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Cell cycle roles for two 14-3-3 proteins during Drosophila development

¹ MCD Biology, University of Colorado, Boulder, CO 80309, USA
² Department of Biochemistry and Biophysics, University of California, San Francisco, CA 94143, USA
³ Institute of Molecular Biology, Academia Sinica, Nankang, Taipei, Taiwan

View larger version (112K): [in a new window]   Fig. 1. Localization of 14-3-3 proteins during cell cycle progression. Wild-type embryos were fixed and stained for DNA and with antibodies to 14-3-3 proteins. Staining with an affinity-purified antibody that is specific to 14-3-3 shows that 14-3-3 is dispersed throughout the embryo in interphase of syncytial cycles (left embryo in A and E). The antigen concentration increased in the nucleus in prophase (embryo on the right in A and E) and persists near chromosomes in metaphase (B,F). The staining intensity decreases in anaphase and telophase, with the remaining signal stretching across the dividing nuclei (D,H). A similar cell cycle profile of staining was seen with a commercial pan-specific antibody to 14-3-3 proteins; only the perichromosomal localization in metaphase is shown here (C,G). During cellular cycles (I-N), 14-3-3-specific antibody detected a similar pattern of localization as in syncytial cycles, but exclusion from the nucleus is more apparent. (I,J) The antigen is cytoplasmic in all cells except those in the midst of M14. Mitotic domains 1,2,5 and 8 are indicated. A single cell in mitotic domain 9 is initiating mitosis and is also indicated (other domains reside in the rest of the embryo). (K-N) The change in localization during M14. The antigen concentrates in the nucleus in prophase (arrowheads in K-M), at which time the nuclear envelope remains (arrowheads show remnant wheat-germ agglutinin (WGA) staining in N). The antigen persists in metaphase (arrow in K-M) when the nuclear envelope is absent (arrow in N). In anaphase and telophase, the antigen disperses (brackets in K-M), with the remaining signal concentrating in the region between the nuclei pair in anaphase (smaller bracket in K-M). A similar profile of localization was also revealed by the commercial pan-specific 14-3-3 antibody (not shown). Bar, 5.5 µm in A-H, 16.5 µm in I,J and 13 µm in K-N.

View larger version (140K): [in a new window]   Fig. 2. Localization of 14-3-3 is dependent on Cdk1 activity. Embryos were fixed and stained for DNA and 14-3-3. (A) Mitotic domains 1, 2 and 3 in the head region of a wild-type embryo is seen initiating M14. Nuclear localization of 14-3-3 is apparent in these cells. (B) In a stg homozygous mutant at a similar embryonic stage, nuclear staining is absent. (C) Expression of Cdk1AF in a stg mutant promotes mitosis (DNA on the left) and concentration of 14-3-3 near chromosomes in metaphase (on the right). (D) In a metaphase arrest induced by non-degradable cyclin A (As) and non-degradable cyclin B (Bs), 14-3-3 (green) remains concentrated near chromosomes (purple). (E) In an irradiated embryo in which cells are delayed from entering M14, 14-3-3 remains cytoplasmic. Bar, 16.5 µm.

