Fig. 2. EAST—GFP fusion proteins during mitosis of live embryos. Two versions
of EAST, EASTFL(1-2336)—GFP (A,C) and EAST
C(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.