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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

A, calculated nanojunctional [Ca2+] transient, [Ca2+]NJ, "measured" inside the volume of the recreated L-SR nanojunction shown inFigure 5. To show the effect of changes in the junctional geometry, we report three transients calculated using different junctional widths of 20, 50 and 100 nm.B, [Ca2+]NJ vs width of junction, concentration values are temporal averages of the transients as in panelA, calculated over an interval of 0.065 s (solid circles) and 0.046 s (empty circles); see text for explanation. The shaded area indicates the approximate threshold values for CICR at RyR3s58.
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f7: A, calculated nanojunctional [Ca2+] transient, [Ca2+]NJ, "measured" inside the volume of the recreated L-SR nanojunction shown inFigure 5. To show the effect of changes in the junctional geometry, we report three transients calculated using different junctional widths of 20, 50 and 100 nm.B, [Ca2+]NJ vs width of junction, concentration values are temporal averages of the transients as in panelA, calculated over an interval of 0.065 s (solid circles) and 0.046 s (empty circles); see text for explanation. The shaded area indicates the approximate threshold values for CICR at RyR3s58.

Mentions: A andB, 3D software reproduction of a lysosome closely apposed to a portion of SR, thereby forming an ≈ 20-nm-wide L-SR nanojunction; this rendering was inspired by a series of observations from micrographs as inFigure 2 (grey object: SR, blue object: lysosome). Included are relevant molecules traversing Brownian motion trajectories produced by the model simulations (see symbol legend below the panels).B, enlarged view of the L-SR junctional region, in which we have displayed the volume object (rust-coloured box) used to measure the [Ca2+]NJ transients like the ones reported inFigure 7. Scale bar: 100 nm. The model geometry and code files are available from the corresponding author.


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

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

A, calculated nanojunctional [Ca2+] transient, [Ca2+]NJ, "measured" inside the volume of the recreated L-SR nanojunction shown inFigure 5. To show the effect of changes in the junctional geometry, we report three transients calculated using different junctional widths of 20, 50 and 100 nm.B, [Ca2+]NJ vs width of junction, concentration values are temporal averages of the transients as in panelA, calculated over an interval of 0.065 s (solid circles) and 0.046 s (empty circles); see text for explanation. The shaded area indicates the approximate threshold values for CICR at RyR3s58.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f7: A, calculated nanojunctional [Ca2+] transient, [Ca2+]NJ, "measured" inside the volume of the recreated L-SR nanojunction shown inFigure 5. To show the effect of changes in the junctional geometry, we report three transients calculated using different junctional widths of 20, 50 and 100 nm.B, [Ca2+]NJ vs width of junction, concentration values are temporal averages of the transients as in panelA, calculated over an interval of 0.065 s (solid circles) and 0.046 s (empty circles); see text for explanation. The shaded area indicates the approximate threshold values for CICR at RyR3s58.
Mentions: A andB, 3D software reproduction of a lysosome closely apposed to a portion of SR, thereby forming an ≈ 20-nm-wide L-SR nanojunction; this rendering was inspired by a series of observations from micrographs as inFigure 2 (grey object: SR, blue object: lysosome). Included are relevant molecules traversing Brownian motion trajectories produced by the model simulations (see symbol legend below the panels).B, enlarged view of the L-SR junctional region, in which we have displayed the volume object (rust-coloured box) used to measure the [Ca2+]NJ transients like the ones reported inFigure 7. Scale bar: 100 nm. The model geometry and code files are available from the corresponding author.

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