Abstract
Herein we demonstrate how nanojunctions between lysosomes and sarcoplasmic reticulum (L-SR junctions) serve to couple lysosomal activation to regenerative, ryanodine receptor-mediated cellular Ca (2+) waves. In pulmonary artery smooth muscle cells (PASMCs) it has been proposed that nicotinic acid adenine dinucleotide phosphate (NAADP) triggers increases in cytoplasmic Ca (2+) via L-SR junctions, in a manner that requires initial Ca (2+) release from lysosomes and subsequent Ca (2+)-induced Ca (2+) release (CICR) via ryanodine receptor (RyR) subtype 3 on the SR membrane proximal to lysosomes. L-SR junction membrane separation has been estimated to be < 400 nm and thus beyond the resolution of light microscopy, which has restricted detailed investigations of the junctional coupling process. The present study utilizes standard and tomographic transmission electron microscopy to provide a thorough ultrastructural characterization of the L-SR junctions in PASMCs. We show that L-SR nanojunctions are prominent features within these cells and estimate that the junctional membrane separation and extension are about 15 nm and 300 nm, respectively. Furthermore, we develop a quantitative model of the L-SR junction using these measurements, prior kinetic and specific Ca (2+) signal information as input data. Simulations of NAADP-dependent junctional Ca (2+) transients demonstrate that the magnitude of these signals can breach the threshold for CICR via RyR3. By correlation analysis of live cell Ca (2+) signals and simulated Ca (2+) transients within L-SR junctions, we estimate that "trigger zones" comprising 60-100 junctions are required to confer a signal of similar magnitude. This is compatible with the 110 lysosomes/cell estimated from our ultrastructural observations. Most importantly, our model shows that increasing the L-SR junctional width above 50 nm lowers the magnitude of junctional [Ca (2+)] such that there is a failure to breach the threshold for CICR via RyR3. L-SR junctions are therefore a pre-requisite for efficient Ca (2+)signal coupling and may contribute to cellular function in health and disease.
Highlights
The importance of cytoplasmic nanojunctions to cellular signaling and to the modulation of cell function was recognised several decades ago1,2, the extent to which cellular nanospaces may contribute to the regulation of cell function received little attention
nicotinic acid adenine dinucleotide phosphate (NAADP)-dependent Ca2+ bursts primarily arise from lysosomes in the perinuclear region of pulmonary artery smooth muscle cells (PASMCs) and appear to promote rapid, local Ca2+ transients that are of sufficient size to activate clusters of sarcoplasmic reticulum (SR) resident ryanodine receptor subtype 3 (RyR3) and initiate, in an all-or-none manner, a propagating global Ca2+ wave10,11
These studies led to the proposal that, for the lysosomal Ca2+ release to trigger Ca2+induced Ca2+ release (CICR), Lysosomes and sarcoplasmic reticulum (L-SR) nanojunctions are required and that they consist of apposing patches of lysosomal and SR membranes separated by a narrow space of nano-scale dimension10,17
Summary
The importance of cytoplasmic nanojunctions to cellular signaling and to the modulation of cell function was recognised several decades ago, the extent to which cellular nanospaces may contribute to the regulation of cell function received little attention. There is a growing recognition of the widespread occurrence and functional significance of cytoplasmic nanospaces in cells from species across several kingdoms. There is a growing recognition of the widespread occurrence and functional significance of cytoplasmic nanospaces in cells from species across several kingdoms3–9 In this respect, membrane-membrane junctions between lysosomes and the sarcoplasmic reticulum (L-SR junctions) are of particular interest; not least because they have been hypothesized to couple calcium signaling between these two organelles. That L-SR junctions may play an important role in cellular Ca2+ signaling was uncovered through early studies on the Ca2+ mobilizing messenger nicotinic acid adenine dinucleotide phosphate (NAADP), which demonstrated that NAADP released Ca2+ from a store other than the sarco/endoplasmic reticulum (S/ER), that could trigger further Ca2+ release from the S/ER by Ca2+induced Ca2+ release (CICR). NAADP-dependent Ca2+ bursts primarily arise from lysosomes in the perinuclear region of PASMCs and appear to promote rapid, local Ca2+ transients that are of sufficient size to activate clusters of SR resident ryanodine receptor subtype 3 (RyR3) and initiate, in an all-or-none manner, a propagating global Ca2+ wave
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