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Generation and characterization of a lysosomally targeted, genetically encoded Ca(2+)-sensor.

McCue HV, Wardyn JD, Burgoyne RD, Haynes LP - Biochem. J. (2013)

Bottom Line: Late endocytic pathway compartments including late-endosomes and lysosomes have recently been observed to sequester Ca2+ to levels comparable with those found within the ER lumen.Lysosomes sequester Ca2+ to a greater extent than any other endocytic compartment, and signalling from this organelle has been postulated to provide 'trigger' release events that can subsequently elicit more extensive Ca2+ signals from stores including the ER.This probe represents a novel tool that will permit detailed investigations examining the impact of lysosomal Ca2+ handling on cellular physiology.

View Article: PubMed Central - PubMed

Affiliation: The Physiological Laboratory, Department of Cellular and Molecular Physiology, Institute of Translational Medicine, University of Liverpool, Crown St, Liverpool L69 3BX, UK.

ABSTRACT
Distinct spatiotemporal Ca2+ signalling events regulate fundamental aspects of eukaryotic cell physiology. Complex Ca2+ signals can be driven by release of Ca2+ from intracellular organelles that sequester Ca2+ such as the ER (endoplasmic reticulum) or through the opening of Ca2+-permeable channels in the plasma membrane and influx of extracellular Ca2+. Late endocytic pathway compartments including late-endosomes and lysosomes have recently been observed to sequester Ca2+ to levels comparable with those found within the ER lumen. These organelles harbour ligand-gated Ca2+-release channels and evidence indicates that they can operate as Ca2+-signalling platforms. Lysosomes sequester Ca2+ to a greater extent than any other endocytic compartment, and signalling from this organelle has been postulated to provide 'trigger' release events that can subsequently elicit more extensive Ca2+ signals from stores including the ER. In order to investigate lysosomal-specific Ca2+ signalling a simple method for measuring lysosomal Ca2+ release is essential. In the present study we describe the generation and characterization of a genetically encoded, lysosomally targeted, cameleon sensor which is capable of registering specific Ca2+ release in response to extracellular agonists and intracellular second messengers. This probe represents a novel tool that will permit detailed investigations examining the impact of lysosomal Ca2+ handling on cellular physiology.

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LAMP1–YCaM FRET responses in intact HeLa cells in response to histamine, TG and bafilomycin A1(A) HeLa cells transfected with LAMP1–YCaM were perfused with LC buffer and perfusion was changed to LC buffer containing 100 μM histamine at the indicated time. FRET output from LAMP1–YCaM is calculated as the ratio of YFP/CFP fluorescence and plotted against time. This is a representative single-cell response taken from eight cells acquired over three independent experiments where oscillatory responses to histamine were observed. (B) HeLa cells transfected with LAMP1–YCaM were perfused with HC buffer to permit loading of internal stores then switched to LC buffer to permit FRET output to return to basal levels. At the indicated times, cells were perfused with 2 μM TG in LC buffer followed by 100 μM histamine in LC buffer. FRET output from LAMP1–YCaM is calculated as the ratio of YFP/CFP fluorescence and plotted against time. This is a representative single-cell response taken from a total of 14 cell responses acquired over three independent experiments. (C) HeLa cells transfected with LAMP1–YCaM were perfused in LC buffer then switched to LC buffer containing 2 μM TG at the indicated time point. The perfusion solution was subsequently switched to LC buffer containing 500 nM bafilomycin A1 (BF) and then to LC buffer containing 100 μM histamine. This trace is a representative response from a total of 17 similar cell responses acquired from three individual experiments (n=20 LAMP1–YCaM-expressing cells in total). FRET output from LAMP1–YCaM is calculated as the ratio of YFP/CFP fluorescence and plotted against time.
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Figure 4: LAMP1–YCaM FRET responses in intact HeLa cells in response to histamine, TG and bafilomycin A1(A) HeLa cells transfected with LAMP1–YCaM were perfused with LC buffer and perfusion was changed to LC buffer containing 100 μM histamine at the indicated time. FRET output from LAMP1–YCaM is calculated as the ratio of YFP/CFP fluorescence and plotted against time. This is a representative single-cell response taken from eight cells acquired over three independent experiments where oscillatory responses to histamine were observed. (B) HeLa cells transfected with LAMP1–YCaM were perfused with HC buffer to permit loading of internal stores then switched to LC buffer to permit FRET output to return to basal levels. At the indicated times, cells were perfused with 2 μM TG in LC buffer followed by 100 μM histamine in LC buffer. FRET output from LAMP1–YCaM is calculated as the ratio of YFP/CFP fluorescence and plotted against time. This is a representative single-cell response taken from a total of 14 cell responses acquired over three independent experiments. (C) HeLa cells transfected with LAMP1–YCaM were perfused in LC buffer then switched to LC buffer containing 2 μM TG at the indicated time point. The perfusion solution was subsequently switched to LC buffer containing 500 nM bafilomycin A1 (BF) and then to LC buffer containing 100 μM histamine. This trace is a representative response from a total of 17 similar cell responses acquired from three individual experiments (n=20 LAMP1–YCaM-expressing cells in total). FRET output from LAMP1–YCaM is calculated as the ratio of YFP/CFP fluorescence and plotted against time.

