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First published online 5 August 2003
doi: 10.1242/jcs.00682

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Barrier-to-autointegration factor plays crucial roles in cell cycle progression and nuclear organization in Drosophila

¹ Department of Chemistry, Faculty of Science, Niigata University, Niigata 950-2181, Japan
² Graduate School of Science and Technology, Niigata University, Niigata 950-2181, Japan
³ Center for Instrumental Analysis, Niigata University, Niigata 950-2181, Japan
⁴ Division of Biological Science, Graduate School of Science, Nagoya University, Nagoya 464-8602, Japan
⁵ The Laboratory of Biochemistry, Aichi Cancer Center Research Institute, Nagoya 464-8681, Japan
⁶ Department of Pharmacological Sciences, School of Medicine, University Medical Center, State University of New York at Stony Brook, Stony Brook, New York 11794-8651, USA

View larger version (86K): [in a new window]   Fig. 1. Localization of BAF in Drosophila cells and tissues. Localization of Drosophila BAF during the cell cycle was determined by double immunostaining with rabbit polyclonal anti-BAF antibodies (green, BAF) and either mouse monoclonal anti-lamin Dm0 antibodies (red, LamDm0) (A,C,E) or rat monoclonal anti-phosphorylated histone H3 Ser28 monoclonal antibodies (HTA28; red, P-H3) (B,D) in tissues from third instar larvae. Both low magnification (A,B) and enlarged images are shown (C,D,E). Colocalization is yellow (Merge). In A, anti-BAF antibodies were also tested for specificity by pre-incubation of antiserum with either thioredoxin-his-tag protein alone (TrH tag) or thioredoxin-his-tag-Drosophila BAF fusion protein (TrH-BAF) as indicated.. White arrows in B and D indicate condensed chromosomes. Scale bars: 20 µm (A,B) and 5 µm (C-E) All images were recorded with a confocal microscope.

View larger version (67K): [in a new window]   Fig. 2. Construction and properties of baf null mutant homozygotes. (A) Alignment of the baf1/baf1 and baf2/baf2 genomic DNAs in comparison with l(2)k10210/l(2)k10210 genomic DNA. Major restriction sites are as follows: B, BamHI; Xa, XbaI; and Xh, XhoI. Deletions generated after imprecise excision of integrated P-element (remnants are indicated by solid black bars). Open reading frames are indicated by either the stippled gray boxes [mouse pancreatic triacylglycerol lipase homolog, TL (entire coding region is shown); and the Cka protein, Cka (only the first exon is shown)] or the open boxes [BAF (entire coding region including both of two exons are shown)] between numbers 3226 (start of the first exon) and 3580 (end of the second exon) in the diagram of the l(2)k10210/l(2)k10210 genome. The insertion position of pP{lacW} in l(2)k10210/l(2)k10210 is shown by the down-pointing arrow in the l(2)k10210 diagram. The mRNA start site of the Cka protein is indicated by the down-pointing arrowhead at nucleotide (nt) 4360 in the l(2)k10210 diagram. The baf1/baf1 genomic DNA is deleted from nt 2991 to nt 3924; in baf2/baf2, the genomic DNA is deleted from nt 864 to nt 3924. Both are replaced with P{lacW} fragments, the lengths of which are indicated in parentheses below the solid boxes. For rescue experiments, the genomic fragment between the BamHI and XhoI sites indicated by asterisks in the l(2)k10210 diagram was recovered from wild-type flies by PCR amplification. (B) Late third instar larval CNS and imaginal discs dissected from baf1/+ or l(2)k10210/l(2)k10210, and baf1/baf1 or baf2/baf2 animals are shown. Br, brain hemispheres; Im, imaginal discs; Ad, abdominal neuroblasts; Th, thoracic neuroblasts. Scale bar: 100 µm and applies to all panels. (C) Cell proliferation and differentiation of third instar larval brain hemispheres from baf1/+ compared with baf1/baf1 and baf2/baf2. A whole brain hemisphere of the baf1/+ is illustrated both at low [baf1/+ (L)], and high [baf1/+ (H)] magnification. Scale bars in baf1/+ (L) and (H), 20 µm. baf1/baf1 and baf2/baf2 are shown at exactly the same magnification as in baf1/+ (H). The complex optic lobe anlagen of baf1/+ larvae is composed of outer anlage, oa; inner optic anlage, ia; medulla neuropil, mn; and medulla cell, mc.

View larger version (104K): [in a new window]   Fig. 7. Comparison of BAF and lamin distribution in cells of larvae lacking the baf gene. The correlation between abnormal lamin distribution and BAF level was investigated by confocal immunofluorescence. (A,B) Third instar larval brain hemispheres and thoracic ganglia of baf1/baf1animals were double immunostained with rabbit polyclonal anti-BAF antibodies (green, BAF) and mouse monoclonal anti-lamin Dm0 antibodies (red, LamDm0). B is a higher magnification from a similar experiment to A. Antigen colocalization is yellow (Merge). White arrows in A indicate convoluted nuclei in which BAF cannot be detected with anti-BAF antibodies. Scale bar: 20 µm (A); 5 µm (B).

