Coilin is essential for Cajal body organization in Drosophila melanogaster

Abstract

Cajal bodies (CBs) are nuclear organelles that occur in a variety of organisms, including vertebrates, insects, and plants. They are most often identified with antibodies against the marker protein coilin. Because the amino acid sequence of coilin is not strongly conserved evolutionarily, coilin orthologues have been difficult to recognize by homology search. Here, we report the identification of Drosophila melanogaster coilin and describe its distribution in tissues of the fly. Surprisingly, we found coilin not only in CBs but also in histone locus bodies (HLBs), calling into question the use of coilin as an exclusive marker for CBs. We analyzed two null mutants in the coilin gene and a piggyBac insertion mutant, which leads to specific loss of coilin from the germline. All three mutants are homozygous viable and fertile. Cells that lack coilin also lack distinct foci of other CB markers, including fibrillarin, the survival motor neuron (SMN) protein, U2 small nuclear RNA (snRNA), U5 snRNA, and the small CB-specific (sca) RNA U85. However, HLBs are not obviously affected in coilin-null flies. Thus, coilin is required for normal CB organization in Drosophila but is not essential for viability or production of functional gametes.

Figure 1.

Ovarian follicle cells. (A) From a transgenic line expressing EGFP-labeled mouse coilin, stained with anti-GFP (green) to enhance the EGFP signal and with an antibody against Drosophila Lsm11 (red), a protein specific for the U7 snRNP. DNA is stained with DAPI (blue). Only a fraction of nuclei express EGFP-coilin. In these, a single CB (green) and a single HLB (red) occur, often in proximity to each other. (B) From a y w stock, stained with antibodies against Drosophila coilin (green) and Lsm11 (red). The CBs (green) and HLBs (red) are similar in size and distribution to those in the mouse coilin transgenic line.

Figure 2.

Clustal analysis (Thompson et al., 1994) of coilin sequences, displayed in Jalview (Clamp et al., 2004). (A) The first 100 amino acids at the amino terminus are moderately well conserved across species. (B) The most conserved motif occurs near the carboxy terminus, as recognized originally by Bellini (2000). The coil gene has been cloned from Drosophila melanogaster, Arabidopsis thaliana, Xenopus laevis, Homo sapiens, and Mus musculus.

Figure 3.

(A) Western blot of D. melanogaster ovary proteins probed with a guinea pig antibody (GP3) against D. melanogaster coilin. Lane 1, y w; lane 2, coil^B220; lane 3, coil²⁰³; and lane 4, coil¹⁹⁹. The major band at ∼90 kDa presumably represents full-length coilin. No bands are present in the mutant ovaries. (B) The same filter after stripping and reprobing with an anti-tubulin antibody.

Figure 4.

Drosophila coilin in CBs of Malpighian tubule cells. (A–C) Coilin (red) colocalizes with a high concentration of snRNPs (green) in a single CB. snRNPs also occur throughout the nucleus. In the overlay, DNA is stained with DAPI (blue). snRNPs are stained with mAb Y12, which recognizes symmetric dimethylarginine (SDMA). (D–F) Coilin (red) colocalizes with fibrillarin (green) in a single CB. The majority of fibrillarin stain is in the nucleolus. In the overlay, DNA is stained with DAPI (blue).

Figure 5.

Germarium from a y w fly stained with antibodies against coilin (red) and Lsm11 (green). DNA is stained with DAPI (blue). (A) Entire germarium with germline stem cells to the left and a stage 1 egg chamber to the right. (B–D) Enlarged view of the two germline stem cells (asterisks), showing a prominent HLB in each nucleus but only diffuse coilin stain without a detectable CB. A CB is present in later stages, often closely associated with the HLB.

Figure 6.