View larger version (88K): [in a new window]   Fig. 3. 14-3-3 mutant embryos enter mitosis prematurely. Wild-type (‘sev’; A,C,E,F) and 14-3-3 mutant (‘14-3-3’; B,D,G,H) embryos in embryonic cycle 14 were fixed and stained to visualize DNA (purple) and with an antibody to phosphorylated histone H3, to visualize mitotic cells (H and green in all others). Embryos in F and G were irradiated for 20 minutes before fixing (+rad). Head regions of embryos in A-D are magnified and shown in A'-D' respectively. The * marks pole cells (enclosed by curved lines) in A-D,G. The numbers refer to mitotic domains (Foe, 1987). Embryos are shown with anterior end to the left. Embryos in A-C have dorsal sides up. Embryos in D-G have dorsal sides rotated towards the viewer. (A-B') Embryos of a developmental stage in which pole cells are still exposed and still close to the posterior end (solid line). At this stage, few cells are in mitosis in wild-type embryos (A,A'), whereas cells of domains 1-5 are in mitosis in 14-3-3 mutants (B; domains 1, 2 and 5 are visible in the view shown in B'). Green stain in the cytoplasm in A' is due to background signal in this sample. (C,D) More advanced embryos in which pole cells, still exposed, have moved away from the posterior end (solid line) owing to germ-band extension. At this stage, cells of domain 1-5 are entering mitosis in wild-type embryos (C,C'). Domains 1 and 5 are visible in C'; only a single mitotic cell in domain 2 (arrow) is seen in this view, which is rotated towards the viewer with respect to the one in B'. In 14-3-3 mutant embryos at similar stages, domains 1-11 are in mitosis (D,D'); many cells of earlier domains have now finished mitosis (e.g. domain 1 in D'). For visual clarity, not all domains visible in these views are numbered. The division pattern in wild-type embryos at this stage (C) is similar to that of 14-3-3 mutant embryos at an earlier stage (B), indicating the advancement of mitotic program in the latter. (E) In yet more advanced embryos, pole cells are internalized and no longer visible. Domains 1-11 are now in mitosis in the wild-type embryo shown here. The division pattern of this embryo is similar to that of 14-3-3 embryos at an earlier stage (D), indicating the advancement of mitotic program in the latter. In wild-type embryos of similar stage that had been irradiated, mitotic cells are absent (F), indicating that DNA damage delayed the entry into mitosis. (G) An irradiated 14-3-3 embryo at similar developmental stage (notice exposed the pole cells) as in D, but with similar division pattern to D and E. Mitotic domains seen in the absence of irradiation (D,E) are also present after irradiation in the 14-3-3 mutant (G), indicating the failure to delay the entry into mitosis in response to DNA damage. (H) In irradiated mutant embryos, cells that enter mitosis execute all stages of mitosis and often show broken or lagging chromosomes (arrowhead), presumably a result of entry into mitosis with damaged DNA. Abbreviations: a/t, anaphase/telophase; m, metaphase; p, prophase. Bar, 30 µm in A-G, 15 µm in A', B' and C', and 4 µm in H. (I) Germ-band extension in live embryos. The anterior lip of the aminoprotodeal fold (e.g. arrow in C) is defined as the extent of the germ band. Its position is measured from the posterior end of the embryo on photographic images of embryos and expressed as a percentage of the total embryo length. Data from three wild-type and 14-3-3 mutants (mutant) embryos are shown. The rate of germ band elongation was 1.38±0.4% embryo length per minute in wild-type and 1.38±0.04% in mutant embryos.

View larger version (94K): [in a new window]   Fig. 4. 14-3-3-deficient embryos fail to execute syncytial divisions properly. Wild-type (A,D) and 14-3-3-deficient (B,C,E-G) embryos were fixed and stained for DNA (red) and an anti--tubulin antibody (C-F) to visualize microtubules (blue). (A) A wild-type embryo at the end of fourth embryonic mitosis. Nuclei are clearly separated. Embryonic cycle number is determined by counting nuclei (n = 2 at the end of M1, n = 4 at the end of M2, etc.). (B) A 14-3-3-deficient embryo at the end of M4 shows nuclei pairs connected by chromosome bridges. One such pair is indicated with a bracket and is shown rotated and magnified in E (arrowhead indicates bridge). (C) A 14-3-3-deficient embryo in a later syncytial cycle. Arrows point to MTOCs that have no obvious nuclei associated with them. Brackets indicate nuclei pairs connected by bridges. These are magnified and shown in F and G. Arrowhead indicates bridges. Notice the presence of robust microtubules around the chromosome bridge in F, the region in which the spindle mid-body normally resides at a similar stage of mitosis in wild-type embryos (D). The red stain in the middle of the embryo in C indicates a large yolk DNA mass. Bar, 15.7 µm in A and B, 8.3 µm in C and 5.5 µm in D-G.

View larger version (16K): [in a new window]   Fig. 5. A model for roles of 14-3-3 proteins in Drosophila cell division. In interphase, 14-3-3 proteins and cyclin A/Cdk1 and cyclin B/Cdk1 complexes are predominantly cytoplasmic. Here, 14-3-3 acts to keep Cdk1 in check, preventing mitosis in normal or irradiated embryos. As Cdk1 becomes active (owing to the accumulation of its activator Stg or after recovery from DNA damage) and cells enter mitosis, accumulating cyclin/Cdk1 activity promotes and maintains 14-3-3 protein localization near chromosomes. Upon the transition to anaphase, the localized 14-3-3 proteins, and 14-3-3 in particular, can contribute to the rapid, effective inactivation of the cyclin/Cdk1. Thus, in interphase, 14-3-3 can act to keep Cdk1 inactive in the cytoplasm but, once Cdk1 is active, it can act in turn to localize 14-3-3 proteins in preparation for their action during the exit from mitosis.