Design and Application of a Class of Sensors to Monitor Ca2+ Dynamics in High Ca2+ Concentration Cellular Compartments

Tang, Shen; Wong, Hing-Cheung; Wang, Zhongmin; Huang, Yun; Zou, Jun; Zhuo, You; Pennati, Andrea; Gadda, Giovanni; Delbono, Osvaldo; Yang, Jenny J.

doi:10.1016/j.bpj.2011.11.1718

Cited by 17 publications

(39 citation statements)

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“…Therefore, the data obtained with the old probe were necessarily an underestimation of the real value. Regarding the data obtained at 22 [19] to near 1 mM [20], with most of the obtained figures around 400-800 M [21][22][23][24][25][26][27]. On the other hand, NMR measurements [21], skeletal muscle cells [25,27] and rabbit heart cells [28] The new probe has also a very important technical and experimental advantage over the previous aequorin probes.…”

Section: Discussionmentioning

confidence: 90%

Ca2+ homeostasis in the endoplasmic reticulum measured with a new low-Ca2+-affinity targeted aequorin

et al. 2013

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a b s t r a c tWe use here a new very low-Ca 2+ -affinity targeted aequorin to measure the [Ca 2+ ] between 0.1 and 3 mM, and was only partially affected by mitochondrial membrane depolarization. Instead, it was significantly reduced by loading cells with chelators, and the fast chelator BAPTA was much more effective than the slow chelator EGTA. This suggests that local [Ca 2+ ] microdomains connecting the store operated Ca 2+ channels with the ER Ca 2+ pumps may be important during refilling.

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Section: Discussionmentioning

confidence: 90%

Ca2+ homeostasis in the endoplasmic reticulum measured with a new low-Ca2+-affinity targeted aequorin

et al. 2013

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“…1E; supplementary material Table S1), we were concerned that potentiated responses to Ins(1,4,5)P 3 might result from enhanced Ca 2+ uptake by the ER. We therefore used a novel lowaffinity Ca 2+ indicator targeted to the ER, CatchER (Tang et al, 2011), to measure directly the free [Ca 2+ ] within the ER of COS-7 cells. These are better suited than HEK cells for these studies because COS-7 cells are larger, flatter and their ER is more easily identified.…”

Section: Camentioning

confidence: 99%

Lysosomes shape Ins(1,4,5)P3-evoked Ca2+ signals by selectively sequestering Ca2+ released from the endoplasmic reticulum

López-Sanjurjo

Tovey

Prole

et al. 2013

Journal of Cell Science

123

111

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Summary Most intracellular Ca2+ signals result from opening of Ca 2+ channels in the plasma membrane or endoplasmic reticulum (ER), and they are reversed by active transport across these membranes or by shuttling Ca 2+ into mitochondria. Ca 2+ channels in lysosomes contribute to endo-lysosomal trafficking and Ca 2+ signalling, but the role of lysosomal Ca 2+ uptake in Ca 2+ signalling is unexplored. Inhibition of lysosomal Ca 2+ uptake by dissipating the H + gradient (using bafilomycin A 1 ), perforating lysosomal membranes (using glycyl-Lphenylalanine 2-naphthylamide) or lysosome fusion (using vacuolin) increased the Ca 2+ signals evoked by receptors that stimulate inositol 1,4,5-trisphosphate [Ins(1,4,5)P 3 ] formation. Bafilomycin A 1 amplified the Ca 2+ signals evoked by photolysis of caged Ins(1,4,5)P 3 or by inhibition of ER Ca 2+ pumps, and it slowed recovery from them. Ca 2+ signals evoked by store-operated Ca 2+ entry were unaffected by bafilomycin A 1 . Video-imaging with total internal reflection fluorescence microscopy revealed that lysosomes were motile and remained intimately associated with the ER. Close association of lysosomes with the ER allows them selectively to accumulate Ca 2+ released by Ins(1,4,5)P 3 receptors.