View larger version (87K): [in a new window]   Fig. 6. Nuclear lamin distribution in cells of larvae lacking the baf gene. The localization of nuclear lamin Dm0-derivatives was evaluated by indirect immunofluorescence microscopy using a highly specific mAb. (A) Immunofluorescence images of the whole CNS from third instar larvae of baf1/+, baf1/baf1 and baf1/baf1 animals rescued with gBAF (gBAF rescue). (B,C) Confocal immunofluorescence images of the third instar larval brain hemispheres of baf1/+, baf1/baf1 rescued with gBAF, and baf1/baf1 animals. Obvious nuclear rim staining is found exclusively in the CNS of baf1/+ and baf1/baf1 animals rescued with gBAF (B). In the case of baf1/baf1 CNS (C), abnormal lamin staining is observed in the left two panels. The convoluted nuclear lamin staining is apparent in brain hemisphere cells of baf1/baf1 animals at higher magnification (rightmost upper and lower panels in C). Br, brain hemisphere; Im, imaginal discs; Th, thoracic neuroblasts. Scale bars: 50 µm (A; all upper panels in and all lower panels are shown at the same magnifications, respectively) 20 µm (B: as in A); 20 µm (C, left) 5 µm (C, right).

View larger version (63K): [in a new window]   Fig. 3. Characterization of mitotic chromosome behavior in the CNS of animals lacking the baf gene: decreased histone H3 phosphorylation. (A,B) Late third instar larval CNS tissues from baf1/+ (A), l(2)k10210/l(2)k10210 (A) or baf1/baf1 animals (B) were labeled with rabbit polyclonal PH10 antibodies, directed against a histone H3 Ser10 phospho-epitope (green). Staining with propidium iodide (PI) identifies DNA (red, PI/DNA). Bracket in baf1/baf1 (B) indicates two consecutive sections of baf1/baf1 tissues. Staining by PH10 was mostly negative; white arrowheads indicate the few positive condensed chromosome masses. (C,D)Behavior of mitotic chromosomes of the baf1/+ (C) or baf1/baf1 (D) tissues was also examined by immunofluorescence staining using PH10 (green, P-H3). The same material was labeled with PI to identify DNA (red, PI/DNA). Colocalization is yellow (Merge). While chromosomes of prophase, prometaphase, metaphase, early anaphase and late anaphase/telophase are clearly visualized by PH10 in the baf1/+ cells (C), chromosomes from baf1/baf1 cells are abnormal (D). Scale bars: 100 µm (A); 50 µm (B); 5 µm (C,D). All images were recorded with a confocal microscope.

View larger version (62K): [in a new window]   Fig. 4. BrdU incorporation in the CNS of third instar larvae lacking the baf gene. (A) Late third instar larvae of baf1/+, baf1/baf1, l(2)k10210/l(2)k10210 and w1/w1 animals were labeled with BrdU for 15-20 hours by feeding just before dissection. Incorporated BrdU was detected with a m-mAb directed against BrdU (green, BrdU). Immunofluorescence images of the entire CNS are shown (B) For baf1/+ and baf1/baf1, immunofluorescence images focused on brain hemispheres were also included. The same fields are labeled with PI to identify nuclei (red, PI/DNA). In both A and B, in baf1/baf1 tissues, two consecutive confocal sections are presented (indicated by brackets). (C) Incorporation of BrdU was also tested with 1.5-hours exposure of dissected late third instar larval CNS tissues in vitro. Individual nuclei of the baf1/baf1 CNS are observed at higher magnification. A merged image demonstrating the overlap of the green (BrdU) and red (PI/DNA) staining is also shown (yellow; Merge). Scale bars: 50 µm (A); 20 µ m (B); 5 µm (C) All images were recorded with a confocal microscope.

View larger version (75K): [in a new window]   Fig. 5. Cyclin levels in the CNS of third instar larvae lacking the baf gene. Cyclins A, B and E were detected with specific antibodies in baf1/baf1 or baf1/+ CNS tissues. In baf1/baf1 tissues, immunofluorescence images are presented in the upper panels, and the same fields labeled with PI, to identify nuclei, are shown in the lower panels. In the case of baf1/+ tissues, only immunofluorescence images are shown. Scale bars: baf1/baf1 panels, 50 µm; baf1/+ panels, 100 µm. All images were recorded with a confocal microscope.

View larger version (115K): [in a new window]   Fig. 8. Indirect immunofluorescence staining for lamin and BrdU in baf1/baf1 cells. Third instar larval CNS tissues from baf1/+ and baf1/baf1 animals were labeled with highly specific rabbit anti-lamin Dm0 antiserum (green; LamDm0) and mouse mAb directed against BrdU (red; BrdU). CNS tissues were labeled with BrdU for 1.5 hours in vitro just after dissection. Merged images are also shown (coincidence of lamin and BrdU labeling shows as yellow; Merge). Scale bar: 5 µm. All images were recorded with a confocal microscope.

View larger version (126K): [in a new window]   Fig. 9. Staining for nucleoporins and histone H2A in baf1/baf1 cells. Nuclear structure in the third instar larval baf1/baf1 CNS was analyzed further with m-mAb414 specific for NPC antigens (mAb414) or rabbit antiserum directed against histone H2A (indicated by the bracket) in addition to either rabbit anti-lamin Dm0 antiserum or a m-mAb directed against lamin Dm0 (LamDm0). White arrows indicate histone H2A staining apart from the abnormal lamin staining but apparently within a single nucleus. Scale bar: 5 µm. All images were recorded with a confocal microscope.

View larger version (173K): [in a new window]   Fig. 10. Ultrastructural analysis of NE and chromatin structures in the CNS of animals lacking the baf gene. Third instar larval of (A) baf1/+ and(BE) baf1/baf1 CNS cell nuclei. (B) Two entire minimally distorted nuclei containing multiple abnormal chromatin clumps are shown. (C) NE folding (distortion) is illustrated in another nucleus. (D,E) Regions of the specimen in C shown at higher magnification, with specific chromatin features indicated by white arrowheads. Black arrowheads indicate nuclear pore complexes. Scale bars: 1 µm (A-C); 0.1 µm (D,E).

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