Stepping stone: a cytohesin adaptor for membrane cytoskeleton restraint in the syncytial Drosophila embryo.
Bottom Line: 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.
Affiliation: Department of Cell and Systems Biology, University of Toronto, Toronto, ON M5S 3G5, Canada.Show MeSH
Mentions: To determine the relationship between Step and the different pools of Sstn, we conducted co-overexpression experiments. Coexpression of GFP-Sstn with mCh-Step induced greater furrow levels for each protein versus single-overexpression controls (Figure 4A; statistical significance shown with quantifications at right), and the proteins colocalized at the membranes. mCh-Step solely localized to the plasma membrane regardless of Sstn coexpression, but the increase in Sstn furrow localization coincided with a decrease in centrosome localization (Figure 4A; line scans from embryos with similar furrow protein levels show the reproducible effect on the centrosome-associated pool). Of interest, the displacement of Sstn from centrosomes was also evident in our proteomic analyses, as centrosomin reproducibly precipitated with GFP-Sstn when expressed alone but not when coexpressed with mCh-Step (Table 1). Thus Sstn-Step associations seem to occur preferentially at plasma membrane furrows.
Affiliation: Department of Cell and Systems Biology, University of Toronto, Toronto, ON M5S 3G5, Canada.