Novel Tool for Imaging in vivo Nuclei in Drosophila Larvae Reveals Unanticipated Peripheral Chromatin Organization Sensitive to Lamin A/C

Background: The current view of chromatin 3D organization suggests that it encompasses the entire nucleus. Repressed, heterochromatic regions preferentially associate with the nuclear periphery, while active, euchromatic regions assume more central location; however, whether this organization reflects live physiological conditions is currently unclear.

Methods: We utilized a custom-made device to visualize chromatin 3D organization in intact live Drosophila larval muscles under a confocal microscope. The chromatin was labeled with His2B-mRFP, nuclear envelope was labeled with muscle-driven Nesprin/klar-GFP, and active chromatin regions were labeled with H3K9Ac-reactive mintbody fused to GFP. To quantify radial chromatin distribution, the nucleus was segmented in 3D and divided into 10 concentric radial shells.

Results: Radial His2B-mRFP 3D distribution profile, in control muscle nuclei (n=27), demonstrated 2.8 fold increase in chromatin density at the outermost shell, compared to the center. Furthermore, active chromatin (H3K9Ac-GFP) also exhibited 2.7 fold increase in chromatin density at the periphery (n=27). Such peripheral chromatin distribution was never observed in fixed nuclei, which loose a significant portion of their volume. Interestingly, over-expression of Lamin A/C, which associates with stiffening of the nuclear envelope, disrupted the peripheral chromatin organization, demonstrating opposite profile with 4.8 fold increase in chromatin density in the center over the nucleus periphery.

Conclusions: We suggest that unveiling global 3D chromatin organization requires preservation of live, physiologic conditions. Significantly, our findings regarding peripheral chromatin distribution in live, intact organism might imply that the balance between chromatin-nuclear lamina versus chromatin-chromatin associations is an essential regulatory factor, which depends on lamin A/C levels.