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Calcium (Ca 2+ ) exerts a pivotal role in controlling both physiological and detrimental cellular processes. This versatility is due to the existence of a cell-specific molecular Ca 2+ toolkit and its fine subcellular compartmentalization. Study of the role of Ca 2+ in cellular physiopathology greatly benefits from tools capable of quantitatively measuring its dynamic concentration ([Ca 2+ ]) simultaneously within organelles and in the cytosol to correlate localized and global [Ca 2+ ] changes. To this aim, as nucleoplasm Ca 2+ changes mirror those of the cytosol, we generated a novel nuclear-targeted version of a Föster resonance energy transfer (FRET)-based Ca 2+ probe. In particular, we modified the previously described nuclear Ca 2+ sensor, H2BD3cpv, by substituting the donor ECFP with mCerulean3, a brighter and more photostable fluorescent protein. The thorough characterization of this sensor in HeLa cells demonstrated that it significantly improved the brightness and photostability compared to the original probe, thus obtaining a probe suitable for more accurate quantitative Ca 2+ measurements. The affinity for Ca 2+ was determined in situ. Finally, we successfully applied the new probe to confirm that cytoplasmic and nucleoplasmic Ca 2+ levels were similar in both resting conditions and upon cell stimulation. Examples of simultaneous monitoring of Ca 2+ signal dynamics in different subcellular compartments in the very same cells are also presented.

Alzheimer's disease (AD) is the most frequent cause of dementia in the elderly. Few cases are familial (FAD), due to autosomal dominant mutations in presenilin-1 (PS1), presenilin-2 (PS2) or amyloid precursor protein (APP). The three proteins are involved in the generation of amyloid-beta (Aβ) peptides, providing genetic support to the hypothesis of Aβ pathogenicity. However, clinical trials focused on the Aβ pathway failed in their attempt to modify disease progression, suggesting the existence of additional pathogenic mechanisms. Ca 2+ dysregulation is a feature of cerebral aging, with an increased frequency and anticipated age of onset in several forms of neurodegeneration, including AD. Interestingly, FAD-linked PS1 and PS2 mutants alter multiple key cellular pathways, including Ca 2+ signaling. By generating novel tools for measuring Ca 2+ in living cells, and combining different approaches, we showed that FAD-linked PS2 mutants significantly alter cell Ca 2+ signaling and brain network activity, as summarized below.

Ca 2+ handling by mitochondria is implicated in energy production, shaping of cytosolic Ca 2+ rises, and determination of cell fate. It is therefore of crucial interest for researchers to directly measure mitochondrial Ca 2+ concentration [Ca 2+ ] in living cells. Synthetic fluorescent Ca 2+ indicators, providing a straightforward loading technique, represents a tempting strategy. Recently, we developed a new highly selective mitochondria-targeted Ca 2+ indicator named mt-fura-2 , obtained by coupling two triphenylphosphonium cation-containing groups to the molecular backbone of the cytosolic ratiometric Ca 2+ indicator fura-2 .The protocols we describe here cover all the significant steps that are necessary to characterize the probe and apply it to biologically relevant contexts. The procedures reported are referred to mt-fura-2 but could in principle be applied to characterize other mitochondria-targeted Ca 2+ probes . More in general, with the due modifications, this chapter can be considered as a handbook for the characterization and/or application of mitochondria-targeted chemical Ca 2+ probes .

