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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 Step coiled-coil domain is needed for Sstn association, Step localization and membrane growth restraint. (A) Furrow corecruitment of mCh-Sstn with GFP-Step but not GFP-Step∆CC. In each case, images are shown after acquisition and adjustment with the same settings. Furrow levels were quantified as in Figure 3. Line scans were performed as in Figure 3. The overall relationships were reproduced with an independent set of crosses. (B) GFP-Step displays enrichment at the base of furrows, but GFP-Step∆CC does not (arrows). Dlg shows cellularization furrows of similar depth. (C) Expression of an RNAi-resistant GFP-Step construct with step shRNA restrains perpendicular membrane expansion at the base of furrows (marked with Amph) more effectively than expression of RNAi-resistant GFP-Step∆CC. Quantifications of furrow base areas in the xy-plane are shown for embryos with 2- to 7-μm-deep furrows and were performed as in Figure 5. Each point in the left graph is one embryo measurement. The right graph compiles all of these points into a histogram.
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Figure 8: The Step coiled-coil domain is needed for Sstn association, Step localization and membrane growth restraint. (A) Furrow corecruitment of mCh-Sstn with GFP-Step but not GFP-Step∆CC. In each case, images are shown after acquisition and adjustment with the same settings. Furrow levels were quantified as in Figure 3. Line scans were performed as in Figure 3. The overall relationships were reproduced with an independent set of crosses. (B) GFP-Step displays enrichment at the base of furrows, but GFP-Step∆CC does not (arrows). Dlg shows cellularization furrows of similar depth. (C) Expression of an RNAi-resistant GFP-Step construct with step shRNA restrains perpendicular membrane expansion at the base of furrows (marked with Amph) more effectively than expression of RNAi-resistant GFP-Step∆CC. Quantifications of furrow base areas in the xy-plane are shown for embryos with 2- to 7-μm-deep furrows and were performed as in Figure 5. Each point in the left graph is one embryo measurement. The right graph compiles all of these points into a histogram.

Mentions: Because we found that the Sstn–Step interaction required the CC domains of each protein, we performed a final set of in vivo experiments interrogating the CC domain of Step. Specifically, we compared GFP-Step with a derivative in which the CC domain was deleted, expressing them from the same genomic site with the Gal-4-UAS system. First, we assessed in vivo associations with a mCh-tagged Sstn construct. Coexpression of GFP-Step with mCh-Sstn led to increased furrow abundance of both proteins versus single-expression controls (Figure 8A), as observed for proteins with the reciprocal tags (Figure 4A). In contrast, coexpression of GFP-Step∆CC with mCh-Sstn did not enhance the furrow level of either protein (Figure 8A).


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 Step coiled-coil domain is needed for Sstn association, Step localization and membrane growth restraint. (A) Furrow corecruitment of mCh-Sstn with GFP-Step but not GFP-Step∆CC. In each case, images are shown after acquisition and adjustment with the same settings. Furrow levels were quantified as in Figure 3. Line scans were performed as in Figure 3. The overall relationships were reproduced with an independent set of crosses. (B) GFP-Step displays enrichment at the base of furrows, but GFP-Step∆CC does not (arrows). Dlg shows cellularization furrows of similar depth. (C) Expression of an RNAi-resistant GFP-Step construct with step shRNA restrains perpendicular membrane expansion at the base of furrows (marked with Amph) more effectively than expression of RNAi-resistant GFP-Step∆CC. Quantifications of furrow base areas in the xy-plane are shown for embryos with 2- to 7-μm-deep furrows and were performed as in Figure 5. Each point in the left graph is one embryo measurement. The right graph compiles all of these points into a histogram.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 8: The Step coiled-coil domain is needed for Sstn association, Step localization and membrane growth restraint. (A) Furrow corecruitment of mCh-Sstn with GFP-Step but not GFP-Step∆CC. In each case, images are shown after acquisition and adjustment with the same settings. Furrow levels were quantified as in Figure 3. Line scans were performed as in Figure 3. The overall relationships were reproduced with an independent set of crosses. (B) GFP-Step displays enrichment at the base of furrows, but GFP-Step∆CC does not (arrows). Dlg shows cellularization furrows of similar depth. (C) Expression of an RNAi-resistant GFP-Step construct with step shRNA restrains perpendicular membrane expansion at the base of furrows (marked with Amph) more effectively than expression of RNAi-resistant GFP-Step∆CC. Quantifications of furrow base areas in the xy-plane are shown for embryos with 2- to 7-μm-deep furrows and were performed as in Figure 5. Each point in the left graph is one embryo measurement. The right graph compiles all of these points into a histogram.
Mentions: Because we found that the Sstn–Step interaction required the CC domains of each protein, we performed a final set of in vivo experiments interrogating the CC domain of Step. Specifically, we compared GFP-Step with a derivative in which the CC domain was deleted, expressing them from the same genomic site with the Gal-4-UAS system. First, we assessed in vivo associations with a mCh-tagged Sstn construct. Coexpression of GFP-Step with mCh-Sstn led to increased furrow abundance of both proteins versus single-expression controls (Figure 8A), as observed for proteins with the reciprocal tags (Figure 4A). In contrast, coexpression of GFP-Step∆CC with mCh-Sstn did not enhance the furrow level of either protein (Figure 8A).

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