CBs and HLBs in mid- and late stage egg chambers, from a transgenic line expressing YFP-coilin, stained with anti-GFP (green) to enhance the YFP signal and with anti-Lsm11 (red). DNA is stained with DAPI (blue). (A) This stage 8 egg chamber has a prominent CB in the GV (arrow). There are one or two CBs (green) and multiple HLBs (red) in the nurse cell nuclei. (B) A single nurse cell nucleus from A at higher magnification. (C) This slightly later stage 8 egg chamber no longer has a detectable CB in the GV (arrow). The CBs in the nurse cell nuclei have begun to fragment. (D) A single nurse cell nucleus from C at higher magnification, showing multiple small CBs. The HLBs are similar to those in younger egg chambers. (E–G) Nurse cell nuclei at the anterior end of a stage 12 egg chamber, after nurse cell ”dumping.“ The HLBs (red in E) now display obvious coilin (green in F), but only a few small CBs remain (smaller green dots in F and G that lack red Lsm11).

Figure 7.

CBs and HLBs in various tissues. (A) Testis from third instar y w larva, stained with antibodies against coilin (green) and Lsm11 (red). DNA is stained with DAPI (blue). The clustered somatic hub cells each display a green CB and a red HLB. The ring of germline stem cells immediately surrounding the hub has a HLB but no evident CB. (B) Anterior end of the larval testis, stained with antibodies against coilin (green) and fibrillarin (red). DNA is stained with DAPI (blue). The large green nuclei on the right are primary spermatocytes in prophase of the first meiotic division. Note the high level of diffuse coilin stain in these nuclei. Fibrillarin stain is prominent in nucleoli through the early spermatocyte stage. (C) Spermatocyte divisions, stained with an antibody against coilin (green) and with DAPI (blue) to show the chromosomes. Note the intense coilin stain in the meiotic spindles. (D) Nuclei from the ejaculatory duct of an adult y w male, stained with antibodies against coilin (green) to show CBs and Lsm 11 (red) to show HLBs. DNA is stained with DAPI (blue). (E) Larval brain stained as in D. In the larval brain, CBs and HLBs are tightly associated with each other. Most of the apparent colocalization of red and green signals is due to orientation of the red and green bodies along the z-axis of the image. (F–H) Nucleus from a cell of the adult Malpighian tubule, stained with antibodies against coilin (red) and Lsm11 (green). DNA is stained with DAPI (blue). Two prominent red CBs are present in this nucleus. Note that the HLB (arrowhead) contains a low level of coilin.

Figure 8.

Coilin on metaphase chromosomes. (A) Metaphase of an ovarian follicle cell, from a fly expressing GFP-labeled polo kinase. Stained with antibodies against coilin (red) and GFP (green). DNA is stained with DAPI (blue). Note that coilin is limited to a single row of dots along the center of the metaphase plate, suggesting that coilin lies between the centromeres of sister chromatids. In contrast, polo kinase occurs in two rows of dots, presumably the sister kinetochores oriented to opposite poles of the spindle. The poles themselves also contain polo kinase (arrows). An arrowhead indicates the plane of the metaphase plate. (B–E) Metaphase from a larval neuroblast stained with antibodies against coilin (red) and CID (green). CID is Drosophila CENP-A and defines the centromeres of the chromosomes. The arrowhead indicates the metaphase plate. Note that coilin is limited to the centromere regions on the metaphase plate and that CID dots lie on either side of the coilin (toward the poles of the spindle), again suggesting that coilin lies between the sister chromatids.

Figure 9.

