A cell-free system of Drosophila egg explants supporting native mitotic cycles

et al. 2020

Preprint

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23Centrioles form centrosomes and cilia. In most proliferating cells, centrioles assemble 24 through canonical duplication, which is spatially, temporally and numerically regulated 25 by the cell cycle and the presence of mature centrioles. However, in certain cell-types, 26 centrioles assemble de novo, yet by poorly understood mechanisms. Here, we 27 established a controlled system to investigate de novo centriole biogenesis, using 28Drosophila melanogaster egg explants overexpressing Polo-like kinase 4 (Plk4), a 29 trigger for centriole biogenesis. At high Plk4 concentration, centrioles form de novo, 30 mature and duplicate, independently of cell cycle progression and of the presence of 31 other centrioles. We show that Plk4 concentration determines the kinetics of centriole 32 assembly. Moreover, our results suggest Plk4 operates in a switch-like manner to control 33

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Plk4 triggers autonomous de novo centriole biogenesis and maturation

Nabais

Pessoa

et al. 2020

Preprint

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“…Molecular crosslinking between astral microtubules of neighboring nuclei during the preblastoderm embryo stage has largely been unexplored due to optical constraints in live imaging. Using an extraction method to generate embryo explants from individual preblastoderm embryos (de-Carvalho et al, 2018) expressing Klp61F::GFP and Feo::mCherry, and injected with Alexa647-labeled Tubulin (Fig. 1A), we visualized the localization of Klp61F and Feo to infer about their binding to spindle microtubules (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…They were dechorionated in 7% sodium hypochlorite solution, aligned and immobilized on a clean coverslip using adhesive dissolved in heptane and covered with halocarbon oil (Voltalef 10S). Extraction of cytoplasm from individual embryos and generation of explants was performed on a custom-made microscope as previously described (de-Carvalho et al, 2018; Telley et al, 2013).…”

Section: Methods Detailsmentioning

confidence: 99%

Astral microtubule crosslinking safeguards uniform nuclear distribution in the Drosophila syncytium

Deshpande

Vieira

et al. 2019

Preprint

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13The early insect embryo develops as multinucleated cell distributing genomes uniformly to the cell 14 cortex. Mechanistic insight for nuclear positioning beyond cytoskeletal requirements is missing to 15 date. Contemporary hypotheses propose actomyosin driven cytoplasmic movement transporting 16 nuclei, or repulsion of neighbor nuclei driven by microtubule motors. Here, we show that 17 microtubule crosslinking by Feo and Klp3A is essential for nuclear distribution and internuclear 18 distance maintenance in Drosophila. RNAi knockdown in the germline causes irregular, less dense 19 nuclear delivery to the embryo cortex and smaller distribution in ex vivo embryo explants. A 20 minimal internuclear distance is maintained in explants from control embryos but not from Feo 21 depleted embryos, following micromanipulation assisted repositioning. A dominant-negative Feo 22 protein abolishes nuclear separation in embryo explants while the full-length protein rescues the 23 genetic knockdown. We conclude that antiparallel microtubule overlap crosslinking by Feo and 24Klp3A generates a length-regulated mechanical link between neighboring microtubule asters. 25Enabled by a novel experimental approach, our study illuminates an essential process of embryonic 26 multicellularity. 27 28 spindle midzone 29,37-40 . One of these motors is Klp3A, a Kinesin-4 homolog, a microtubule 63 depolymerase with chromatin binding affinity 35,[41][42][43][44] . PRC1 and Kinesin-4 are sufficient to form a 64 stable microtubule overlap in vitro 35 . Kinesin-5 is able to reduce overlapping, antiparallel 65 microtubules crosslinked by PRC1 in vitro 36 , which was proposed to contribute to force balance in 66 the spindle midzone during anaphase B 30 . Here, we investigated whether these three proteins are 67 required for nuclear separation, lending support to an aster-aster interaction model 15,45 . We 68 performed a combination of gene knockdown, micromanipulation and perturbation by exogenous 69 protein addition in embryo explants which enable time-lapse visualization of nuclear and 70 cytoskeletal dynamics previously unachieved. 71

Astral microtubule cross-linking safeguards uniform nuclear distribution in the Drosophila syncytium

Deshpande

Journal of Cell Biology

Vieira

et al. 2021

Self Cite

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 cross-linking 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 cross-linking 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.