Triple-Color Live Imaging of Mouse Oocytes

Courtois, Aurélien; Solc, P; Kitajima, Tomoya S.

doi:10.1007/978-1-4939-8603-3_10

Cited by 1 publication

(2 citation statements)

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“…Having confirmed that IPZ disrupts both the chromosomal RanGTP and importin b cargo gradients in oocytes, we were interested in testing how IPZ affects the MI progression in mouse oocytes. To do so, we isolated oocytes from the histone H2B-EGFP-expressing transgenic mice, stained the microtubules with the fluorogenic SiRtubulin dye (Lukinavicius et al, 2014;Balboula et al, 2016;Courtois et al, 2018) To examine in more detail how the inhibition of RanGTP signaling by IPZ affects MI spindle assembly, we performed 3D three-color live imaging (Balboula et al, 2016;Courtois et al, 2018)…”

Section: Treatment With Ipz Blocks MI Spindle Formation and Functionmentioning

confidence: 99%

“…Our results suggest that the MI spindle assembly and function continuously require RanGTP, which enables the activation of importin b-regulated SAFs throughout the oocytes. Advances in live-cell imaging methods, including multi-color time-lapse imaging (Courtois et al, 2018) and the quantitative FLIM imaging with optimized FRET biosensors, were important for clarifying the role of RanGTP in mouse MI spindle function.…”

Section: Regulation Of Mtoc Dynamics By Ran and Importin Bmentioning

confidence: 99%

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Ran GTP and importin β regulate meiosis I spindle assembly and function in mouse oocytes

Drutovič

Duan

et al. 2019

The EMBO Journal

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Homologous chromosome segregation during meiosis I (MI) in mammalian oocytes is carried out by the acentrosomal MI spindles. Whereas studies in human oocytes identified Ran GTPase as a crucial regulator of the MI spindle function, experiments in mouse oocytes questioned the generality of this notion. Here, we use livecell imaging with fluorescent probes and Förster resonance energy transfer (FRET) biosensors to monitor the changes in Ran and importin b signaling induced by perturbations of Ran in mouse oocytes while examining the MI spindle dynamics. We show that unlike RanT24N employed in previous studies, a RanT24N, T42A double mutant inhibits RanGEF without perturbing cargo binding to importin b and disrupts MI spindle function in chromosome segregation. Roles of Ran and importin b in the coalescence of microtubule organizing centers (MTOCs) and MI spindle assembly are further supported by the use of the chemical inhibitor importazole, whose effects are partially rescued by the GTP hydrolysisresistant RanQ69L mutant. These results indicate that RanGTP is essential for MI spindle assembly and function both in humans and mice.

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