title: Stepping stone: a cytohesin adaptor for membrane cytoskeleton restraint in the syncytial Drosophila embryo.
abstract: Cytohesin Arf-GEFs are conserved plasma membrane regulators. The sole Drosophila cytohesin, Steppke, restrains Rho1-dependent membrane cytoskeleton activity at the base of plasma membrane furrows of the syncytial embryo. By mass spectrometry, we identified a single major Steppke-interacting protein from syncytial embryos, which we named Stepping stone (Sstn). By sequence, Sstn seems to be a divergent homologue of the mammalian cytohesin adaptor FRMD4A. Our experiments supported this relationship. Specifically, heterophilic coiled-coil interactions linked Sstn and Steppke in vivo and in vitro, whereas a separate C-terminal region was required for Sstn localization to furrows. Sstn mutant and RNAi embryos displayed abnormal, Rho1-dependent membrane cytoskeleton expansion from the base of pseudocleavage and cellularization furrows, closely mimicking Steppke loss-of-function embryos. Elevating Sstn furrow levels had no effect on the steppke phenotype, but elevating Steppke furrow levels reversed the sstn phenotype, suggesting that Steppke acts downstream of Sstn and that additional mechanisms can recruit Steppke to furrows. Finally, the coiled-coil domain of Steppke was required for Sstn binding and in addition homodimerization, and its removal disrupted Steppke furrow localization and activity in vivo. Overall we propose that Sstn acts as a cytohesin adaptor that promotes Steppke activity for localized membrane cytoskeleton restraint in the syncytial Drosophila embryo.
authors.affiliation: Department of Cell and Systems Biology, University of Toronto, Toronto, ON M5S 3G5, Canada.
authors.forename: Jiangshu
authors.initials: J
authors.lastname: Liu
authors.affiliation: Department of Cell and Systems Biology, University of Toronto, Toronto, ON M5S 3G5, Canada.
authors.forename: Donghoon M
authors.initials: DM
authors.lastname: Lee
authors.affiliation: Department of Cell and Systems Biology, University of Toronto, Toronto, ON M5S 3G5, Canada.
authors.forename: Cao Guo
authors.initials: CG
authors.lastname: Yu
authors.affiliation: Department of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy, University of Toronto, Toronto, ON M5S 3G5, Canada Department of Biochemistry, University of Toronto, Toronto, ON M5S 3G5, Canada.
authors.forename: Stephane
authors.initials: S
authors.lastname: Angers
authors.affiliation: Department of Cell and Systems Biology, University of Toronto, Toronto, ON M5S 3G5, Canada tony.harris@utoronto.ca.
authors.forename: Tony J C
authors.initials: TJ
authors.lastname: Harris
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D000595:Amino Acid Sequence; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism*; D003599:Cytoskeleton / Q000378:metabolism* / Q000648:ultrastructure; D029721:Drosophila Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D004331:Drosophila melanogaster / Q000196:embryology / Q000378:metabolism / Q000648:ultrastructure*; D004625:Embryo, Nonmammalian / Q000378:metabolism; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism; D008969:Molecular Sequence Data; D016415:Sequence Alignment
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D029721:Drosophila Proteins; D020662:Guanine Nucleotide Exchange Factors; C000603870:Sstn protein, Drosophila; C516026:Step protein, Drosophila