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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, open probabilityPo of the TPC2-dependent Ca2+ conductance reproduced from a quadratic fit to the data reported in the literature45.B, Ca2+ release rate calculated as explained in the text, based on thePo inA.
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f6: A, open probabilityPo of the TPC2-dependent Ca2+ conductance reproduced from a quadratic fit to the data reported in the literature45.B, Ca2+ release rate calculated as explained in the text, based on thePo inA.

Mentions: A recent electrophysiological study of the Ca2+ conductance of TPC2 signaling complex provides valuable information regarding its biophysical properties and, importantly, highlights the fact that, for a given relatively low activating concentration of NAADP (10 nM, as inFigure 1), the channel open probability appears to depend on [Ca2+]lys (this is likely due to a partial neutralization of the electrochemical potential across the lysosomal membrane)45. We used the Ca2+ conductivity measured in that study and values of the lysosome membrane potential49 to calculate the maximal Ca2+ current,Imax, as 2.4 × 106 ions/s. We then applied a weighted quadratic fit to the channel’s open probability (Po) as a function of the reported [Ca2+]lys data with constraints that thePo would tend to zero at [Ca2+]lys = 80µM, based on the observation that below [Ca2+]lys = 100µM essentially no single channel openings were observed45. Another constraint for the fit was that the curve be within the standard deviation value ofPo at the highest reported [Ca2+]lys = 1 mM. From the quadratic fit, we then obtained our ownPo-vs-[Ca2+]lys table (plotted inFigure 6A) and assumed that at the beginning of Ca2+ release, the Ca2+ current would bePo,max ×Imax at the maximal luminal concentration until the luminal concentration decreased to the next point in thePo-vs-[Ca2+]lys relationship. At this point, the release rate decreases to a new (lower)Po ×Imax until the luminal concentration reaches the next lower point in the table, and so on untilPo = 0 at [Ca2+]lys = 80µM. The Ca2+ release rate as a function of time obtained in this manner is shown inFigure 6B.


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, open probabilityPo of the TPC2-dependent Ca2+ conductance reproduced from a quadratic fit to the data reported in the literature45.B, Ca2+ release rate calculated as explained in the text, based on thePo inA.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f6: A, open probabilityPo of the TPC2-dependent Ca2+ conductance reproduced from a quadratic fit to the data reported in the literature45.B, Ca2+ release rate calculated as explained in the text, based on thePo inA.
Mentions: A recent electrophysiological study of the Ca2+ conductance of TPC2 signaling complex provides valuable information regarding its biophysical properties and, importantly, highlights the fact that, for a given relatively low activating concentration of NAADP (10 nM, as inFigure 1), the channel open probability appears to depend on [Ca2+]lys (this is likely due to a partial neutralization of the electrochemical potential across the lysosomal membrane)45. We used the Ca2+ conductivity measured in that study and values of the lysosome membrane potential49 to calculate the maximal Ca2+ current,Imax, as 2.4 × 106 ions/s. We then applied a weighted quadratic fit to the channel’s open probability (Po) as a function of the reported [Ca2+]lys data with constraints that thePo would tend to zero at [Ca2+]lys = 80µM, based on the observation that below [Ca2+]lys = 100µM essentially no single channel openings were observed45. Another constraint for the fit was that the curve be within the standard deviation value ofPo at the highest reported [Ca2+]lys = 1 mM. From the quadratic fit, we then obtained our ownPo-vs-[Ca2+]lys table (plotted inFigure 6A) and assumed that at the beginning of Ca2+ release, the Ca2+ current would bePo,max ×Imax at the maximal luminal concentration until the luminal concentration decreased to the next point in thePo-vs-[Ca2+]lys relationship. At this point, the release rate decreases to a new (lower)Po ×Imax until the luminal concentration reaches the next lower point in the table, and so on untilPo = 0 at [Ca2+]lys = 80µM. The Ca2+ release rate as a function of time obtained in this manner is shown inFigure 6B.

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