Mentions: We first wanted to test whether LAMP1–YCaM was capable of responding to intracellular Ca2+ fluxes generated by agonists known to mobilize Ca2+ in HeLa cells [31]. In initial studies we utilised histamine as a Ca2+-releasing extracellular agonist in intact cell experiments to assess the sensitivity of LAMP1–YCaM (Figure 4). Application of histamine elicited robust increases in FRET output from the cameleon construct, consistent with an elevation in cytosolic Ca2+ in close proximity to the lysosomal surface (Figure 4A). These responses were oscillatory in nature and the magnitude of each oscillatory FRET response decreased progressively in LC external buffer. In HeLa cells, histamine has previously been shown to liberate ER Ca2+ through generation of the second messenger IP3 [32]. There is also evidence that histamine is capable of generating NAADP directly in some cell types [33], and we probed the possible existence of such a pathway in HeLa cells in related experiments. In this protocol, cells were first perfused in HC buffer to permit loading of internal stores (HeLa cell responses were highly variable and inclusion of a loading step with HC buffer was found to maximize signal-to-noise in many of our assays). Cells were then switched back to LC buffer before perfusion with the SERCA pump inhibitor TG [34] to first deplete ER Ca2+ before perfusion with histamine (Figure 4B). TG responses were relatively slow to develop and exhibited an average FRET response that was 27.01±1.06% (mean±S.E.M.) of the maximal response induced in response to incubation with HC external buffer (n=14 cells from three independent experiments). In the same experiments we were able to observe reproducible post-TG treatment histamine-induced FRET responses that were on average 40.32±5.81% (mean±S.E.M.) of the maximal FRET signal observed in response to HC buffer application. Interestingly, these histamine responses failed to exhibit oscillatory behaviour and instead gave rise to a single short-lived Ca2+ spike. It was not possible to test the well-characterized antagonist of NAADP signalling Ned-19 [35] in these experiments as it was found to fluoresce strongly in the blue region of the visible spectra and therefore interfered with the emission spectra of the CFP tag in LAMP1–YCaM. Consistent with these observations, treatment of LAMP1–YCaM-transfected cells with TG followed by bafilomycin A1 before the application of histamine (Figure 4C) inhibited any detectable histamine-evoked FRET responses in 85% of cells imaged (n=20 cells from three independent experiments).


Generation and characterization of a lysosomally targeted, genetically encoded Ca(2+)-sensor.

McCue HV, Wardyn JD, Burgoyne RD, Haynes LP - Biochem. J. (2013)

LAMP1–YCaM FRET responses in intact HeLa cells in response to histamine, TG and bafilomycin A1(A) HeLa cells transfected with LAMP1–YCaM were perfused with LC buffer and perfusion was changed to LC buffer containing 100 μM histamine at the indicated time. FRET output from LAMP1–YCaM is calculated as the ratio of YFP/CFP fluorescence and plotted against time. This is a representative single-cell response taken from eight cells acquired over three independent experiments where oscillatory responses to histamine were observed. (B) HeLa cells transfected with LAMP1–YCaM were perfused with HC buffer to permit loading of internal stores then switched to LC buffer to permit FRET output to return to basal levels. At the indicated times, cells were perfused with 2 μM TG in LC buffer followed by 100 μM histamine in LC buffer. FRET output from LAMP1–YCaM is calculated as the ratio of YFP/CFP fluorescence and plotted against time. This is a representative single-cell response taken from a total of 14 cell responses acquired over three independent experiments. (C) HeLa cells transfected with LAMP1–YCaM were perfused in LC buffer then switched to LC buffer containing 2 μM TG at the indicated time point. The perfusion solution was subsequently switched to LC buffer containing 500 nM bafilomycin A1 (BF) and then to LC buffer containing 100 μM histamine. This trace is a representative response from a total of 17 similar cell responses acquired from three individual experiments (n=20 LAMP1–YCaM-expressing cells in total). FRET output from LAMP1–YCaM is calculated as the ratio of YFP/CFP fluorescence and plotted against time.
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Related In: Results  -  Collection

