Rab1 interacts with GOLPH3 and controls Golgi structure and contractile ring constriction during cytokinesis in Drosophila melanogaster

Creators: Sechi, Stefano; Frappaolo, Anna; Fraschini, Roberta; Capalbo, Luisa; Gottardo, Marco; Belloni, Giorgio; Glover, David M.; Wainman, Alan; Giansanti, Maria Grazia

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Abstract

Cytokinesis requires a tight coordination between actomyosin ring constriction and new membrane addition along the ingressing cleavage furrow. However, the molecular mechanisms underlying vesicle trafficking to the equatorial site and how this process is coupled with the dynamics of the contractile apparatus are poorly defined. Here we provide evidence for the requirement of Rab1 during cleavage furrow ingression in cytokinesis. We demonstrate that the gene omelette (omt) encodes the Drosophila orthologue of human Rab1 and is required for successful cytokinesis in both mitotic and meiotic dividing cells of Drosophila melanogaster. We show that Rab1 protein colocalizes with the conserved oligomeric Golgi (COG) complex Cog7 subunit and the phosphatidylinositol 4-phosphate effector GOLPH3 at the Golgi stacks. Analysis by transmission electron microscopy and 3D-SIM super-resolution microscopy reveals loss of normal Golgi architecture in omt mutant spermatocytes indicating a role for Rab1 in Golgi formation. In dividing cells, Rab1 enables stabilization and contraction of actomyosin rings. We further demonstrate that GTP-bound Rab1 directly interacts with GOLPH3 and controls its localization at the Golgi and at the cleavage site. We propose that Rab1, by associating with GOLPH3, controls membrane trafficking and contractile ring constriction during cytokinesis.

Additional Information

© 2017 The Authors. Published by the Royal Society under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/4.0/, which permits unrestricted use, provided the original author and source are credited. Received: 9 September 2016. Accepted: 12 December 2016. We thank R. Karess, A. Paululat and O. Papoulas for antibodies, J. Raff for antibodies and fly stocks and M. T. Fuller for the pCaSpeR-sa plasmid. We thank G. Colotti for assisting in purifying GST-tagged proteins. We thank R. Piergentili and P. D'Avino for helpful discussion. This work was supported by a grant from Associazione Italiana per la Ricerca sul Cancro (AIRC) (grant no. IG14671) and a grant from Fondazione Telethon-Italy (grant no. GEP14076) to M.G.G. A.W. was supported by a Wellcome Trust Strategic Award to the Micron Oxford Advanced Bioimaging Unit (107457). L.C. and D.M.G. are grateful for support from Cancer Research UK (RG78567). Funding to pay the Open Access publication charges for this article was provided by Telethon-Italy. Authors' contributions: S.S., A.F., R.F., L.C., M.G., G.B., D.M.G., A.W. and M.G.G. analysed the data. A.F, M.G.G. and A.W. designed and carried out the Golgi analysis by immunofluorescence. M.G.G. and A.F. carried out the PLA experiments. A.W. designed and carried out the studies by 3D-SIM super-resolution microscopy. M.G. carried out the TEM analysis. M.G.G., G.B. and A.W. performed the genetic mapping and rescue experiments. L.C. designed and carried out the RNAi experiments in S2 cells. S.S. developed experiments aimed at constructing GST-Rab1/GST-Rab11 and at purifying the GST-Rab1 to raise anti-Rab1 antibodies; S.S. performed the molecular biology experiments aimed at constructing the YFP/RFP transgenes. S.S. also performed the Co-IP and the GST pull-down experiments. R.F. designed and performed the two-hybrid experiments. M.G.G., A.W., R.F. and A.F. carried out the statistical analyses. M.G.G. wrote the paper and A.W., L.C., R.F. and D.M.G helped draft the manuscript. All authors gave final approval for publication. We have no competing interests.