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“…2D). We next focused on the expression of GAP in the ER, where available Ca 2+ indicators pose serious limitations, namely a small dynamic range (D1ER) (27) or not being ratiometric (catchER) (14). As [Ca 2+ ] inside ER ([Ca 2+ ] ER ) is higher than [Ca 2+ ] C , we modified GAP to reduce its affinity for Ca 2+ .…”

Section: Significancementioning

confidence: 99%

“…We compared the performance of erGAP1 to that obtained with two other ER sensors, namely catchER (14) or D1ER (27), in two cell types under identical experimental conditions. In HeLa cells, stimulation with ATP + histamine produced a 14.4 ± 2.1% (n = 7) ratio change for D1ER in comparison with 58.8 ± 7.5% (n = 28) for erGAP1 (Fig.…”

Section: Significancementioning

confidence: 99%

“…They are generally composed of one or two fluorescent proteins, most of them variants of GFP, fused to a Ca 2+ -binding protein (13). Recently, a single EF-hand motif has been inserted in the GFP moiety to generate a Ca 2+ fluorescent probe (14). Since the first cameleon based on FRET (15), the number of GECIs has exponentially increased, attempting optimization of critical features such as adequate expression, signal strength, or dynamic range.…”

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GAP, an aequorin-based fluorescent indicator for imaging Ca ²⁺ in organelles

Rodrı́guez-Garcı́a

Rojo-Ruiz

Navas-Navarro

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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Genetically encoded calcium indicators allow monitoring subcellular Ca 2+ signals inside organelles. Most genetically encoded calcium indicators are fusions of endogenous calcium-binding proteins whose functionality in vivo may be perturbed by competition with cellular partners. We describe here a novel family of fluorescent Ca 2+ sensors based on the fusion of two Aequorea victoria proteins, GFP and apo-aequorin (GAP). GAP exhibited a unique combination of features: dual-excitation ratiometric imaging, high dynamic range, good signal-to-noise ratio, insensitivity to pH and Mg 2+ , tunable Ca 2+ affinity, uncomplicated calibration, and targetability to five distinct organelles. Moreover, transgenic mice for endoplasmic reticulum-targeted GAP exhibited a robust long-term expression that correlated well with its reproducible performance in various neural tissues. This biosensor fills a gap in the actual repertoire of Ca 2+ indicators for organelles and becomes a valuable tool for in vivo Ca 2+ imaging applications.calcium signalling | Golgi apparatus | hippocampus | motor neuron

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Design and Application of a Class of Sensors to Monitor Ca2+ Dynamics in High Ca2+ Concentration Cellular Compartments

Cited by 17 publications

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Ca2+ homeostasis in the endoplasmic reticulum measured with a new low-Ca2+-affinity targeted aequorin

Ca2+ homeostasis in the endoplasmic reticulum measured with a new low-Ca2+-affinity targeted aequorin

Lysosomes shape Ins(1,4,5)P3-evoked Ca2+ signals by selectively sequestering Ca2+ released from the endoplasmic reticulum

GAP, an aequorin-based fluorescent indicator for imaging Ca ²⁺ in organelles

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Design and Application of a Class of Sensors to Monitor Ca2+ Dynamics in High Ca2+ Concentration Cellular Compartments

Cited by 17 publications

References 0 publications

Ca2+ homeostasis in the endoplasmic reticulum measured with a new low-Ca2+-affinity targeted aequorin

Ca2+ homeostasis in the endoplasmic reticulum measured with a new low-Ca2+-affinity targeted aequorin

Lysosomes shape Ins(1,4,5)P3-evoked Ca2+ signals by selectively sequestering Ca2+ released from the endoplasmic reticulum

GAP, an aequorin-based fluorescent indicator for imaging Ca 2+ in organelles

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GAP, an aequorin-based fluorescent indicator for imaging Ca ²⁺ in organelles