Reorganization of the nuclear architecture in the Drosophila melanogaster Lamin B mutant lacking the CaaX box

Abstract

Lamins interact with the nuclear membrane and chromatin but the precise players and mechanisms of these interactions are unknown. Here, we tested whether the removal of the CaaX motif from Lamin B disrupts its attachment to the nuclear membrane and affects chromatin distribution. We usedDrosophila melanogaster Lam^A25 homozygous mutants that lack the CaaX box. We found that the mutant Lamin B was not confined to the nuclear periphery but was distributed throughout the nuclear interior, colocalizing with chromosomes in salivary gland and proventriculus. The peripheral position of Lamin C, nuclear pore complex (NPC), heterochromatin protein 1a (HP1a), H3K9me2- and H3K27me3-associated chromatin remained intact. The fluorescence intensity of the DAPI-stained peripheral chromatin significantly decreased and that of the central chromatin significantly increased in the proventriculus nuclei of the mutantflies compared to wild-type. However, the mutation had little effect on chromatin radial distribution inside highly polytenized salivary gland nuclei.

Keywords: Dm0; Drosophila; Lama25 mutant; B-type lamin; Lamin B; Nuclear lamina; chromatin; confocal microscopy; nuclear envelope; proventriculus nuclei; salivary gland nuclei.

Figure 1.

Crossing scheme and analysis workflow to characterize homozygous Lam^A25 D. melanogaster mutants.

Figure 2.

The structure of the Lam gene and Lam^A25 allele in D. melanogaster. (a) The location of the Lam^A25 frameshift mutation in the Lam gene. NLS – nuclear localization signal; ADL67.10 and ADL84.12 – epitopes recognized by the Lamin B antibodies used in this study; CDS – coding sequence. Grey numbers indicate genomic coordinates on chromosome 2L in FlyBase, v. 6.29. Black numbers correspond to nucleotides of the Lam gene sequence. The first genomic coordinate and gene nucleotide number as well as the first and last amino acid number of the Lam^A25 substitution are shown in red. The tail domain is from 411 to 622 amino acids. (b) The fragment of wild-type (wt) Lamin B nucleotide sequence (from ²⁹⁸⁸C to ³⁰⁰⁸C) is shown in comparison with the corresponding sequence of the Lam^A25 mutant. The sequences involved in the frameshift are shown in blue and red.

Figure 3.

Whole-mount immunostaining of salivary gland (a, b) and proventriculus (c, d) nuclei of wt (a, c) and Lam^A25 (A25) mutant (b, d) D. melanogaster larvae. Chromatin (blue) is stained by DAPI. Lamin B (green) is stained by the specific antibody ADL67.10. Scale bar = 10 µm.

Figure 4.

Radial fluorescence intensity of DAPI-stained chromatin in polytene nuclei of the salivary gland and the proventriculus in D. melanogaster. The X-axis is the relative position from the nuclear center to the periphery (0%-100%). The Y-axis is the intensity of DAPI fluorescence normalized by the maximum intensity in the nucleus. The green line represents wt data, the red line represents Lam^A25 mutant data. Error bars show standard deviation. Asterisks indicate 5% intervals with statistically significant p-values. Δ quantifies how the relative intensity in the Lam^A25 group changes in comparison with wt in each ⅓ interval of the X-axis.

Figure 5.

Immunostaining of proventriculus nuclei of wt and Lam^A25 (A25) mutant D. melanogaster larvae. Chromatin (blue) is stained by DAPI. Lamin B (green) is stained by the specific antibody ADL67.10. Nucleolus is stained by the fibrillarin antibody ab6785. Scale bar = 10 µm.

Figure 6.

Localization of Lamin C in salivary gland and proventriculus nuclei from wt and Lam^A25 D. melanogaster larvae. Chromatin (blue) is stained by DAPI. Lamin C (green) is stained by the specific antibody LC28.26. Scale bar = 10 µm.

Figure 7.

Localization of HP1a in proventriculus nuclei from wt (top) and Lam^A25 mutant (bottom) D. melanogaster larvae. Chromatin (blue) is stained by DAPI. HP1a (green) is stained by the specific antibody C1A9. Scale bar = 10 µm.

Figure 8.

Localization of H3K9me2 in salivary gland and proventriculus nuclei from wt and Lam^A25 D. melanogaster larvae. Chromatin (blue) is stained by DAPI. H3K9me2 (red) is stained by the specific antibody A-4035-025. Scale bar = 10 µm.

Figure 9.

Scheme demonstrating the role of the CaaX box of Lamin B in nuclear organization. (a) Organization of the nuclear periphery in the wt nucleus. (b) The effect of CaaX box removal on nuclear architecture in the Lam^A25 mutant. Chromatin fibers (thin gray lines) are shown interacting with Lamin B (thick green lines). CaaX boxes (red circles) are shown interacting with the inner nuclear membrane (INM) in wt flies. ONM stands for the outer nuclear membrane. The scheme reflects data obtained from Drosophila nuclei with low levels of polyteny found in proventriculus.