Proceedings of The Physiological Society

Physiology 2016 (Dublin, Ireland) (2016) Proc Physiol Soc 37, PCA303

Poster Communications

Strategical positioning of chromatin anchoring NET protein complexes in the nuclear invaginations of pulmonary arterial smooth muscle cells

J. Duan1, J. Navarro-Dorado1, A. M. Evans1

1. Biomedical Science, The University of Edinburgh, Edinburgh, United Kingdom.

Nuclear invaginations (NIs) have been identified in multiple cell types, yet their function remains unclear. Because NIs extend the nuclear envelope (NE) into the nucleoplasm, they were proposed to increase the surface area available to support Ca2+ entry into the nucleus and thus regulated gene expression(1). Consistent with this view we have identified that NIs contain chromatin attachment points formed by nuclear envelope transmembrane (NET) proteins that are targeted to the inner and/or outer nuclear membrane (NM) of NI. Pulmonary arterial smooth muscle cells (PASMCs) were isolated from rats, fixed either immediately or after 7 days of culture. Cells were stained with ER tracker or labelled for lamin A, nesprin-1, SUN2 and emerin using immunocytochemistry methods described previously(2). Confocal images were acquired (Nikon A1R), deconvolved (Huygens) and reconstructed in 3D (Imaris, Bitplane). All data are quoted as mean±SEM, and compared by t-test. Both the Lamin A labelling (n=7) and ER-tracker staining (n=6) revealed trans-nuclear networks of NIs in acutely isolated PASMCs. 61% of cells exhibited deep NIs and 100% of cells harboured superficial NIs. The number of NIs identified per cell was 4±0.41. Labelling for nesprin-1, a trans-outer NM protein, was widely distributed across the entire surface of the NE including NIs, albeit in a punctate manner, and colocalised (Pearson's correlation coefficient=0.47) with 60% of the lamin A labelling (n=2). SUN2, which combines with nesprin-1 to form the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex, colocalised with 46±2% of nesprin-1 labelling (Pearson's correlation coefficient=0.26±0.09, n=3), and SUN2 labelling appeared to be absent from the centre of some deep NIs. This indicates that the LINC complex, which serves to connect the cytoplasm to the nucleoplasm, may be strategically positioned at the entry point to NIs(3). By contrast, emerin labelling extended across most of the NE and was present along the entire length of the NIs (n=5). Given that this trans-inner NM protein may bind to nesprins directly or through SUN proteins and may either way anchor chromatin to the vicinity of the NE via its binding partners(4), NIs might confer different loci for chromatin attachment through observed targeting of NET proteins to the peripheral NE and to different regions of NIs. Surprisingly, no NIs were found in cultured, proliferating PASMCs and the NE surface area to volume ratio (µm2/µm3) dropped from 5±0.8 (acutely isolated, n=4) to 2±0.4 (7-day culture, n=4; p=0.01). Given this finding and the fact that the switch from a contractile to proliferative phenotype is underpinned by changes in gene expression(5), it is likely that the NI provides greater surface area for chromatin attachment via NET proteins and thus enhanced suppression of gene expression.

Where applicable, experiments conform with Society ethical requirements