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First published online 11 March 2003
doi: 10.1242/jcs.00379

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The Drosophila EAST protein associates with a nuclear remnant during mitosis and constrains chromosome mobility

¹ Institute of Molecular and Cell Biology, National University of Singapore, 30 Medical Drive, Singapore 117609
² MRC Centre for Developmental Neurobiology, King's College London, 4th Floor, New Hunts House, Guy's Hospital Campus, London SE1 1UL, UK

View larger version (59K): [in a new window]   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.

View larger version (94K): [in a new window]   Fig. 5. Loss of east leads to abnormal movements of chromosomes during prometaphase. Time-lapse recordings of cell divisions in mitotic domains of live embryos expressing histone—GFP reveal irregular patterns of congression to the metaphase plate (see Movies 2 and 3, http://jcs.biologists.org/supplemental). The beginning of prometaphase is chosen as timepoint zero. (A) In control embryos, on nuclear envelope breakdown, condensed chromosomes remain clustered in the center of the cell and rapidly orient themselves perpendicular to the future axis of division (+02:00). (B) In east(mat)hop-1, chromosomes were seen to break up into distinct clumps of DNA or (C) to stray to the periphery of the cell. Compare A and C at timepoint +02:00 minutes: chromosomes in the wt cell are aligned centrally perpendicular to the future axis of division, whereas the chromosomes in east mutant cell are positioned near the cell cortex parallel to the future axis of division. Compare the three live recordings at timepoint +04:30 minutes: while chromosomes in the control cell are undergoing anaphase (A), chromosomes in the east cells are still split up into distinct groups (B) or located near the cortex in an abnormal orientation (C). However, with a time delay, chromosomes in mutant cells eventually manage to arrive in the center and complete mitosis. Stippled lines outline the boundaries of the cells.

View larger version (89K): [in a new window]   Fig. 1. Localization of EAST protein in dividing cells. The distribution of EAST in cells of mitotic domains was revealed by triple labeling post-syncytial embryos with anti-EAST antibody (green), anti-tubulin antibody (white) and DNA dye (red). Throughout interphase (A,H) and prophase (B), EAST shows nuclear localization. At prometaphase (C), metaphase (D) and early anaphase (E), EAST remains in the center of the cell associated with the remnant of the interphase nucleus. EAST labeling decays at late anaphase (F) and is barely detectable at telophase (G). Intense staining is again seen in early interphase (H).

View larger version (25K): [in a new window]   Fig. 3. Mutations in east cause nondisjunction (ND) of achiasmate X-chromosomes in female meiosis. Females carrying different X-chromosomes over the FM7c balancer were crossed with yw males. The bar chart compares the rates of ND of X-chromosomes from wt (CS), the viable east insertion allele ETX3, the viable revertant P86 and six lethal reversion alleles (Wasser and Chia, 2000). To determine frequencies of ND, the numbers of nullo-X (yw males) and diplo-X (w+/FM7c) exceptional offspring were scored. The original insertion line ETX3 shows normal segregation, whereas some of the lethal alleles show ND rates of up to 20%. Note that the partial deletion easthop-7 is associated with more than 20x higher frequency of ND than the complete deletion easthop-1 of the east transcription unit. n, total number of flies scored.

View larger version (102K): [in a new window]   Fig. 4. Loss of maternal east results in pleiotropic mitotic and developmental defects. Loss of maternal east expression in easthop-1 germline clones coupled with zygotic expression from one wt copy of east can lead to a variety of phenotypes, ranging from fertile female escapers without discernible morphological abnormalities (A), to adults showing a loss of various morphological structures (B), like tergites on the abdomen (arrowheads) or appendages (arrow). Lethality, along with variable developmental defects, was also observed during embryonic development. In late control embryos (C), the in vivo GFP marker of the balancer FM7i-pAct—GFP labels the midgut. (D) Loss of maternal east can cause the GFP expression to appear in the anterior regions of the embryo. (E,F) Nuclei of the syncytial blastoderm stage in wt (E) and east(mat) (F) embryos stained with anti-histone antibody. Loss of maternal east can lead to the elimination of nuclei from the surface (F). (G,H) Syncytial blastoderm embryos stained with anti-tubulin (green) and anti-histone (red) antibodies. (G) Polyploid nuclei at metaphase and (H) orphan centrosomes in anaphase (arrowheads) without daughter nuclei (arrows) indicate mitotic errors. Bars, 100 µm (C-F) and 10 µm (G-J).

