The cell-wide web coordinates cellular processes by directing site-specific Ca2+ flux across cytoplasmic nanocourses

Abstract

Ca 2+ coordinates diverse cellular processes, yet how function-specific signals arise is enigmatic. We describe a cell-wide network of distinct cytoplasmic nanocourses with the nucleus at its centre, demarcated by sarcoplasmic reticulum (SR) junctions (≤400 nm across) that restrict Ca 2+ diffusion and by nanocourse-specific Ca 2+ -pumps that facilitate signal segregation. Ryanodine receptor subtype 1 (RyR1) supports relaxation of arterial myocytes by unloading Ca 2+ into peripheral nanocourses delimited by plasmalemma-SR junctions, fed by sarco/endoplasmic reticulum Ca 2+ ATPase 2b (SERCA2b). Conversely, stimulus-specified increases in Ca 2+ flux through RyR2/3 clusters selects for rapid propagation of Ca 2+ signals throughout deeper extraperinuclear nanocourses and thus myocyte contraction. Nuclear envelope invaginations incorporating SERCA1 in their outer nuclear membranes demarcate further diverse networks of cytoplasmic nanocourses that receive Ca 2+ signals through discrete RyR1 clusters, impacting gene expression through epigenetic marks segregated by their associated invaginations. Critically, this circuit is not hardwired and remodels for different outputs during cell proliferation.

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“…Supporting the view that the wider network of cytoplasmic nanocourses may represent a circuit for cell-wide communication, it was evident that a subpopulation of LysoTracker Red labelled endolysosomes migrated through this network of cytoplasmic nanocourses, while, consistent with our previous observations [ 108 , 116 ], a larger, static cluster was evident in the perinuclear region of these cells [ 144 ]. By contrast, all MitoTracker Red labelled mitochondria formed static clusters in acutely isolated cells [ 144 ], as reported previously by others [ 191 ], which sit within the nanocourse network where they too likely form nanojunctions with the SR [ 192 ].…”

“…Site- and function-specific calcium signalling was confirmed using a membrane-traversing peptide from scorpion venom that selectively activates RyR1, namely maurocalcine [ 193 ]. Consistent with the distribution of RyR1s, maurocalcine preferentially increased calcium flux into subplasmalemmal and nuclear nanocourses ( Figure 14 [ 144 ]). Maurocalcine also evoked concomitant myocyte relaxation, confirming our previous proposal that RyR1s might coordinate vasodilation [ 25 , 103 ].…”

“…They demonstrate that junctional membrane complexes, variations in RyR subtype, and variations in the spatial distribution of RyR clusters contribute to regional variations in the pattern of calcium flux from local release sites in smooth muscles. Accordingly, distances of separation between hotspots for subplasmalemmal (~350 nm) and nuclear nanocourses (350 nm) [ 144 ] are consistent with those for skeletal muscle RyR1s [ 166 ], while distances of separation for extra-perinuclear (~400 nm) and perinuclear (~450 nm) nanocourses are significantly greater [ 144 ] and closer to those reported for cardiomyocyte RyR2s (0.6–0.8 µm) [ 115 ]. This is significant, because these distances of separation are entirely consistent with the regional distribution of RyR1 and RyR2 in pulmonary arterial myocytes previously reported by my laboratory and others [ 103 , 108 , 137 ].…”

“…I asked Jill if she’d retained the nuclear fraction removed from the homogenates used for her previous Western blots. Like all good scientists she had, and when this fraction was run it revealed a very faint band for SERCA1 [ 144 ].…”

“…In the meantime, a new Nikon A1R+ confocal microscope had been installed in our imaging facility (IMPACT) at Edinburgh, which was critical to our further investigations due to the much lower signal-to-noise and greater spatial resolution afforded by the Galvano scanner supplied with this system (for details see [ 144 ]). Jorge and Duan spent the first 2–3 years focused on studying calcium signals within raw images of nuclear nanocourses.…”