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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, Snapshot from a TEM tomogram of a L-SR region of rat pulmonary artery smooth muscle, illustrating, among other things, a single SR extension apparently forming junctions with several lysosomes. Magnification: 62,000×.B, Same snapshot shown inA, but with a lysosome (orange) and a portion of SR (turquoise) partially traced out in 3D. These pseudocolour tracings underscore how the SR can appear as a large cistern in a given plane, but can actually branch out in different directions when viewed in 3D. Scale bars ≈ 100 nm.
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f4: A, Snapshot from a TEM tomogram of a L-SR region of rat pulmonary artery smooth muscle, illustrating, among other things, a single SR extension apparently forming junctions with several lysosomes. Magnification: 62,000×.B, Same snapshot shown inA, but with a lysosome (orange) and a portion of SR (turquoise) partially traced out in 3D. These pseudocolour tracings underscore how the SR can appear as a large cistern in a given plane, but can actually branch out in different directions when viewed in 3D. Scale bars ≈ 100 nm.

Mentions: To gather more direct information on the 3D morphology of L-SR junctions, we acquired a set of tomograms of those regions from the same sample blocks used to obtain the images inFigure 2. InFigure 4, we report snapshots from one of the tomograms; in these stills, we have also traced out parts of one lysosome and the closely apposed SR region that together form a L-SR junction. These tomograms are very helpful in clarifying the detailed morphology of L-SR junctions and informing on the spatial variability of the SR network. For example, while the SR segment shown in a single 2D tomographic scan (Figure 4A) would appear to be continuous and part of a large SR compartment, it actually branches out into narrower cisterns as revealed by 3D tomographic reconstruction (Figure 4B). Furthermore, it is interesting to observe the fact that one extension of the SR appears to couple with multiple organelles. Thus, the 3D views generated by tomography are paramount for demonstrating the presence of a true junctional complex and for the design of a prototypical L-SR environment as a software mesh object, on which we may simulate the NAADP-mediated localized Ca2+ release.


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, Snapshot from a TEM tomogram of a L-SR region of rat pulmonary artery smooth muscle, illustrating, among other things, a single SR extension apparently forming junctions with several lysosomes. Magnification: 62,000×.B, Same snapshot shown inA, but with a lysosome (orange) and a portion of SR (turquoise) partially traced out in 3D. These pseudocolour tracings underscore how the SR can appear as a large cistern in a given plane, but can actually branch out in different directions when viewed in 3D. Scale bars ≈ 100 nm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: A, Snapshot from a TEM tomogram of a L-SR region of rat pulmonary artery smooth muscle, illustrating, among other things, a single SR extension apparently forming junctions with several lysosomes. Magnification: 62,000×.B, Same snapshot shown inA, but with a lysosome (orange) and a portion of SR (turquoise) partially traced out in 3D. These pseudocolour tracings underscore how the SR can appear as a large cistern in a given plane, but can actually branch out in different directions when viewed in 3D. Scale bars ≈ 100 nm.
Mentions: To gather more direct information on the 3D morphology of L-SR junctions, we acquired a set of tomograms of those regions from the same sample blocks used to obtain the images inFigure 2. InFigure 4, we report snapshots from one of the tomograms; in these stills, we have also traced out parts of one lysosome and the closely apposed SR region that together form a L-SR junction. These tomograms are very helpful in clarifying the detailed morphology of L-SR junctions and informing on the spatial variability of the SR network. For example, while the SR segment shown in a single 2D tomographic scan (Figure 4A) would appear to be continuous and part of a large SR compartment, it actually branches out into narrower cisterns as revealed by 3D tomographic reconstruction (Figure 4B). Furthermore, it is interesting to observe the fact that one extension of the SR appears to couple with multiple organelles. Thus, the 3D views generated by tomography are paramount for demonstrating the presence of a true junctional complex and for the design of a prototypical L-SR environment as a software mesh object, on which we may simulate the NAADP-mediated localized Ca2+ release.

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