Cell-wide web of cytoplasmic nanocourses coordinates calcium signalling
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Ca2+ signals determine smooth muscle contraction and the switch from a contractile to a migratory-proliferative phenotype(s), which requires changes in gene expression. However, the mechanism by which different Ca2+ signals are selective for these processes is enigmatic. In the thesis, I built on the “panjunctional sarcoplasmic reticulum” hypothesis, and described the evidence in support of the view that a variety of Ca2+ pumps and release channels, with different kinetics and affinities for Ca2+, are strategically positioned within the cytoplasmic nanocourses of pulmonary arterial smooth muscle cells (PASMCs), and they serve to demarcate different Ca2+ signalling. Nanocourses of the SR are formed in the perinuclear, extraperinuclear, subplasmalemmal regions and the nucleus. Different subtypes of ryanodine receptors (RyRs) are targeted to those nanocourses. Immunocytochemistry results suggest that RyR1s was preferentially targeted to the subplasmalemmal and nuclear nanocourses of PASMCs, they gave rise to a spatially restricted Ca2+ signal within the nanocourses upon stimulation, without affecting global Ca2+ concentration. The Ca2+ signals in the subplasmalemmal nanocourses were shown to induce arterial smooth muscle cell relaxation. On the other hand, the RyR2 and 3 were shown to target to the perinuclear and extraperinuclear nanocourses. Upon stimulation, they generate propagating Ca2+ waves in the cytoplasmic nanocourses, which trigger arterial smooth muscle cell contraction. However, during this process, no Ca2+ transient was observed within the subplasmalemmal nanocourses, suggesting that the regulation of both contraction and relaxation of smooth muscle cells are achieved by spatially restricted Ca2+ signals within different nanocourses. Invaginations of the nucleoplasmic reticulum in arterial myocytes form trans-nuclear networks of cytoplasmic nanospaces, generate Ca2+ signals by strategically positioned Ca2+ pumps (SERCA1) and release channels (RyR1). Within a subpopulation of nuclear invaginations, evoked Ca2+ signals via ryanodine receptors exhibited spatial and temporal separation from adjacent Ca2+ signals within a single “activated” nuclear invagination, and also from those Ca2+ signals arising within different nuclear invaginations. Moreover, nuclear invaginations provide sites for transcriptional suppression, because lamin A and/or emerin line the entire surface of their inner nuclear membranes and co-localise with nesprin-1 positive puncta. More intriguing still, a subpopulation of these nuclear invaginations harboured punctate regions of colocalisation between lamin A and the suppressive heterochromatin mark H3K9me2, while emerin-positive invaginations harboured puncta of BAF (Barrior to autointegration factor) co-localisation and thus an alternative pathway to the regulation of gene expression. I propose that nuclear invaginations form cytoplasmic nanotubes within which nano-patterning of Ca2+ signals may support stochastic modulation of transcriptional suppressors. Together, the cytoplasmic nanocourses form a cell-wide web for Ca2+ signalling and the regulation of various arterial smooth muscle functions, ranging from the regulation of blood pressure by vasodilation and vasoconstriction to gene expression.