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

Fameli N, Ogunbayo OA, van Breemen C, Evans AM - F1000Res (2014)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Anesthesiology, Pharmacology, and Therapeutics, University of British Columbia, Vancouver, V6T 1Z3, Canada ; Centre for Integrative Physiology, University of Edinburgh, Edinburgh, EH8 9XD, UK ; Current address: Institute for Biophysics, Medical University of Graz, Graz, 8010, Austria.

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.

No MeSH data available.


Related in: MedlinePlus

Upper panel shows a series of pseudocolour images of the Fura-2 fluorescence ratio (F340/F380) recorded in an isolated pulmonary artery smooth muscle cell during intracellular dialysis of 10 nM NAADP. Note the spatially localized ‘Ca2+ burst’ (time point 1). Lower panel shows the record of the Fura-2 fluorescence ratio against time corresponding to the upper panel of pseudocolours images; note the discrete shoulder in the rising phase of the F340/F380 ratio that corresponds to the initial ‘Ca2+ burst’. Scale bar: 10μm.
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f1: Upper panel shows a series of pseudocolour images of the Fura-2 fluorescence ratio (F340/F380) recorded in an isolated pulmonary artery smooth muscle cell during intracellular dialysis of 10 nM NAADP. Note the spatially localized ‘Ca2+ burst’ (time point 1). Lower panel shows the record of the Fura-2 fluorescence ratio against time corresponding to the upper panel of pseudocolours images; note the discrete shoulder in the rising phase of the F340/F380 ratio that corresponds to the initial ‘Ca2+ burst’. Scale bar: 10μm.

Mentions: The work presented in this article is based on earlier Ca2+ fluorescence imaging observations by optical microscopy, of which the data inFigure 1 is a representative example, and immunofluorescence results. These studies led to the proposal that, for the lysosomal Ca2+ release to trigger CICR, 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. The relevant background findings that stimulated the development of the work exposed here were first reported by Evans’ group10,17, and are summarized inFigure 1.


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

Fameli N, Ogunbayo OA, van Breemen C, Evans AM - F1000Res (2014)

Upper panel shows a series of pseudocolour images of the Fura-2 fluorescence ratio (F340/F380) recorded in an isolated pulmonary artery smooth muscle cell during intracellular dialysis of 10 nM NAADP. Note the spatially localized ‘Ca2+ burst’ (time point 1). Lower panel shows the record of the Fura-2 fluorescence ratio against time corresponding to the upper panel of pseudocolours images; note the discrete shoulder in the rising phase of the F340/F380 ratio that corresponds to the initial ‘Ca2+ burst’. Scale bar: 10μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4126599&req=5

f1: Upper panel shows a series of pseudocolour images of the Fura-2 fluorescence ratio (F340/F380) recorded in an isolated pulmonary artery smooth muscle cell during intracellular dialysis of 10 nM NAADP. Note the spatially localized ‘Ca2+ burst’ (time point 1). Lower panel shows the record of the Fura-2 fluorescence ratio against time corresponding to the upper panel of pseudocolours images; note the discrete shoulder in the rising phase of the F340/F380 ratio that corresponds to the initial ‘Ca2+ burst’. Scale bar: 10μm.
Mentions: The work presented in this article is based on earlier Ca2+ fluorescence imaging observations by optical microscopy, of which the data inFigure 1 is a representative example, and immunofluorescence results. These studies led to the proposal that, for the lysosomal Ca2+ release to trigger CICR, 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. The relevant background findings that stimulated the development of the work exposed here were first reported by Evans’ group10,17, and are summarized inFigure 1.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Anesthesiology, Pharmacology, and Therapeutics, University of British Columbia, Vancouver, V6T 1Z3, Canada ; Centre for Integrative Physiology, University of Edinburgh, Edinburgh, EH8 9XD, UK ; Current address: Institute for Biophysics, Medical University of Graz, Graz, 8010, Austria.

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.

No MeSH data available.


Related in: MedlinePlus