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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 coiled-coil domain and conserved region have distinct effects on subcellular localization. (A) Images of GFP-Sstn constructs acquired and adjusted with the same settings. Dlg staining shows cellularization furrows of similar depths. GFP-Sstn and Sstn∆CC are enriched at the base of furrows (arrows). All three constructs localize around the centrosomes (two per cell). (B) Quantifications of construct signals at the base of furrows. Each point is an average of five background corrected measurements from one confocal section of one cellularizing embryo with 2- to 5-μm-deep furrows. Results are shown from one set of crosses, and the overall results were reproduced with an independent set of crosses. (C) Quantifications of construct signal along lines from the centrosome to the furrow. Each curve represents the mean ± SD of three line scans with a similar spacing of centrosome and furrow in a single section from one embryo. To illustrate differences in furrow and cytosolic levels, three embryos with similar centrosome intensity are shown for each construct. The overall relationships were reproduced with an independent set of crosses.
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Figure 3: The Sstn coiled-coil domain and conserved region have distinct effects on subcellular localization. (A) Images of GFP-Sstn constructs acquired and adjusted with the same settings. Dlg staining shows cellularization furrows of similar depths. GFP-Sstn and Sstn∆CC are enriched at the base of furrows (arrows). All three constructs localize around the centrosomes (two per cell). (B) Quantifications of construct signals at the base of furrows. Each point is an average of five background corrected measurements from one confocal section of one cellularizing embryo with 2- to 5-μm-deep furrows. Results are shown from one set of crosses, and the overall results were reproduced with an independent set of crosses. (C) Quantifications of construct signal along lines from the centrosome to the furrow. Each curve represents the mean ± SD of three line scans with a similar spacing of centrosome and furrow in a single section from one embryo. To illustrate differences in furrow and cytosolic levels, three embryos with similar centrosome intensity are shown for each construct. The overall relationships were reproduced with an independent set of crosses.

Mentions: If Sstn acts as a cytohesin adaptor, then one region of the protein would be expected to bind Step, and a separate region would be expected mediate other interactions. To test this hypothesis, we generated GFP-tagged versions of Sstn, expressed them from the same genomic site with the Gal-4-UAS system, and localized them in the early embryo during peripheral nuclear divisions and early cellularization. Full-length Sstn accumulated at two main sites within the cell compartments: to pericentrosomal regions and along the basal end of pseudocleavage and early cellularization furrows (Figure 3A; arrow at basal furrow tip). We attempted to generate antibodies against both the CC domain and the CR of Sstn, but neither detected Sstn specifically in early embryos.


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 coiled-coil domain and conserved region have distinct effects on subcellular localization. (A) Images of GFP-Sstn constructs acquired and adjusted with the same settings. Dlg staining shows cellularization furrows of similar depths. GFP-Sstn and Sstn∆CC are enriched at the base of furrows (arrows). All three constructs localize around the centrosomes (two per cell). (B) Quantifications of construct signals at the base of furrows. Each point is an average of five background corrected measurements from one confocal section of one cellularizing embryo with 2- to 5-μm-deep furrows. Results are shown from one set of crosses, and the overall results were reproduced with an independent set of crosses. (C) Quantifications of construct signal along lines from the centrosome to the furrow. Each curve represents the mean ± SD of three line scans with a similar spacing of centrosome and furrow in a single section from one embryo. To illustrate differences in furrow and cytosolic levels, three embryos with similar centrosome intensity are shown for each construct. The overall relationships were reproduced with an independent set of crosses.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 3: The Sstn coiled-coil domain and conserved region have distinct effects on subcellular localization. (A) Images of GFP-Sstn constructs acquired and adjusted with the same settings. Dlg staining shows cellularization furrows of similar depths. GFP-Sstn and Sstn∆CC are enriched at the base of furrows (arrows). All three constructs localize around the centrosomes (two per cell). (B) Quantifications of construct signals at the base of furrows. Each point is an average of five background corrected measurements from one confocal section of one cellularizing embryo with 2- to 5-μm-deep furrows. Results are shown from one set of crosses, and the overall results were reproduced with an independent set of crosses. (C) Quantifications of construct signal along lines from the centrosome to the furrow. Each curve represents the mean ± SD of three line scans with a similar spacing of centrosome and furrow in a single section from one embryo. To illustrate differences in furrow and cytosolic levels, three embryos with similar centrosome intensity are shown for each construct. The overall relationships were reproduced with an independent set of crosses.
Mentions: If Sstn acts as a cytohesin adaptor, then one region of the protein would be expected to bind Step, and a separate region would be expected mediate other interactions. To test this hypothesis, we generated GFP-tagged versions of Sstn, expressed them from the same genomic site with the Gal-4-UAS system, and localized them in the early embryo during peripheral nuclear divisions and early cellularization. Full-length Sstn accumulated at two main sites within the cell compartments: to pericentrosomal regions and along the basal end of pseudocleavage and early cellularization furrows (Figure 3A; arrow at basal furrow tip). We attempted to generate antibodies against both the CC domain and the CR of Sstn, but neither detected Sstn specifically in early embryos.

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
Related in: MedlinePlus