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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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With Sstn loss membranes expand perpendicularly from the base of furrows. (A) Normal early-cellularization furrow organization with a control shRNA construct and perpendicular expansions of furrow bases with sstn shRNA. Disks large (Dlg) is enriched at lateral membranes, and amphiphysin (Amph) is enriched at the furrow base (the furrow canal). Note that the upper furrows stained with Dlg are minimally affected and that the membrane expansion occurs from the furrow base (seen with Amph). Embryos with moderate and strong membrane expansion are shown. Images were deconvolved. (B) Quantifications of furrow base areas (in xy-sections) for control; sstn shRNA and sstn mutant cellularization embryos with 2- to 5.5-μm-deep furrows. In xy images of furrow bases, the percentage of total area occupied by Amph signal was calculated (Lee and Harris, 2013). The control data in each graph are a compilation of data from mCh shRNA embryos and Histone-GFP embryos collected across the experiments. Each point is from one embryo (total embryo numbers bracketed).
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Figure 5: With Sstn loss membranes expand perpendicularly from the base of furrows. (A) Normal early-cellularization furrow organization with a control shRNA construct and perpendicular expansions of furrow bases with sstn shRNA. Disks large (Dlg) is enriched at lateral membranes, and amphiphysin (Amph) is enriched at the furrow base (the furrow canal). Note that the upper furrows stained with Dlg are minimally affected and that the membrane expansion occurs from the furrow base (seen with Amph). Embryos with moderate and strong membrane expansion are shown. Images were deconvolved. (B) Quantifications of furrow base areas (in xy-sections) for control; sstn shRNA and sstn mutant cellularization embryos with 2- to 5.5-μm-deep furrows. In xy images of furrow bases, the percentage of total area occupied by Amph signal was calculated (Lee and Harris, 2013). The control data in each graph are a compilation of data from mCh shRNA embryos and Histone-GFP embryos collected across the experiments. Each point is from one embryo (total embryo numbers bracketed).

Mentions: If Sstn contributes positively to Step activity, then removal of Sstn should result in the membrane cytoskeleton expansion defect that occurs with Step loss (Lee and Harris, 2013). To test the function of Sstn in vivo, we generated three sstn short hairpin RNA (shRNA) constructs unique to distinct sequences in sstn and acquired available sstn P-element insertions and deletions (Supplemental Figure S1). Maternal expression of the sstn shRNA constructs effectively depleted GFP-Sstn (Supplemental Figure S2) and led to the same specific change to the plasma membrane furrows of the early embryo. Both pseudocleavage and cellularization furrows extended effectively into the embryo, but, in contrast to controls, they then underwent abnormal perpendicular expansion at their basal tips (in embryo surface views, the nuclear spaces gained the appearance of stepping stones more widely separated across a pond; Figure 5A, quantified in Figure 5B; pseudocleavage furrows shown in Supplemental Figure S3). The expanded membranes were coated with a cytoskeleton marker (the septin Peanut) and were observed to encroach into space normally occupied by nuclei, resulting in nuclear indentations and bottle-like shapes (Supplemental Figure S4). The pseudocleavage and cellularization furrow defects also occurred in progeny of mothers transheterozygous for a sstn promoter P-element insertion and a large deletion removing sstn (quantified in Figure 5B; pseudocleavage furrows shown in Supplemental Figure S3) and for the sstn promoter P-element insertion and an sstn exon P-element insertion (unpublished data). These defects were very similar to, although somewhat milder than, those observed with Step loss (Lee and Harris, 2013). In addition, we observed preadult lethality of zygotic mutants transheterozygous for the large deletion and the sstn exon P-element insertion, indicating that Sstn functions in later development as well.


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)

With Sstn loss membranes expand perpendicularly from the base of furrows. (A) Normal early-cellularization furrow organization with a control shRNA construct and perpendicular expansions of furrow bases with sstn shRNA. Disks large (Dlg) is enriched at lateral membranes, and amphiphysin (Amph) is enriched at the furrow base (the furrow canal). Note that the upper furrows stained with Dlg are minimally affected and that the membrane expansion occurs from the furrow base (seen with Amph). Embryos with moderate and strong membrane expansion are shown. Images were deconvolved. (B) Quantifications of furrow base areas (in xy-sections) for control; sstn shRNA and sstn mutant cellularization embryos with 2- to 5.5-μm-deep furrows. In xy images of furrow bases, the percentage of total area occupied by Amph signal was calculated (Lee and Harris, 2013). The control data in each graph are a compilation of data from mCh shRNA embryos and Histone-GFP embryos collected across the experiments. Each point is from one embryo (total embryo numbers bracketed).
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Related In: Results  -  Collection

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Figure 5: With Sstn loss membranes expand perpendicularly from the base of furrows. (A) Normal early-cellularization furrow organization with a control shRNA construct and perpendicular expansions of furrow bases with sstn shRNA. Disks large (Dlg) is enriched at lateral membranes, and amphiphysin (Amph) is enriched at the furrow base (the furrow canal). Note that the upper furrows stained with Dlg are minimally affected and that the membrane expansion occurs from the furrow base (seen with Amph). Embryos with moderate and strong membrane expansion are shown. Images were deconvolved. (B) Quantifications of furrow base areas (in xy-sections) for control; sstn shRNA and sstn mutant cellularization embryos with 2- to 5.5-μm-deep furrows. In xy images of furrow bases, the percentage of total area occupied by Amph signal was calculated (Lee and Harris, 2013). The control data in each graph are a compilation of data from mCh shRNA embryos and Histone-GFP embryos collected across the experiments. Each point is from one embryo (total embryo numbers bracketed).
Mentions: If Sstn contributes positively to Step activity, then removal of Sstn should result in the membrane cytoskeleton expansion defect that occurs with Step loss (Lee and Harris, 2013). To test the function of Sstn in vivo, we generated three sstn short hairpin RNA (shRNA) constructs unique to distinct sequences in sstn and acquired available sstn P-element insertions and deletions (Supplemental Figure S1). Maternal expression of the sstn shRNA constructs effectively depleted GFP-Sstn (Supplemental Figure S2) and led to the same specific change to the plasma membrane furrows of the early embryo. Both pseudocleavage and cellularization furrows extended effectively into the embryo, but, in contrast to controls, they then underwent abnormal perpendicular expansion at their basal tips (in embryo surface views, the nuclear spaces gained the appearance of stepping stones more widely separated across a pond; Figure 5A, quantified in Figure 5B; pseudocleavage furrows shown in Supplemental Figure S3). The expanded membranes were coated with a cytoskeleton marker (the septin Peanut) and were observed to encroach into space normally occupied by nuclei, resulting in nuclear indentations and bottle-like shapes (Supplemental Figure S4). The pseudocleavage and cellularization furrow defects also occurred in progeny of mothers transheterozygous for a sstn promoter P-element insertion and a large deletion removing sstn (quantified in Figure 5B; pseudocleavage furrows shown in Supplemental Figure S3) and for the sstn promoter P-element insertion and an sstn exon P-element insertion (unpublished data). These defects were very similar to, although somewhat milder than, those observed with Step loss (Lee and Harris, 2013). In addition, we observed preadult lethality of zygotic mutants transheterozygous for the large deletion and the sstn exon P-element insertion, indicating that Sstn functions in later development as well.

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