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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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The Sstn and Step coiled-coil domains are needed for their interaction in vivo. (A–C) Furrow corecruitment of mCh-Step with GFP-Sstn (A) and GFP-Sstn∆CR (C) but not GFP-Sstn∆CC (B). In each case, images are shown after acquisition and adjustment with the same settings. Furrow levels were quantified as in Figure 3B, with GFP-Sstn construct levels on the y-axis and mCh-Step levels on the x-axis (the extension of the signal distributions with coexpression [blue points] beyond single-expression controls [green or red points] indicates corecruitment [each point is one embryo quantification]). Line scans were performed as in Figure 3C (each line is one embryo quantification). The overall relationships were reproduced with an independent set of crosses. (D) Co-IPs of mCh-Step with GFP-Sstn and GFP-Sstn∆CR, but not GFP-Sstn∆CC, from 0.5- to 2.5-h embryo lysates. The results were reproduced with an independent set of crosses.
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Figure 4: The Sstn and Step coiled-coil domains are needed for their interaction in vivo. (A–C) Furrow corecruitment of mCh-Step with GFP-Sstn (A) and GFP-Sstn∆CR (C) but not GFP-Sstn∆CC (B). In each case, images are shown after acquisition and adjustment with the same settings. Furrow levels were quantified as in Figure 3B, with GFP-Sstn construct levels on the y-axis and mCh-Step levels on the x-axis (the extension of the signal distributions with coexpression [blue points] beyond single-expression controls [green or red points] indicates corecruitment [each point is one embryo quantification]). Line scans were performed as in Figure 3C (each line is one embryo quantification). The overall relationships were reproduced with an independent set of crosses. (D) Co-IPs of mCh-Step with GFP-Sstn and GFP-Sstn∆CR, but not GFP-Sstn∆CC, from 0.5- to 2.5-h embryo lysates. The results were reproduced with an independent set of crosses.

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.


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)

The Sstn and Step coiled-coil domains are needed for their interaction in vivo. (A–C) Furrow corecruitment of mCh-Step with GFP-Sstn (A) and GFP-Sstn∆CR (C) but not GFP-Sstn∆CC (B). In each case, images are shown after acquisition and adjustment with the same settings. Furrow levels were quantified as in Figure 3B, with GFP-Sstn construct levels on the y-axis and mCh-Step levels on the x-axis (the extension of the signal distributions with coexpression [blue points] beyond single-expression controls [green or red points] indicates corecruitment [each point is one embryo quantification]). Line scans were performed as in Figure 3C (each line is one embryo quantification). The overall relationships were reproduced with an independent set of crosses. (D) Co-IPs of mCh-Step with GFP-Sstn and GFP-Sstn∆CR, but not GFP-Sstn∆CC, from 0.5- to 2.5-h embryo lysates. The results were reproduced with an independent set of crosses.
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Related In: Results  -  Collection

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Figure 4: The Sstn and Step coiled-coil domains are needed for their interaction in vivo. (A–C) Furrow corecruitment of mCh-Step with GFP-Sstn (A) and GFP-Sstn∆CR (C) but not GFP-Sstn∆CC (B). In each case, images are shown after acquisition and adjustment with the same settings. Furrow levels were quantified as in Figure 3B, with GFP-Sstn construct levels on the y-axis and mCh-Step levels on the x-axis (the extension of the signal distributions with coexpression [blue points] beyond single-expression controls [green or red points] indicates corecruitment [each point is one embryo quantification]). Line scans were performed as in Figure 3C (each line is one embryo quantification). The overall relationships were reproduced with an independent set of crosses. (D) Co-IPs of mCh-Step with GFP-Sstn and GFP-Sstn∆CR, but not GFP-Sstn∆CC, from 0.5- to 2.5-h embryo lysates. The results were reproduced with an independent set of crosses.
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.

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