crosslinking of antiparallel microtubule overlap generates a length-regulated mechanical link between neighboring microtubule asters. Enabled by a novel experimental approach, our study illuminates an essential process of embryonic multicellularity.

cell biology↗