Absence of coilin from nuclei of coil¹⁹⁹ and coil²⁰³ flies. (A) Stage 8 egg chamber from a coil²⁰³ female, stained with antibodies against coilin (green) and Lsm11 (red). DNA is stained with DAPI (blue). No coilin stain is seen in any nuclei, whereas the Lsm11 stain in the HLBs is normal. (B) Nurse cell nuclei at the anterior end of a stage 12 egg chamber from a coil²⁰³ female, after nurse cell dumping. The prominent HLBs (red) fail to stain with the antibody against coilin. Compare with Figure 6, E–G, which shows a comparable stage from a nonmutant ovary. (C and D) Primary spermatocytes stained with antibodies against coilin (green) and fibrillarin (red). In C, from a fly heterozygous for coil¹⁹⁹, the entire nucleus stains green for coilin, except for the nucleolus, which stains red for fibrillarin. In D, from a fly homozygous for coil¹⁹⁹, only the nucleoli stain. In primary spermatocytes, the DAPI stain is weak, because the DNA is dispersed throughout a very large nucleus. (E and F) Cells of the ejaculatory duct of male flies stained for coilin (green) and Lsm11 (red). CBs (green) and HLBs (red) are evident in E, from a fly heterozygous for coil¹⁹⁹, whereas only HLBs (red) are seen in F, from a fly homozygous for coil¹⁹⁹. (G and H) Cells of the ejaculatory duct of male flies after in situ hybridization for U7 snRNA (green) and U85 scaRNA (red). HLBs (green) and CBs (red) are evident in G, from a y w fly, whereas only HLBs (green) are seen in H, from a fly homozygous for coil¹⁹⁹.

Figure 10.

In situ hybridization of U7 snRNA and U85 scaRNA. (A) Stage 8 egg chamber from a y w fly. In the giant nurse cell nuclei, HLBs hybridize with the U7 snRNA probe (green) and CBs with the U85 scaRNA probe (red). The green bodies in the nurse cell cytoplasm are U bodies (Liu and Gall, 2007), whereas most of the green in the oocyte at the right end of the egg chamber is due to autofluorescence of yolk. DNA is stained with DAPI (blue). (B) A single nurse cell nucleus from A. (C) Stage 8 egg chamber from a coil²⁰³ fly. HLBs hybridize with U7 snRNA (green), but no hybridization is seen with the U85 scaRNA (red) probe. DNA is stained with DAPI (blue). Arrow points to the GV. (D) A single nurse cell nucleus from C.

Figure 11.

CB and HLB components in Malpighian tubule nuclei of wild type (y w, top row) and coilin-null flies (coil¹⁹⁹ and coil²⁰³, bottom row). CBs are present in wild-type cells but missing from coilin-null cells, whereas HLBs are present in both wild type and coilin-null cells. (A and B) FISH for U7 snRNA (green) and U85 scaRNA (red). In A, the CB and HLB are closely associated. (C and D) FISH for U2 snRNA (green) and U85 scaRNA (red). In the wild-type nucleus in C, U2 is colocalized with U85 in a distinct CB, but it is also diffusely present throughout the nucleus. In the coilin-null nucleus in D, there is no distinct CB, but diffuse U2 snRNA is present. (E and F) FISH for U5 snRNA and U85 scaRNA. In the wild-type nucleus in E, there are two CBs in which U5 and U85 are colocalized. U5 is diffusely distributed in both the wild-type and coilin-null nuclei. No CB is present in the coilin-null nucleus. (G and H) Immunostain for Lsm11 (red) and symmetric dimethylarginine (SDMA, green). A red HLB and a green CB are present in the wild-type nucleus (G) but only a red HLB is seen in the coilin-null nucleus (H). snRNP proteins (SDMA) are diffusely distributed in both wild-type and mutant nuclei. (I and J) Immunostain for Lsm11 and fibrillarin. In the wild-type nucleus (I), most of the fibrillarin is in the large nucleolus (green), with a small amount in a separate CB (green). The single HLB (red) is positive for Lsm11. In the coilin-null nucleus (J), there is a single HLB (red). The nucleolus is stained for fibrillarin, but there is no fibrillarin-positive CB.

Figure 12.

Follicle cells of a stage 11–12 egg chamber, from a transgenic fly that expressed EYFP-labeled short isoform of coilin. EYFP-labeled protein was enhanced by staining with an antibody against GFP (green). Endogenous coilin was detected with an antibody specific for the long isoform (red). Note that red stain is limited to CBs in the nuclei, whereas green stain, corresponding to the EYFP short isoform, is limited to the cytoplasm. Furthermore, expression of the short isoform is heterogeneous. Endogenous long isoform (red) is detectable only in nuclei of cells that have little or no transgenic short isoform (green) in the cytoplasm.