Fig. 2. EAST—GFP fusion proteins during mitosis of live embryos. Two versions of EAST, EASTFL(1-2336)—GFP (A,C) and EASTC(1-1573)—GFP (B,D), were ectopically expressed using the GAL4 system and studied in vivo using confocal microscopy (also see Movie 1, http://jcs.biologists.org/supplemental). (A,B) In larval salivary glands, the two EAST—GFP species (green) preferentially localize to extrachromosomal and extranucleolar regions of the nucleus. Chromosomes were labeled with the in vivo nucleic acid marker Syto-17 (red), which also labels ribosomal RNA in the nucleolus (*) and cytoplasmic RNA. (C,D) In dividing embryonic cells during germband extension, images of EAST—GFP fusion proteins were acquired at 15 second intervals. (E) The live recording of a third embryo expressing histone-H2A—GFP is displayed for comparison. The onset of prometaphase, when GFP-tagged proteins begin to leak into the cytoplasm, presumably due to nuclear envelope breakdown, was chosen as timepoint zero (+00:00). Both full-length and truncated versions of EAST show nuclear localization in interphase (-01:00). However, they differ in their behavior during mitosis. EASTFL (C) remains enriched in the central part of the cell until anaphase (+04:45), whereas its truncated counterpart EASTC (D) rapidly disperses at prometaphase (+01:00) and shows diffuse distribution until telophase (+06:15). After cytokinesis (arrows indicate cleavage furrow), both forms of EAST—GFP are recruited back to the daughter nuclei (+07:45). Bars, 10 µm; bar in B also applies to A; bar in E also applies to C and D.

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