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Cytoplasmic nanojunctions between lysosomes and sarcoplasmic reticulum are required for specific calcium signaling.

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.

SUBMITTER: Fameli N

PROVIDER: S-EPMC4126599 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Cytoplasmic nanojunctions between lysosomes and sarcoplasmic reticulum are required for specific calcium signaling.

Fameli Nicola N Ogunbayo Oluseye A OA van Breemen Cornelis C Evans A Mark AM

F1000Research 20140422

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 ...[more]

PMID: 25126414

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Project description:Although nitric oxide (NO) signaling modulates cardiac function and excitation-contraction coupling, opposing results because of inconsistent experimental conditions, particularly with respect to temperature, confound the ability to elucidate NO signaling pathways. Here, we show that temperature significantly modulates NO effects.To test the hypothesis that temperature profoundly affects nitroso-redox equilibrium, thereby affecting sarcoplasmic reticulum (SR) calcium (Ca(2+)) leak.We measured SR Ca(2+) leak in cardiomyocytes from wild-type (WT), NO/redox imbalance (neuronal nitric oxide synthase-deficient mice-1 [NOS1(-/-)]), and hyper S-nitrosoglutathione reductase-deficient (GSNOR(-/-)) mice. In WT cardiomyocytes, SR Ca(2+) leak increased because temperature decreased from 37°C to 23°C, whereas in NOS1(-/-) cells, the leak suddenly increased when the temperature surpassed 30°C. GSNOR(-/-) cardiomyocytes exhibited low leak throughout the temperature range. Exogenously added NO had a biphasic effect on NOS1(-/-) cardiomyocytes; reducing leak at 37°C but increasing it at subphysiological temperatures. Oxypurinol and Tempol diminished the leak in NOS1(-/-) cardiomyocytes. Cooling from 37°C to 23°C increased reactive oxygen species generation in WT but decreased it in NOS1(-/-) cardiomyocytes. Oxypurinol further reduced reactive oxygen species generation. At 23°C in WT cells, leak was decreased by tetrahydrobiopterin, an essential NOS cofactor. Cooling significantly increased SR Ca(2+) content in NOS1(-/-) cells but had no effect in WT or GSNOR(-/-).Ca(2+) leak and temperature are normally inversely proportional, whereas NOS1 deficiency reverses this effect, increasing leak and elevating reactive oxygen species production because temperature increases. Reduced denitrosylation (GSNOR deficiency) eliminates the temperature dependence of leak. Thus, temperature regulates the balance between NO and reactive oxygen species which in turn has a major effect on SR Ca(2+).

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Cytoplasmic nanojunctions between lysosomes and sarcoplasmic reticulum are required for specific calcium signaling.

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Cytoplasmic nanojunctions between lysosomes and sarcoplasmic reticulum are required for specific calcium signaling.

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