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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

Representative electron micrographs of rat pulmonary artery SMC regions containing lysosomes (L), several SR cisterns, and including several examples of L-SR junctions (arrows). Also indicated are nuclei (N), Golgi apparatus (G), mitochondria (M), a multivesicular body (MVB) and extra-cellular space (ECS). Scale bars: 500 nm. Magnifications:A,C 80,000×,B, 60,000×,D, 70,000×.
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f2: Representative electron micrographs of rat pulmonary artery SMC regions containing lysosomes (L), several SR cisterns, and including several examples of L-SR junctions (arrows). Also indicated are nuclei (N), Golgi apparatus (G), mitochondria (M), a multivesicular body (MVB) and extra-cellular space (ECS). Scale bars: 500 nm. Magnifications:A,C 80,000×,B, 60,000×,D, 70,000×.

Mentions: To identify lysosomes, SR regions and L-SR nanojunctions, we recorded and surveyed 74 electron micrographs of rat pulmonary arterial smooth muscle taken from samples prepared as described in the Materials and methods section. The images inFigure 2 provide a set of examples. Since we were aiming to detect L-SR junctions, namely close appositions of the lysosomal and SR membranes, immuno-gold labeling of lysosomes was prohibited, since this technique compromises membranes definition by electron microscopy to the extent that we would be unable to assess junctional architecture. Instead, in images like those inFigure 2, lysosome identification was accomplished by relying on the knowledge of lysosomal ultrastructural features, which has accumulated over the past 50 years since the discovery of the lysosomes40. In standard (2D) transmission electron microscopy (TEM) images, lysosomes typically appear as elliptical structures bound by a single lipid bilayer, a feature that distinguishes them from mitochondria. Depending on the lysosomal system stage, they also tend to have a more or less uniformly electron-dense interior as compared to the surrounding cytosol41. They can be distinguished from endosomes by their larger size and darker lumen and they differ from peroxisomes, since the latter usually display a geometrically distinct and markedly darker structure called “crystalloid” in their luminal area. Moreover, it would appear that peroxisomes are found far more frequently in liver, kidney, bronchioles and odontoblasts than in other cell types40,42 (see, for example,http://www.uni-mainz.de/FB/Medizin/Anatomie/workshop/EM/EMPeroxisomE.html).


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

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

Representative electron micrographs of rat pulmonary artery SMC regions containing lysosomes (L), several SR cisterns, and including several examples of L-SR junctions (arrows). Also indicated are nuclei (N), Golgi apparatus (G), mitochondria (M), a multivesicular body (MVB) and extra-cellular space (ECS). Scale bars: 500 nm. Magnifications:A,C 80,000×,B, 60,000×,D, 70,000×.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: Representative electron micrographs of rat pulmonary artery SMC regions containing lysosomes (L), several SR cisterns, and including several examples of L-SR junctions (arrows). Also indicated are nuclei (N), Golgi apparatus (G), mitochondria (M), a multivesicular body (MVB) and extra-cellular space (ECS). Scale bars: 500 nm. Magnifications:A,C 80,000×,B, 60,000×,D, 70,000×.
Mentions: To identify lysosomes, SR regions and L-SR nanojunctions, we recorded and surveyed 74 electron micrographs of rat pulmonary arterial smooth muscle taken from samples prepared as described in the Materials and methods section. The images inFigure 2 provide a set of examples. Since we were aiming to detect L-SR junctions, namely close appositions of the lysosomal and SR membranes, immuno-gold labeling of lysosomes was prohibited, since this technique compromises membranes definition by electron microscopy to the extent that we would be unable to assess junctional architecture. Instead, in images like those inFigure 2, lysosome identification was accomplished by relying on the knowledge of lysosomal ultrastructural features, which has accumulated over the past 50 years since the discovery of the lysosomes40. In standard (2D) transmission electron microscopy (TEM) images, lysosomes typically appear as elliptical structures bound by a single lipid bilayer, a feature that distinguishes them from mitochondria. Depending on the lysosomal system stage, they also tend to have a more or less uniformly electron-dense interior as compared to the surrounding cytosol41. They can be distinguished from endosomes by their larger size and darker lumen and they differ from peroxisomes, since the latter usually display a geometrically distinct and markedly darker structure called “crystalloid” in their luminal area. Moreover, it would appear that peroxisomes are found far more frequently in liver, kidney, bronchioles and odontoblasts than in other cell types40,42 (see, for example,http://www.uni-mainz.de/FB/Medizin/Anatomie/workshop/EM/EMPeroxisomE.html).

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