Optogenetic dissection of mitotic spindle positioning in vivo
- Utrecht University, Netherlands
- University of North Carolina at Chapel Hill, United States
Abstract
The position of the mitotic spindle determines the plane of cell cleavage, and thereby daughter cell location, size, and content. Spindle positioning is driven by dynein-mediated pulling forces exerted on astral microtubules, which requires an evolutionarily conserved complex of Gα-GDP, GPR-1/2Pins/LGN, and LIN-5Mud/NuMA proteins. To examine individual functions of the complex components, we developed a genetic strategy for light-controlled localization of endogenous proteins in C. elegans embryos. By replacing Gα and GPR-1/2 with a light-inducible membrane anchor, we demonstrate that Gα-GDP, Gα-GTP, and GPR-1/2 are not required for pulling-force generation. In the absence of Gα and GPR-1/2, cortical recruitment of LIN-5, but not dynein itself, induced high pulling forces. The light-controlled localization of LIN-5 overruled normal cell-cycle and polarity regulation and provided experimental control over the spindle and cell-cleavage plane. Our results define Gα∙GDP-GPR-1/2 Pins/LGN as a regulatable membrane anchor, and LIN-5Mud/NuMA as a potent activator of dynein-dependent spindle-positioning forces.
Data availability
Our design algorithm is accessible via a web interface at http://104.131.81.59/, and the source code can be found at https://github.com/dannyhmg/germline. The data that support the findings of this study are included in the supplementary information. All key plasmids and strains will be deposited with Addgene and the CGC upon publication.
Article and author information
Author details
Lars-Eric Fielmich
Sander Van den Heuvel
Funding
Nederlandse Organisatie voor Wetenschappelijk Onderzoek (NWO TOP/ECHO grant 711.015.001)
- Lars-Eric Fielmich
- Sander Van den Heuvel
National Institutes of Health (NIH R01 GM083071)
- Bob Goldstein
Helen Hay Whitney Foundation
- Daniel J Dickinson
European Research Council (Synergy grant 609822)
- Ruben Schmidt
- Anna Akhmanova
National Institutes of Health (NIH K99 GM115964)
- Daniel J Dickinson
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Copyright
© 2018, Fielmich et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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Optogenetic dissection of mitotic spindle positioning in vivo
eLife 7:e38198.
https://doi.org/10.7554/eLife.38198
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