View larger version (17K): [in a new window]   Fig. 6. Duration (minutes) of prometaphase and metaphase in mitotic domains of easthop-1 (n=68 divisions) and control (n=43 divisions) germline clones. Note that the maxima in distribution of controls and mutants are close together. However, although control cells show a symmetric distribution, mutant cells display a more skewed pattern, reflecting a greater variability in the duration of chromosome congression.

View larger version (45K): [in a new window]   Fig. 7. Abnormal behaviour of chromosomes in east spermatocytes. Wild-type and easthop-7 testes were labeled with antibodies to PH3 (green) and -tubulin (cyan). DNA was detected with TO-PRO-3 (red). (A,B) Primary spermatocytes in early prophase. The stage is inferred from the nucleation of astral microtubules (left panels). The central panels show the nuclei of the same cells in focal planes 3 µm below. Chromosomal DNA in wt is highly condensed and organized into three distinct clusters. DNA in the east spermatocytes is less condensed and does not show discernible clustering. Chromosomal DNA does not show any H3 phosphorylation (right panels, same focal planes as middle panels). The green label is due to a cross-reaction of the antimouse secondary with the rat primary antitubulin antibody. In C-E: top panels, PH3 labeling (green); middle panels, PH3+DNA labelings; bottom panels, PH3+DNA+-tubulin labelings. (C) As wt cells progress towards late prophase (left cell) and prometaphase (right cell), PH3 labeling can be detected, apparently coinciding with nuclear envelope breakdown (right cell). The organization of chromosomes into three clumps of DNA is maintained. (D) In east prometaphase, the number of DNA clumps can exceed four; here, two major and four minor clusters can be observed. (E) Two neighbouring spermatocytes of the same cyst in an east testis display different intensities of PH3 labeling (weak in the left, strong in right cell), suggesting that these cells are at the beginning of prometaphase. The chromosomes appear scattered and their arms become exposed and visible. By contrast, chromosome arms are not discernible in wt primary spermatocytes (C). (F,G) Primary spermatocytes during metaphase of wt (F) and east (G) testes. Left and right panels show focal planes of the same cells 2 µm apart. (F) In wt, chromosomes congregate to a single cluster in the middle of the spindle. (G) In east, some of the chromosomes (right panel) disperse away from the main DNA cluster (left panel). (H,J) Testes of wt (H) and east (J) pupae containing PH3-positive cells (arrows) that are shown in higher magnifications in (I) for wt and (K) for east. In (I,K): top left panels, PH3; top right, PH3+DNA; bottom left, tubulin; bottom right, PH3+DNA+tubulin. (J,K) Testes in east pupae contain small cells with PH3-positive nuclei (arrow), presumably spermatogonia. (I,H) In wt, small PH3-positive cells located near the tip of the testis are stem cells and spermatogonia that undergo mitosis. Note that the abnormal PH3-positive cells in east are not associated with mitotic spindles as in wt (I,K; compare bottom panels). Bar in (A) represents 10 µm and applies to all panels except (H,J). Bar in (H) equals 50 µm and also applies to (J).

View larger version (27K): [in a new window]   Fig. 8. Model proposing how EAST, as part of an internal nucleoskeleton, could facilitate the congression of chromosomes at prometaphase. Even after nuclear envelope breakdown, chromosomes remain embedded in a nucleoskeleton that constrains their mobility and helps them to remain clustered in the center of the cell. Loss of east might disrupt this structure, causing condensed chromosomes to stray away from the center of the cell or from one another.