Monitoring ER Ca by Luminescence with Low Affinity GFP-Aequorin Protein (GAP).

The endoplasmic reticulum (ER) is the main cellular reservoir of Ca, able to accumulate high amounts of calcium close to the millimolar range and to release it upon cell activation. Monitoring of Ca dynamics within the ER lumen is best achieved using genetically encoded and targeted reporters. Luminescent probes based on the photoprotein aequorin have provided significant insight to measure subcellular Ca.

Direct measurements of luminal Ca with endo-lysosomal GFP-aequorin reveal functional IP receptors.

Endo-lysosomes are considered acidic Ca stores but direct measurements of luminal Ca within them are limited. Here we report that the Ca -sensitive luminescent protein aequorin does not reconstitute with its cofactor at highly acidic pH but that a significant fraction of the probe is functional within a mildly acidic compartment when targeted to the endo-lysosomal system. We leveraged this probe (ELGA) to report Ca dynamics in this compartment.

Using Fluorescent GAP Indicators to Monitor ER Ca.

The endoplasmic reticulum (ER) is the main reservoir of Ca of the cell. Accurate and quantitative measuring of Ca dynamics within the lumen of the ER has been challenging. In the last decade a few genetically encoded Ca indicators have been developed, including a family of fluorescent Ca indicators, dubbed GFP-Aequorin Proteins (GAPs).

In vitro and in vivo calibration of low affinity genetic Ca indicators.

Calcium is a universal intracellular messenger and proper Caconcentrations ([Ca]) both in the cytosol and in the lumen of cytoplasmic organelles are essential for cell functions. Ca homeostasis is achieved by a delicate pump/leak balance both at the plasma membrane and at the endomembranes, and improper Ca levels result in malfunction and disease. Selective intraorganellar Cameasurements are best achieved by using targeted genetically encoded Ca indicators (GECIs) but to calibrate the luminal fluorescent signals into accurate [Ca] is challenging, especially in vivo, due to the difficulty to normalize and calibrate the fluorescent signal in various tissues or conditions.

Use of aequorin-based indicators for monitoring Ca in acidic organelles.

Over the last years, there is accumulating evidence that acidic organelles can accumulate and release Ca upon cell activation. Hence, reliable recording of Ca dynamics in these compartments is essential for understanding the physiopathological aspects of acidic organelles. Genetically encoded Ca indicators (GECIs) are valuable tools to monitor Ca in specific locations, although their use in acidic compartments is challenging due to the pH sensitivity of most available fluorescent GECIs.

Mitochondrial cristae-remodeling protein OPA1 in POMC neurons couples Ca homeostasis with adipose tissue lipolysis.

Appropriate cristae remodeling is a determinant of mitochondrial function and bioenergetics and thus represents a crucial process for cellular metabolic adaptations. Here, we show that mitochondrial cristae architecture and expression of the master cristae-remodeling protein OPA1 in proopiomelanocortin (POMC) neurons, which are key metabolic sensors implicated in energy balance control, is affected by fluctuations in nutrient availability. Genetic inactivation of OPA1 in POMC neurons causes dramatic alterations in cristae topology, mitochondrial Ca handling, reduction in alpha-melanocyte stimulating hormone (α-MSH) in target areas, hyperphagia, and attenuated white adipose tissue (WAT) lipolysis resulting in obesity.

Imaging of Endoplasmic Reticulum Ca in the Intact Pituitary Gland of Transgenic Mice Expressing a Low Affinity Ca Indicator.

The adenohypophysis contains five secretory cell types (somatotrophs, lactotrophs, thyrotrophs, corticotrophs, and gonadotrophs), each secreting a different hormone, and controlled by different hypothalamic releasing hormones (HRHs). Exocytic secretion is regulated by cytosolic Ca signals ([Ca]), which can be generated either by Ca entry through the plasma membrane and/or by Ca release from the endoplasmic reticulum (ER). In addition, Ca entry signals can eventually be amplified by ER release calcium-induced calcium release (CICR).

Direct monitoring of ER Ca dynamics reveals that Ca entry induces ER-Ca release in astrocytes.

Excitability in astroglia is controlled by Ca fluxes from intracellular organelles, mostly from the endoplasmic reticulum (ER). Astrocytic ER possesses inositol 1,4,5-trisphosphate receptors (InsPR) that can be activated upon stimulation through a vast number of metabotropic G-protein-coupled receptors. By contrast, the role of Ca-gated Ca release channels is less explored in astroglia.

