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: Together these data indicated that the Step CC domain was required for Sstn association, Step localization, and membrane cytoskeleton restraint. However, it seemed that the lack of Sstn association could not solely explain the effects of deleting the CC domain from Step. Specifically, GFP-Step localization and function were disrupted with deletion of the CC domain, but GFP-Step could localize and function with the depletion of Sstn. One explanation for this discrepancy would be additional interactions promoted by the Step CC domain. Suggesting one possible interaction, the CC domains of mammalian cytohesins have been shown to homodimerize (DiNitto et al., 2007, 2010). To test for such homodimerization for Step, we purified bacterially expressed Step proteins with or without the CC domain. Using blot overlays, we detected Step–Step dimerization dependent on the CC domain (Figure 9). If this Step dimerization occurs in vivo, then the CC domain of Step may have two roles at plasma membranes: 1) direct interaction with Sstn and 2) indirect promotion of other membrane interactions through avidity effects with dimerization (see Discussion).
Affiliation: Department of Cell and Systems Biology, University of Toronto, Toronto, ON M5S 3G5, Canada.