Lamin B1 (Lmnb1) is a major component of the nuclear lamina and together with other lamins (lamin A/C) plays a key role in the structure and function of the nucleus. Mutations in lamins and lamin-associated proteins have been indeed shown to cause various human diseases. There are indications that mammalian lamins A and B and drosophila Lamin Dm₀ are involved in nuclear envelope formation and nuclear morphology (Prüfert et al. J Cell Sci 117, 6105). To explore the specific role of Lamin B1 in defining nuclear morphology we applied electron microscopy, electron tomography and correlative light electron microscopy (CLEM) on different cell types in which murine lamin B1 (Lmnb1) was silenced or human lamin B1 (LMNB1) was over-expressed.
To investigate how Lmnb1 deficiency affects nuclei of post-mitotic neurons, we analyzed the nuclear morphology of primary mouse cortical neurons derived from Lmnb1-null (Lmnb1^Δ/Δ) embryos (Giacomini et al. Mol Biol Cell 27, 35). Nuclei of cultured Lmnb1^Δ/Δ neurons were smaller and rounder than those of Lmnb1^+/+ embryos (the area of Lmnb1^Δ/Δ nuclei was 43% less than that of Lmnb1^+/+). Moreover the Lmnb1^Δ/Δ nuclei presented a rounder shape than those of Lmnb1^+/+, as confirmed by increased circularity values (mean circularity was 0.62±0.16 n=28 and 0.9±0.87 n=39 for respectively Lmnb1^+/+ and Lmnb1^Δ/Δ nuclei). Taken together, these results indicate that optimal Lmnb1 levels are essential to maintain neuronal nuclear size and shape. Lmnb1 deficiency also affected the distribution and composition of nuclear pore complexes (NPCs) in mouse cortical neurons. Indeed in Lmnb1^Δ/Δ neurons, the NPCs were distributed irregularly, with some regions of the nuclear envelope hosting groups of NPCs located close to each other and other areas devoid of NPCs (Fig.1A-C). Electron tomography performed on serial semi-thin sections showed that in Lmnb1^Δ/Δ neurons the NPCs are distributed in parallel, closely packed rows (Fig.1D).
The effect of the LMNB1 over-expression on nuclear morphology was assessed in transfected HEK 293 and HeLa cells. HEK 293 cells transfected with a bicistronic plasmid containing LMNB1 and EGFP downstream of an IRES sequence were sorted by EGFP expression using fluorescence-activated cell sorting (FACS) and high pressure frozen and freeze substituted to maintain structural integrity as much as possible. To increase contrast at the nuclear membranes we set up a freeze substitution recipe with a pre-incubation step at -90°C in 2% hydrated acetone containing small percent of tannic acid and glutaraldehyde. The cell nuclei of HEK 293 over-expressing LMNB1 were lobulated, with highly folded nuclear membranes often associated with multi-membrane stacks and intra-nuclear membranes (Fig.2A). As in primary cortical neurons lacking Lmnb1, we observed clusters of NPCs at the nuclear membrane (Fig.2B). Immunolabeling performed on cryo-sections revealed the presence of LMNB1 on the membrane stacks associated with the nuclear envelope (Fig.2C). This result supports the idea that these membrane stacks are effectively multi-membrane-layered nuclear membranes. To better understand the fine localization of Lmnb1 we performed correlative light electron microscopy (CLEM) experiments on HEK 293 and HeLa cells overexpressing LMNB1 in fusion with EGFP and tdTomato downstream of an IRES sequence. The cells, grown on sapphire disks carbon coated with a finder grid pattern, were live imaged at the confocal laser scanning microscope and then high pressure frozen and freeze substituted (Fig.3). As for HEK 293, the cell nuclei of HeLa cells over-expressing LMNB1 were lobulated with highly folded nuclear membranes. The HeLa nuclei differed from HEK 293 nuclei for the absence of membrane stacks associated to the nuclear membranes and for the presence of peculiar morphologies (Fig.3A). Indeed our CLEM approach revealed the presence of highly folded intra-nuclear membranes delimiting large cytoplasmic regions (Fig.3B,D). This approach also revealed the presence of nuclei that branched apically in thin and long nuclear rods delimited by conventional nuclear membranes (Fig.3B,C). Altogether, our results indicate that proper levels of Lamin B1 are required to maintain structural integrity and morphology of the nuclear envelope.
Acknowledgement
We thank Roberta Ruffilli and all the EM Lab members for their support. Special thanks to Prof. Liberato Manna for supporting this research.

Figures:

« Back to The 16th European Microscopy Congress 2016

EMC Abstracts - https://emc-proceedings.com/abstract/role-of-lamin-b1-in-structuring-the-cell-nucleus-in-eukaryotic-cells/