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.

Dynamic Culture of Mesenchymal Stromal/Stem Cell Spheroids and Secretion of Paracrine Factors.

In recent years, conditioned medium (CM) obtained from the culture of mesenchymal stromal/stem cells (MSCs) has been shown to effectively promote tissue repair and modulate the immune response and in different animal models, with potential for application in regenerative medicine. Using CM offers multiple advantages over the implantation of MSCs themselves: 1) simpler storage, transport, and preservation requirements, 2) avoidance of the inherent risks of cell transplantation, and 3) potential application as a ready-to-go biologic product. For these reasons, a large amount of MSCs research has focused on the characterization of the obtained CM, including soluble trophic factors and vesicles, preconditioning strategies for enhancing paracrine secretion, such as hypoxia, a three-dimensional (3D) environment, and biochemical stimuli, and potential clinical applications.

Functional Properties of Human-Derived Mesenchymal Stem Cell Spheroids: A Meta-Analysis and Systematic Review.

Mesenchymal stem cells (MSC) are adult multi-potent cells that can be isolated from many types of tissues including adipose tissue, bone marrow, and umbilical cord. They show great potential for cell therapy-based treatments, which is why they are being used in numerous clinical trials for a wide range of diseases. However, the success of placebo-controlled clinical trials has been limited, so new ways of improving the therapeutic effects of MSC are being developed, such as their assembly in a 3D conformation.

Fluoride exposure alters Ca signaling and mitochondrial function in enamel cells.

Fluoride ions are highly reactive, and their incorporation in forming dental enamel at low concentrations promotes mineralization. In contrast, excessive fluoride intake causes dental fluorosis, visually recognizable enamel defects that can increase the risk of caries. To investigate the molecular bases of dental fluorosis, we analyzed the effects of fluoride exposure in enamel cells to assess its impact on Ca signaling.

Differential regulation of Ca influx by ORAI channels mediates enamel mineralization.

Store-operated Ca entry (SOCE) channels are highly selective Ca channels activated by the endoplasmic reticulum (ER) sensors STIM1 and STIM2. Their direct interaction with the pore-forming plasma membrane ORAI proteins (ORAI1, ORAI2, and ORAI3) leads to sustained Ca fluxes that are critical for many cellular functions. Mutations in the human gene result in immunodeficiency, anhidrotic ectodermal dysplasia, and enamel defects.

Ca-Dependent Transcriptional Repressors KCNIP and Regulation of Prognosis Genes in Glioblastoma.

Glioblastomas (GBMs) are the most aggressive and lethal primary astrocytic tumors in adults, with very poor prognosis. Recurrence in GBM is attributed to glioblastoma stem-like cells (GSLCs). The behavior of the tumor, including proliferation, progression, invasion, and significant resistance to therapies, is a consequence of the self-renewing properties of the GSLCs, and their high resistance to chemotherapies have been attributed to their capacity to enter quiescence.

Quiescence status of glioblastoma stem-like cells involves remodelling of Ca signalling and mitochondrial shape.

Quiescence is a reversible cell-cycle arrest which allows cancer stem-like cells to evade killing following therapies. Here, we show that proliferating glioblastoma stem-like cells (GSLCs) can be induced and maintained in a quiescent state by lowering the extracellular pH. Through RNAseq analysis we identified Ca signalling genes differentially expressed between proliferating and quiescent GSLCs.

Altered Ca signaling in enamelopathies.

Biomineralization requires the controlled movement of ions across cell barriers to reach the sites of crystal growth. Mineral precipitation occurs in aqueous phases as fluids become supersaturated with specific ionic compositions. In the biological world, biomineralization is dominated by the presence of calcium (Ca) in crystal lattices.

Bisacodyl and its cytotoxic activity on human glioblastoma stem-like cells. Implication of inositol 1,4,5-triphosphate receptor dependent calcium signaling.

Glioblastoma is the most common malignant brain tumor. The heterogeneity at the cellular level, metabolic specificities and plasticity of the cancer cells are a challenge for glioblastoma treatment. Identification of cancer cells endowed with stem properties and able to propagate the tumor in animal xenografts has opened a new paradigm in cancer therapy.

Calcium signaling orchestrates glioblastoma development: Facts and conjunctures.

While it is a relatively rare disease, glioblastoma multiform (GBM) is one of the more deadly adult cancers. Following current interventions, the tumor is never eliminated whatever the treatment performed; whether it is radiotherapy, chemotherapy, or surgery. One hypothesis to explain this poor outcome is the "cancer stem cell" hypothesis.

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.

Differential calcium handling by the cis and trans regions of the Golgi apparatus.

High Ca2+ content in the Golgi apparatus (Go) is essential for protein processing and sorting. In addition, the Go can shape the cytosolic Ca2+ signals by releasing or sequestering Ca2+. We generated two new aequorin-based Ca2+ probes to specifically measure Ca2+ in the cis/cis-to-medial-Go (cGo) or the trans-Go (tGo).

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).

Two distinct calcium pools in the endoplasmic reticulum of HEK-293T cells.

Agonist-sensitive intracellular Ca2+ stores may be heterogeneous and exhibit distinct functional features. We have studied the properties of intracellular Ca2+ stores using targeted aequorins for selective measurements in different subcellular compartments. Both, HEK-293T [HEK (human embryonic kidney)-293 cells expressing the large T-antigen of SV40 (simian virus 40)] and HeLa cells accumulated Ca2+ into the ER (endoplasmic reticulum) to near millimolar concentrations and the IP3-generating agonists, carbachol and ATP, mobilized this Ca2+ pool.