The detachment of lamin Dm0 from the nuclear envelope increases variability in 3D positioning of LADs within Drosophila melanogaster nuclei
Authors: Simon Bondarenko 1,2; Igor Sharakhov 1,2
Affiliations: 1) Department of Entomology and the Fralin Life Sciences Institute, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061, USA; 2) Department of Genetics and Cell Biology, Tomsk State University, Tomsk, Russian Federation
Keywords: k. nuclear organization; a. chromatin structure
The attachment of chromatin to the nuclear envelope correlates with gene repression, which may play an important regulatory role in the interphase nucleus. Lamins interact with the internal nuclear membrane (INM) and together with other proteins form nuclear lamina. Lamina-associated domains (LADs) of chromatin are shown to interact with the nuclear lamina, however the role of lamins in the 3D position of LADs inside the nucleus is not well understood. In this study, we developed LAD- and nonLAD-specific fluorescent oligo-probes for the chromosome 3 of Drosophila melanogaster and hybridized them with the wild type (wt) Canton S and Lam[A25] mutant nuclei. This mutant lamin lacks the hydrophobic CaaX box responsible for tethering the lamin Dm0 to the INM. The mutant Dm0 protein is not confined to the nuclear periphery but is distributed throughout the nuclear interior, colocalizing with chromosomes. We performed the confocal microscopy of highly-polytenized nuclei of salivary glands (SG) and low-polytenized nuclei of proventriculus (PV) labeled with LAD- and nonLAD-specific probes using Zeiss LSM 880 with Airyscan module. The radial distribution of DAPI intensity was measured by the radial profile plugin for Fiji software and the radial distribution of LADs and nonLADs was analyzed with the TANGO plugin for Fiji. The radial distribution of chromatin in Lam[A25] nuclei was significantly shifted toward the center of the nucleus in comparison with the wt, which suggests that the lamin Dm0 is necessary for the attachment of chromatin to the NE. Although, we did not detect a significant difference in the radial distribution of LADs and nonLADs between the mutants and wt (PCA analysis), the variability in LADs in the PV nuclei was 29% higher in Lam[A25] than in wt (significant, t-test, p≤0.5), whereas the variability in the radial position of LADs in SG was only 10.5% higher in Lam[A25] than in wt (significant, t-test, p≤0.5). At the same time, the variability of the radial position of nonLADs was not significantly different among all groups. The results suggest that the Dm0 protein plays an important role in the 3D position of LADs inside the nucleus.