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Stepping stone: a cytohesin adaptor for membrane cytoskeleton restraint in the syncytial Drosophila embryo.

Liu J, Lee DM, Yu CG, Angers S, Harris TJ - Mol. Biol. Cell (2014)

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.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Systems Biology, University of Toronto, Toronto, ON M5S 3G5, Canada.

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Mapping a direct interaction between the coiled-coil domains of Sstn and Step. (A) Sstn and Step constructs used in the binding assays. (B) Blot overlays showing binding of GST-Step to major MBP-Sstn proteolytic fragments (asterisks) but not to higher levels of MBP or MBP-Sstn∆CC fragments. GST and GST-Step∆CC showed no binding to the MBP-Sstn proteolytic fragments, despite incubation at higher levels than GST-Step (see left blot for the relative GST, GST-Step, and GST-Step∆CC protein levels used for the overlays shown). The overlay blots were probed and imaged side by side with identical reagents and settings. The overall results were replicated in a separate complete analysis.
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Figure 2: Mapping a direct interaction between the coiled-coil domains of Sstn and Step. (A) Sstn and Step constructs used in the binding assays. (B) Blot overlays showing binding of GST-Step to major MBP-Sstn proteolytic fragments (asterisks) but not to higher levels of MBP or MBP-Sstn∆CC fragments. GST and GST-Step∆CC showed no binding to the MBP-Sstn proteolytic fragments, despite incubation at higher levels than GST-Step (see left blot for the relative GST, GST-Step, and GST-Step∆CC protein levels used for the overlays shown). The overlay blots were probed and imaged side by side with identical reagents and settings. The overall results were replicated in a separate complete analysis.

Mentions: The CC domain of FRMD4A binds to the CC domains of cytohesins (Ikenouchi and Umeda, 2010). If Sstn is a homologue of FRMD4A, a similar interaction with Step would be expected. Our IP-MS data implicated a direct Sstn–Step interaction. To test further for the interaction and map the sequences involved, we designed full-length and deletion constructs for Sstn and Step (Figure 2A), purified the proteins from bacteria, and pursued blot overlays. Maltose-binding protein (MBP)–Sstn was separated by SDS–PAGE, blotted, and then probed with purified glutathione S-transferase (GST)–Step, followed by GST antibodies. A very strong signal for GST-Step was detected where major proteolytic fragments of the MBP-Sstn protein were positioned on the blot (Figure 2B). Slightly higher levels of GST showed no binding to MBP-Sstn, and slightly higher levels of MBP showed no binding to GST-Step (Figure 2B). Thus Sstn and Step can interact directly in vitro, and the interacting MBP-Sstn fragment sizes suggested that the interaction site was in the N-terminal half of Sstn.


Stepping stone: a cytohesin adaptor for membrane cytoskeleton restraint in the syncytial Drosophila embryo.

Liu J, Lee DM, Yu CG, Angers S, Harris TJ - Mol. Biol. Cell (2014)

Mapping a direct interaction between the coiled-coil domains of Sstn and Step. (A) Sstn and Step constructs used in the binding assays. (B) Blot overlays showing binding of GST-Step to major MBP-Sstn proteolytic fragments (asterisks) but not to higher levels of MBP or MBP-Sstn∆CC fragments. GST and GST-Step∆CC showed no binding to the MBP-Sstn proteolytic fragments, despite incubation at higher levels than GST-Step (see left blot for the relative GST, GST-Step, and GST-Step∆CC protein levels used for the overlays shown). The overlay blots were probed and imaged side by side with identical reagents and settings. The overall results were replicated in a separate complete analysis.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4325841&req=5

Figure 2: Mapping a direct interaction between the coiled-coil domains of Sstn and Step. (A) Sstn and Step constructs used in the binding assays. (B) Blot overlays showing binding of GST-Step to major MBP-Sstn proteolytic fragments (asterisks) but not to higher levels of MBP or MBP-Sstn∆CC fragments. GST and GST-Step∆CC showed no binding to the MBP-Sstn proteolytic fragments, despite incubation at higher levels than GST-Step (see left blot for the relative GST, GST-Step, and GST-Step∆CC protein levels used for the overlays shown). The overlay blots were probed and imaged side by side with identical reagents and settings. The overall results were replicated in a separate complete analysis.
Mentions: The CC domain of FRMD4A binds to the CC domains of cytohesins (Ikenouchi and Umeda, 2010). If Sstn is a homologue of FRMD4A, a similar interaction with Step would be expected. Our IP-MS data implicated a direct Sstn–Step interaction. To test further for the interaction and map the sequences involved, we designed full-length and deletion constructs for Sstn and Step (Figure 2A), purified the proteins from bacteria, and pursued blot overlays. Maltose-binding protein (MBP)–Sstn was separated by SDS–PAGE, blotted, and then probed with purified glutathione S-transferase (GST)–Step, followed by GST antibodies. A very strong signal for GST-Step was detected where major proteolytic fragments of the MBP-Sstn protein were positioned on the blot (Figure 2B). Slightly higher levels of GST showed no binding to MBP-Sstn, and slightly higher levels of MBP showed no binding to GST-Step (Figure 2B). Thus Sstn and Step can interact directly in vitro, and the interacting MBP-Sstn fragment sizes suggested that the interaction site was in the N-terminal half of Sstn.

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.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Systems Biology, University of Toronto, Toronto, ON M5S 3G5, Canada.

Show MeSH