Sarcoplasmic reticulum Ca decreases with age and correlates with the decline in muscle function in .

Sarcopenia, the loss of muscle mass and strength associated with age, has been linked to impairment of the cytosolic Ca peak that triggers muscle contraction, but mechanistic details remain unknown. Here we explore the hypothesis that a reduction in sarcoplasmic reticulum (SR) Ca concentration ([Ca]) is at the origin of this loss of Ca homeostasis. We engineered to express the Ca indicator GAP3 targeted to muscle SR, and we developed a new method to calibrate the signal into [Ca] [Ca] fell with age from ∼600 µM to 50 µM in close correlation with muscle function, which declined monotonically when [Ca] was <400 µM.

Caffeine chelates calcium in the lumen of the endoplasmic reticulum.

Cytosolic Ca signals are often amplified by massive calcium release from the endoplasmic reticulum (ER). This calcium-induced calcium release (CICR) occurs by activation of an ER Ca channel, the ryanodine receptor (RyR), which is facilitated by both cytosolic- and ER Ca levels. Caffeine sensitizes RyR to Ca and promotes ER Ca release at basal cytosolic Ca levels.

Using aequorin probes to measure Ca in intracellular organelles.

Aequorins are excellent tools for measuring intra-organellar Ca and assessing its role in physiological and pathological functions. Here we review targeting strategies to express aequorins in various organelles. We address critical topics such as probe affinity tuning as well as normalization and calibration of the signal.

Measuring Ca inside intracellular organelles with luminescent and fluorescent aequorin-based sensors.

GFP-Aequorin Protein (GAP) can be used to measure [Ca] inside intracellular organelles, both by luminescence and by fluorescence. The low-affinity variant GAP3 is adequate for ratiometric imaging in the endoplasmic reticulum and Golgi apparatus, and it can be combined with conventional synthetic indicators for simultaneous measurements of cytosolic Ca. GAP is bioorthogonal as it does not have mammalian homologues, and it is robust and functionally expressed in transgenic flies and mice, where it can be used for Ca measurements ex vivo and in vivo to explore animal models of health and disease.

GFP-Aequorin Protein Sensor for Ex Vivo and In Vivo Imaging of Ca(2+) Dynamics in High-Ca(2+) Organelles.

Proper functioning of organelles such as the ER or the Golgi apparatus requires luminal accumulation of Ca(2+) at high concentrations. Here we describe a ratiometric low-affinity Ca(2+) sensor of the GFP-aequorin protein (GAP) family optimized for measurements in high-Ca(2+) concentration environments. Transgenic animals expressing the ER-targeted sensor allowed monitoring of Ca(2+) signals inside the organelle.

A new low-Ca²⁺ affinity GAP indicator to monitor high Ca²⁺ in organelles by luminescence.

We have recently described a new class of genetically encoded Ca(2+) indicators composed of two jellyfish proteins, a variant of green fluorescent protein (GFP) and the calcium binding protein apoaequorin, named GAP (Rodriguez-García et al., 2014). GAP is a unique dual-mode Ca(2+) indicator, able to function either as a fluorescent or a luminescent probe, depending on whether the photoprotein aequorin is in its apo-state or reconstituted with its cofactor coelenterazine.

GAP, an aequorin-based fluorescent indicator for imaging Ca2+ in organelles.

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).

Leptin counteracts the hypoxia-induced inhibition of spontaneously firing hippocampal neurons: a microelectrode array study.

Besides regulating energy balance and reducing body-weight, the adipokine leptin has been recently shown to be neuroprotective and antiapoptotic by promoting neuronal survival after excitotoxic and oxidative insults. Here, we investigated the firing properties of mouse hippocampal neurons and the effects of leptin pretreatment on hypoxic damage (2 hours, 3% O(2)). Experiments were carried out by means of the microelectrode array (MEA) technology, monitoring hippocampal neurons activity from 11 to 18 days in vitro (DIV).

Nimodipine inhibits AP firing in cultured hippocampal neurons predominantly due to block of voltage-dependent potassium channels.

L-type calcium channels (LTCC) are important functional elements of hippocampal neurons contributing to processes like memory formation and gene expression. Mice lacking the Ca(V)1.2 channel in hippocampal pyramidal cells exhibited defects in spatial memory (Moosmang et al.