Abstract
Correct placement and orientation of the mitotic spindle is essential for segregation of localized components and positioning of daughter cells. Although these processes are important in many cells, few factors that regulate spindle placement are known. Previous work has shown that GPB-1, the Gβ subunit of a heterotrimeric G protein, is required for orientation of early cell division axes in C. elegans embryos. Here we show that GOA-1 (a Gαo) and the related GPA-16 are the functionally redundant Gα subunits and that GPC-2 is the relevant Gγ subunit that is required for spindle orientation in the early embryo. We show that Gα and Gβγ are involved in controlling distinct microtubule-dependent processes. Gβγ is important in regulating migration of the centrosome around the nucleus and hence in orientating the mitotic spindle. Gα is required for asymmetric spindle positioning in the one-celled embryo.
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Acknowledgements
We thank M. Koelle, J. Kilmartin, K. Kemphues and S. Strome for antibodies, Y. Kohara for cDNAs, and E. Cuppen, G. Jansen and R. Plasterk for plasmids, strains and suggestions. Some strains used in this study were from the Caenorhabditis Genetics Centre, which is supported by the NIH National Center for Research Resources (NCRR). We also thank A. H. Brand, A. G. Fraser, R. Kamath, M. Martinez-Campos, J. W. Raff, and M. Sohrmann for comments on the manuscript. This work was supported by a Wellcome Senior Research Fellowship (to J.A.), an HFSP long-term fellowship (to M.G.) and an EC TMR network grant.
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Figure S1. Heterotrimeric G proteins are not required for embryonic polarity. a–c, Localization of P granules (green) in wild-type (a), Gα(RNAi) (b) and Gβ(RNAi) (c) two-cell embryos. P granules are segregated to the posterior cell, P1, in both wild-type and mutant embryos. DNA is counterstained with DAPI (blue). d–f, Localization of PAR-2 (red) and PAR-3 (green) in wild-type (d), Ga(RNAi) (e) and Gβ(RNAi) (f) one-cell embryos. In wild-type embryos PAR-3 and PAR-2 proteins are enriched at the anterior and posterior cortices, respectively. Localization of both PAR-3 and PAR-2 is normal in both Gα(RNAi) (n = 25), and Gβ(RNAi) (n = 20) embryos. Localization of PAR-2 and PAR-3 is also normal in two-cell embryos (data not shown). Posterior is to the right in each case; scale bar represents 10 μm. (PDF 356 kb)
Figure S2. Tubulin distribution is abnormal in Gα(RNAi) embryos. Tubulin staining (green) in two-cell (a–c) and four-cell (d–f) embryos. a, d, Wild-type embryos; b, c, e, f, Gα(RNAi) embryos. This figure provides more examples for comparison of tubulin distribution and aster morphology in wild-type embryos and Gα(RNAi) embryos. Arrows indicate centrosomes. DNA is counterstained with DAPI (blue). In a one aster of AB (the anterior cell) is out of focus. Posterior is to the right in each case; scale bar represents 10 –m.
Table S1. Centrosome migration paths and spindle orientations in wild-type, Gß(RNAi) and Ga(RNAi) embryos. Centrosome positions were monitored in wild- type and gpb-1(RNAi) AB, P1, ABa and ABp cells from birth until division, from 4D video recordings of embryonic development (15 focal planes per 30 s). ‚Migration’ indicates the axis defined by the migration path. As the d/v axis is not defined until the division of AB, wild-type migration in AB and P1 cells is designated as ‚transverse’, meaning transverse to the a/p axis. ‚Rotation’ indicates whether the nucleocentrosomal complex rotated (+) or not (−); an asterisk indicates that the spindle rotated. ‚Spindle orientation’ shows the final orientation of the mitotic spindle. In wild-type, Gß(RNAi), and Ga(RNAi) embryos, centrosomes migrate in opposite directions. The wild-type row shows data from 11 separate embryos. a, anterior; p, posterior; d, dorsal; v, ventral; l, left; r, right; ns, not scorable. †Centrosome starting position was incorrect. ††Centrosomes did not migrate apart from each other; this is probably the axis of centrosome duplication.
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Gotta, M., Ahringer, J. Distinct roles for Gα and Gβγ in regulating spindle position and orientation in Caenorhabditis elegans embryos. Nat Cell Biol 3, 297–300 (2001). https://doi.org/10.1038/35060092
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DOI: https://doi.org/10.1038/35060092
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