Fig. 3. Mutations in the KEN-box of PIM result in mitotic stabilization and inhibit sister chromatid separation only at high-expression levels. (A) An alignment of the N-terminal regions of PIM with fission (SP CUT2) and budding (SC PDS1) yeast securins, as well as human securin (HS PTTG1), reveals the presence of KEN-boxes in addition to D-boxes. KEN- and D-boxes are underligned. (B-I) PIM-myc (B-E) or PIM^kena-myc with a mutant KEN-box (F-I) were expressed in the anterior region of embryos during the stage of mitosis 14. Embryos were labeled with anti-myc (B,C,F,G; myc), anti-Cyclin B (D,H; CYCB) and a DNA stain (E,I; DNA). Whole embryos are shown in B and F, whereas high-magnification views from the anterior region are presented in C-E and G-I. Arrowheads indicate normal telophase figures in regions of PIM-myc-expressing embryos lacking anti-myc and anti-Cyclin B labeling (C-E), whereas arrows mark bridged telophase nuclei in regions of PIM^kena-myc-expressing embryos that lack anti-Cyclin B but not anti-myc labeling (G-I). (J-L) Using prd-GAL4, UAS-pim^kena-myc was expressed in alternating segments of pim-mutant embryos. DNA (J, red in L) and anti-Cyclin B labeling (K, green in L) indicated that PIM^kena-myc can promote sister chromatid separation in pim mutants initially when expression levels are still low. The horizontal dashed line in the high-magnification view of the embryonic epidermis separates the upper dorsal epidermis, where cells have already progressed through mitosis 15 and re-accumulated some Cyclin B, from the lower ventral epidermis, where cells are in the process of mitosis 15 and therefore either still have high levels Cyclin B before metaphase or no Cyclin B after metaphase. The vertical dashed lines separate outer PIM^kena-myc-expressing regions from a middle region without PIM^kena-myc. Although the failure of sister chromatid separation in this middle region results in large undivided nuclei in the dorsal epidermis and in the absence of normal anaphase and telophase figures in the ventral epidermis, the outer PIM^kena-myc-expressing regions display an almost normal nuclear density in the dorsal epidermis and normal late mitotic figures (anaphase indicated by white arrowhead) in the ventral epidermis.

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