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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, High magnification (150,000×) electron micrograph of a region ofFigure 2B containing 3 L-SR junctions (arrows); coloured tracings as shown were used to measure lysosome dimensions, L-SR widths and extensions. Scale bar: 100 nm.B–D, Histograms showing distribution of several relevant lysosomal and L-SR junctional parameters, used to characterize the junctions and inform the quantitative model.B, lysosomal dimensions as major and minor axes of oval shapes in micrographs;C, L-SR junctional width;D, percentage apposition between junctional SR and lysosome perimeter as projected in 2D micrographs.
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f3: A, High magnification (150,000×) electron micrograph of a region ofFigure 2B containing 3 L-SR junctions (arrows); coloured tracings as shown were used to measure lysosome dimensions, L-SR widths and extensions. Scale bar: 100 nm.B–D, Histograms showing distribution of several relevant lysosomal and L-SR junctional parameters, used to characterize the junctions and inform the quantitative model.B, lysosomal dimensions as major and minor axes of oval shapes in micrographs;C, L-SR junctional width;D, percentage apposition between junctional SR and lysosome perimeter as projected in 2D micrographs.

Mentions: The SR and lysosomal membranes were outlined, highlighted and measured in pixels using the Inkscape software program (open source, available athttp://www.inkscape.org), converting the pixel measurements to nm using the scale bar in the recorded micrographs. By modifying the Inkscape script for measuring lengths, we were able to output the measurements directly into a text file, which we used to produce the histograms inFigure 3B–D. We used the Gnuplot program (open source, available athttp://www.gnuplot.info) to produce the histograms and plots presented herein.


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, High magnification (150,000×) electron micrograph of a region ofFigure 2B containing 3 L-SR junctions (arrows); coloured tracings as shown were used to measure lysosome dimensions, L-SR widths and extensions. Scale bar: 100 nm.B–D, Histograms showing distribution of several relevant lysosomal and L-SR junctional parameters, used to characterize the junctions and inform the quantitative model.B, lysosomal dimensions as major and minor axes of oval shapes in micrographs;C, L-SR junctional width;D, percentage apposition between junctional SR and lysosome perimeter as projected in 2D micrographs.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: A, High magnification (150,000×) electron micrograph of a region ofFigure 2B containing 3 L-SR junctions (arrows); coloured tracings as shown were used to measure lysosome dimensions, L-SR widths and extensions. Scale bar: 100 nm.B–D, Histograms showing distribution of several relevant lysosomal and L-SR junctional parameters, used to characterize the junctions and inform the quantitative model.B, lysosomal dimensions as major and minor axes of oval shapes in micrographs;C, L-SR junctional width;D, percentage apposition between junctional SR and lysosome perimeter as projected in 2D micrographs.
Mentions: The SR and lysosomal membranes were outlined, highlighted and measured in pixels using the Inkscape software program (open source, available athttp://www.inkscape.org), converting the pixel measurements to nm using the scale bar in the recorded micrographs. By modifying the Inkscape script for measuring lengths, we were able to output the measurements directly into a text file, which we used to produce the histograms inFigure 3B–D. We used the Gnuplot program (open source, available athttp://www.gnuplot.info) to produce the histograms and plots presented herein.

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