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First published online 14 September 2004
doi: 10.1242/jcs.01334

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Novel nuclear defects in KLP61F-deficient mutants in Drosophila are partially suppressed by loss of Ncd function

Georgia State University, Department of Biology, 24 Peachtree Center, Atlanta, GA 30303, USA

View larger version (49K): [in a new window]   Fig. 1. Immunoblot analysis of Ncd and KLP61F expression. (A) Crude extracts of ovaries of wild-type females probed with antibodies from immune (I) and preimmune (P) antibodies against Ncd. (B) Crude extracts of larval brains of wild type (wt), ncd1 and klp61F4 ncd1 mutants fractionated in three wide lanes (underscores) of a 10% SDS-PAGE gel. Parallel channels of an Immunetics manifold were probed with antibodies against Ncd (Ab Ncd) and KLP61F (Ab KLP61F). Positions of Ncd (*) and KLP61F (**) proteins are indicated.

View larger version (85K): [in a new window]   Fig. 2. Spindle organization in klp61Fand klp61F ncd mutants. Larval brains of wild type (A), klp61F3 (B,C) and klp61F3 ncd1 (D) animals were stained with antibodies against -tubulin (green), -tubulin (red) and a fluorescent chromatin dye (blue). (C) Arrow indicates a tiny dot of -tubulin at a nearly anastral spindle pole and asterisk indicates chromosomes at opposing pole. Bar, 2 µm.

View larger version (120K): [in a new window]   Fig. 3. Nuclear lamina organization in klp61F and klp61F ncd mutants. Larval brains of wild type (A) and klp61F3 (B), klp61F3 ncd1/Df(3L)bab PG (C) and klp61F3 ncd1/klp61F4 (D) mutants stained with antibodies against lamins (grayscale), -tubulin (red) and a chromatin dye (blue). Arrows in (B, C) indicate involutions of the nuclear lamina that extend toward centrosomes. (D) Cell with bipolar positioning of centrosomes and metaphase alignment of chromosomes, showing regions of (arrow) disorganization and blebbing of nuclear lamina. Images of -tubulin and chromatin are single projected images of a stack of deconvolved sections. Images of lamin staining are single deconvolved sections chosen to show involutions in nuclear lamina. Bar, 5 µm.

View larger version (159K): [in a new window]   Fig. 4. TEM of nuclear lamina defects in klp61F ncd mutants. Somatic cells in larval brains of wild-type (A) and klp61F4 ncd1/klp61F3 ncd1 mutant (B-D) animals with nuclear lamina indicated with red arrows, condensed chromatin indicated by blue arrows and mitochondria indicated by green arrows. (A) Nuclear lamina in a wild-type cell lies between condensed chromatin and cytoplasmic mitochondria, shown in the insert at 2x magnification. (B) Somatic cell in klp61F4 ncd1/klp61F3 ncd1 larval brain lacks detectable nuclear lamina lying between mitochondrion and condensed chromatin. A region between a mitochondrion and chromatin is shown in the inset at 2x magnification. (C) Nuclear lamina surrounding separate masses of condensed chromatin. (D) Region lying between marked lamina is shown at 3x magnification. Fenestration of the nuclear lamina in prometaphase or metaphase cells was inferred from uniform staining throughout mitotic cells whereas interphase cells showed lighter staining of nucleoplasm in comparison to darker staining of the cytoplasm and by position of condensed chromatin near the cell center rather than the nuclear periphery. Interphase cells were more than tenfold more frequent than cells in mitosis. Bars, 2 µm.

View larger version (229K): [in a new window]   Fig. 5. Atypical localization of KLP61F in spermatocytes. Wild-type testes stained with antibodies against KLP61F (red), a fluorescent chromatin dye (blue) and -tubulin (green, A-C) and -tubulin (lavender, D). (A-D) Single projection of a stack of optical sections. Spermatocytes at the G2/M transition failed to show KLP61F enrichment at centrosomes (A) or spindle localization at metaphase (B). Spermatocytes are shown at telophase of meiosis II (C) and meiosis I (D) with septin-like localization of KLP61F. (E) Low magnification wide-field image of a cyst. Asterisks indicate spermatocytes at higher magnification in metaphase (F), anaphase (G) and late anaphase/telophase (H). Bar (A-D), 2 µm; (E), 10 µm; (F-H), 5 µm.