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Figure 4: LAMP1–YCaM FRET responses in intact HeLa cells in response to histamine, TG and bafilomycin A1(A) HeLa cells transfected with LAMP1–YCaM were perfused with LC buffer and perfusion was changed to LC buffer containing 100 μM histamine at the indicated time. FRET output from LAMP1–YCaM is calculated as the ratio of YFP/CFP fluorescence and plotted against time. This is a representative single-cell response taken from eight cells acquired over three independent experiments where oscillatory responses to histamine were observed. (B) HeLa cells transfected with LAMP1–YCaM were perfused with HC buffer to permit loading of internal stores then switched to LC buffer to permit FRET output to return to basal levels. At the indicated times, cells were perfused with 2 μM TG in LC buffer followed by 100 μM histamine in LC buffer. FRET output from LAMP1–YCaM is calculated as the ratio of YFP/CFP fluorescence and plotted against time. This is a representative single-cell response taken from a total of 14 cell responses acquired over three independent experiments. (C) HeLa cells transfected with LAMP1–YCaM were perfused in LC buffer then switched to LC buffer containing 2 μM TG at the indicated time point. The perfusion solution was subsequently switched to LC buffer containing 500 nM bafilomycin A1 (BF) and then to LC buffer containing 100 μM histamine. This trace is a representative response from a total of 17 similar cell responses acquired from three individual experiments (n=20 LAMP1–YCaM-expressing cells in total). FRET output from LAMP1–YCaM is calculated as the ratio of YFP/CFP fluorescence and plotted against time.
Mentions: We first wanted to test whether LAMP1–YCaM was capable of responding to intracellular Ca2+ fluxes generated by agonists known to mobilize Ca2+ in HeLa cells [31]. In initial studies we utilised histamine as a Ca2+-releasing extracellular agonist in intact cell experiments to assess the sensitivity of LAMP1–YCaM (Figure 4). Application of histamine elicited robust increases in FRET output from the cameleon construct, consistent with an elevation in cytosolic Ca2+ in close proximity to the lysosomal surface (Figure 4A). These responses were oscillatory in nature and the magnitude of each oscillatory FRET response decreased progressively in LC external buffer. In HeLa cells, histamine has previously been shown to liberate ER Ca2+ through generation of the second messenger IP3 [32]. There is also evidence that histamine is capable of generating NAADP directly in some cell types [33], and we probed the possible existence of such a pathway in HeLa cells in related experiments. In this protocol, cells were first perfused in HC buffer to permit loading of internal stores (HeLa cell responses were highly variable and inclusion of a loading step with HC buffer was found to maximize signal-to-noise in many of our assays). Cells were then switched back to LC buffer before perfusion with the SERCA pump inhibitor TG [34] to first deplete ER Ca2+ before perfusion with histamine (Figure 4B). TG responses were relatively slow to develop and exhibited an average FRET response that was 27.01±1.06% (mean±S.E.M.) of the maximal response induced in response to incubation with HC external buffer (n=14 cells from three independent experiments). In the same experiments we were able to observe reproducible post-TG treatment histamine-induced FRET responses that were on average 40.32±5.81% (mean±S.E.M.) of the maximal FRET signal observed in response to HC buffer application. Interestingly, these histamine responses failed to exhibit oscillatory behaviour and instead gave rise to a single short-lived Ca2+ spike. It was not possible to test the well-characterized antagonist of NAADP signalling Ned-19 [35] in these experiments as it was found to fluoresce strongly in the blue region of the visible spectra and therefore interfered with the emission spectra of the CFP tag in LAMP1–YCaM. Consistent with these observations, treatment of LAMP1–YCaM-transfected cells with TG followed by bafilomycin A1 before the application of histamine (Figure 4C) inhibited any detectable histamine-evoked FRET responses in 85% of cells imaged (n=20 cells from three independent experiments).

Bottom Line: Late endocytic pathway compartments including late-endosomes and lysosomes have recently been observed to sequester Ca2+ to levels comparable with those found within the ER lumen.Lysosomes sequester Ca2+ to a greater extent than any other endocytic compartment, and signalling from this organelle has been postulated to provide 'trigger' release events that can subsequently elicit more extensive Ca2+ signals from stores including the ER.This probe represents a novel tool that will permit detailed investigations examining the impact of lysosomal Ca2+ handling on cellular physiology.

View Article: PubMed Central - PubMed

Affiliation: The Physiological Laboratory, Department of Cellular and Molecular Physiology, Institute of Translational Medicine, University of Liverpool, Crown St, Liverpool L69 3BX, UK.

ABSTRACT
Distinct spatiotemporal Ca2+ signalling events regulate fundamental aspects of eukaryotic cell physiology. Complex Ca2+ signals can be driven by release of Ca2+ from intracellular organelles that sequester Ca2+ such as the ER (endoplasmic reticulum) or through the opening of Ca2+-permeable channels in the plasma membrane and influx of extracellular Ca2+. Late endocytic pathway compartments including late-endosomes and lysosomes have recently been observed to sequester Ca2+ to levels comparable with those found within the ER lumen. These organelles harbour ligand-gated Ca2+-release channels and evidence indicates that they can operate as Ca2+-signalling platforms. Lysosomes sequester Ca2+ to a greater extent than any other endocytic compartment, and signalling from this organelle has been postulated to provide 'trigger' release events that can subsequently elicit more extensive Ca2+ signals from stores including the ER. In order to investigate lysosomal-specific Ca2+ signalling a simple method for measuring lysosomal Ca2+ release is essential. In the present study we describe the generation and characterization of a genetically encoded, lysosomally targeted, cameleon sensor which is capable of registering specific Ca2+ release in response to extracellular agonists and intracellular second messengers. This probe represents a novel tool that will permit detailed investigations examining the impact of lysosomal Ca2+ handling on cellular physiology.

Show MeSH