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. 2016 Oct 15;27(20):3143–3155. doi: 10.1091/mbc.E16-05-0272

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© 2016 Rodrigues et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

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FIGURE 3: — Asap localizes to multiple subcellular sites and relocates during the cell cycle. (A) A GFP-Asap–expressing embryo imaged live over a syncytial cell cycle. A side view and a projection of the full peripheral nuclear layer are shown as a mitotic wave spreads across the embryo. At interphase (top left), GFP-Asap is enriched at punctate structures resembling apical microvilli (blue arrows) and at early PM furrows (white arrows) and has a cytoplasmic pool detectable by exclusion from nuclei (yellow asterisks). At the onset of mitosis (bottom right), GFP-Asap is depleted from the PM and becomes enriched at nuclear regions surrounding condensed chromosomes (yellow arrows). After mitosis, GFP-Asap returns to the PM (not shown). (B) Single cross-section at the apical PM surface at interphase. GFP-Asap puncta closely colocalize with F-actin–rich microvilli (arrows). (C) A side view and single section at the nuclear level at interphase shows GFP-Asap most prominently at the PM, more weakly in the cytosol, with slight enrichment around the nuclear periphery, and exclusion from the nucleus. Staining with the Golgi marker AP-1 reveals no enrichment of GFP-Asap at Golgi puncta. (D) GFP-Asap becomes enriched within the nuclear region at prometaphase. RFP-KDEL marks the periphery of the nuclear region. Inset, separation of GFP-Asap from condensed chromosomes (marked with histone RFP). (A–D) Constructs expressed as in Figure 1. (E) Ectoderm of a postsyncytial, stage 8–9 histone-GFP embryo stained for endogenous Asap and the Golgi marker AP-1. The oblique section from the embryo surface (right) inward (left) reveals endogenous Asap strongly at the PM of each cell and weakly in the cytoplasm, with no enrichment at AP-1–positive Golgi elements. (F) Mitotic domain within the ectoderm of a stage 8–9 histone-mRFP embryo stained for endogenous Asap and the PM marker Dlg. At metaphase, endogenous Asap localizes to the nuclear region (yellow arrows) surrounding condensed mitotic chromosomes (histone-mRFP). Endogenous Asap is excluded from the nucleus in interphase cells (yellow asterisks). Bars, 5 μm.