View larger version (99K): [in a new window]   Fig. 6. Spindle organization in klp61F mutant spermatocytes. Testes of klp61F3 mutants stained with antibodies against -tubulin (green), -tubulin (red) and a fluorescent chromatin dye (blue). Arrows in (B) indicate an acentrosomal spermatocyte, (E) bivalent at a metaphase position in microtubule bundle, and (F) a cleavage furrow forming an asymmetric telophase spindle. Images are representative spindle organization in klp61F3/klp61F3, klp61F3/klp61F1 transheterozygotes and klp61F3/Df(3L)bab PG mutants. Bar, 5 µm.

View larger version (124K): [in a new window]   Fig. 7. Nuclear lamina organization in klp61F mutant spermatocytes. Testes from wild type (A-C) and klp61F3 mutants (D-F) stained with antibodies against -tubulin (red), lamin (green) and a fluorescent chromatin dye (blue). Images are single projections of a stack of optical sections. (A) Antibodies against -tubulin crossreact with a mitochondrial antigen(s) in testes, providing a useful marker for mitochondrial derivatives and the progression of spermatocyte development. (E,F) Arrows indicate acentrosomal spermatocytes. (E) To show the apposition of centrosomes and chromatin relative to invaginations of nuclear lamina, the lasso tool of Photoshop was used to replace the projected stack of lamin staining with lamin staining in a single optical section in the spermatocyte outlined in dots. Bar, 5 µm.

View larger version (80K): [in a new window]   Fig. 8. Micronuclei in klp61F ncd spermatocytes at telophase. Testes in klp61F ncd mutants (A-F), wild-type animals (G) and ncd1 mutants (H,I) were stained with antibodies against -tubulin (green) and a fluorescent chromatin dye (blue) and -tubulin (green, A-C) or lamins (green, D-I). (A,D) Multiple nuclei were not detected in metaphase of meiosis, but were detected at the poles of telophase spindles (B, arrows) and in spermatids (C,F arrows). Multiple nuclei were not detected in wild-type (G) or in ncd1 (H) spermatocytes in telophase of meiosis or in post-meiotic spermatocytes (I). Bar, 2 µm.

View larger version (138K): [in a new window]   Fig. 9. Ncd localization in klp61F and ncd mutant spermatocytes. Spermatocytes in wild type (A-D), ncd1 (E) and klp61F3/klp61F1 (F) mutants stained with a blue fluorescent chromatin dye and antibodies against Ncd (red), -tubulin (green, A,C,E), -tubulin (green, B,D) and lamin (green, F). Arrows in (A) indicate enriched Ncd near heterochromatin in primary spermatocyte. (C) Arrow indicates overexposure of nuclear staining of Ncd in primary spermatocyte for comparison with spindle staining marked by asterisks. (F) Arrow indicates lamina around bivalent in spermatocyte near prometaphase. Bar, 5 µm.

View larger version (96K): [in a new window]   Fig. 10. Skeletor localization in ncd, klp61F and klp61F ncd mutant. Testes of the wild type (A-C), ncd1 (E), klp61F3 (F), klp61F3 ncd1/Df(3L)bab PG ncd1 (klp61F3/Df ncd1) (G) and klp61F3 ncd1/klp61F4 ncd1 (klp61F3/4 ncd1) (D) mutants stained with antibodies against Skeletor (green), -tubulin (red) and a chromatin dye (blue). (A) Arrows indicate spermatocytes near the G2/M transition with low levels of Skeletor. (C) Arrow show enriched Skeletor localization near chromatin and (F) aggregates of Skeletor in klp61F3 spermatocytes near prometaphase. Bar, 5 µm.