The endoplasmic reticulum (ER) is a highly dynamic network whose shape is thought to be actively regulated by membrane resident proteins. Mutation of several such morphology regulators cause the neurological disorder Hereditary Sp astic Paraplegia (HSP), suggesting a critical role of ER shape maintenance in neuronal activity and function. Human Atlastin-1 mutations are responsible for SPG3A, the earliest onset and one of the more severe forms of dominant HSP. Atlastin has been initially identified in Drosophila as the GTPase responsible for the homotypic fusion of ER membrane. The majority of SPG3A-linked Atlastin-1 mutations map to the GTPase domain, potentially interfering with atlastin GTPase activity, and to the three-helix-bundle (3HB) domain, a region critical for homo-oligomerization. Here we have examined the in vivo effects of four pathogenetic missense mutations (two mapping to the GTPase domain and two to the 3HB domain) using two complementary approaches: CRISPR/Cas9 editing to introduce such variants in the endogenous atlastin gene and transgenesis to generate lines overexpressing atlastin carrying the same pathogenic variants. We found that all pathological mutations examined reduce atlastin activity in vivo although to different degrees of severity. Moreover, overexpression of the pathogenic variants in a wild type atlastin background does not give rise to the loss of function phenotypes expected for dominant negative mutations. These results indicate that the four pathological mutations investigated act through a loss of function mechanism., (Copyright © 2020 Montagna, Vajente, Pendin and Daga.)

Presenilin-2 (PS2) is one of the three proteins that are dominantly mutated in familial Alzheimer's disease (FAD). It forms the catalytic core of the γ-secretase complex-a function shared with its homolog presenilin-1 (PS1)-the enzyme ultimately responsible of amyloid-β (Aβ) formation. Besides its enzymatic activity, PS2 is a multifunctional protein, being specifically involved, independently of γ-secretase activity, in the modulation of several cellular processes, such as Ca 2+ signalling, mitochondrial function, inter-organelle communication, and autophagy. As for the former, evidence has accumulated that supports the involvement of PS2 at different levels, ranging from organelle Ca 2+ handling to Ca 2+ entry through plasma membrane channels. Thus FAD-linked PS2 mutations impact on multiple aspects of cell and tissue physiology, including bioenergetics and brain network excitability. In this contribution, we summarize the main findings on PS2, primarily as a modulator of Ca 2+ homeostasis, with particular emphasis on the role of its mutations in the pathogenesis of FAD. Identification of cell pathways and molecules that are specifically targeted by PS2 mutants, as well as of common targets shared with PS1 mutants, will be fundamental to disentangle the complexity of memory loss and brain degeneration that occurs in Alzheimer's disease (AD).

Drosophila melanogaster, colloquially known as the fruit fly, is one of the most commonly used model organisms in scientific research. Although the final architecture of a fly and a human differs greatly, most of the fundamental biological mechanisms and pathways controlling development and survival are conserved through evolution between the two species. For this reason, Drosophila has been productively used as a model organism for over a century, to study a diverse range of biological processes, including development, learning, behavior and aging. Ca 2+ signaling comprises complex pathways that impact on virtually every aspect of cellular physiology. Within such a complex field of study, Drosophila offers the advantages of consolidated molecular and genetic techniques, lack of genetic redundancy and a completely annotated genome since 2000. These and other characteristics provided the basis for the identification of many genes encoding Ca 2+ signaling molecules and the disclosure of conserved Ca 2+ signaling pathways. In this review, we will analyze the applications of Ca 2+ imaging in the fruit fly model, highlighting in particular their impact on the study of normal brain function and pathogenesis of neurodegenerative diseases.

The endoplasmic reticulum (ER) extends as a network of interconnected tubules and sheet-like structures in eukaryotic cells. ER tubules dynamically change their morphology and position within the cells in response to physiological stimuli and these network rearrangements depend on the microtubule (MT) cytoskeleton. Store-operated calcium entry (SOCE) relies on the repositioning of ER tubules to form specific ER-plasma membrane junctions. Indeed, the tips of polymerizing MTs are supposed to provide the anchor for ER tubules to move toward the plasma membrane, however the precise role of the cytoskeleton during SOCE has not been conclusively clarified. Here we exploit an in vivo approach involving the manipulation of MT dynamics in Drosophila melanogaster by neuronal expression of a dominant-negative variant of the MT-severing protein spastin to induce MT hyper-stabilization. We show that MT stabilization alters ER morphology, favoring an enrichment in ER sheets at the expense of tubules. Stabilizing MTs has a negative impact on the process of SOCE and results in a reduced ER Ca 2+ content, affecting the flight ability of the flies. Restoring proper MT organization by administering the MT-destabilizing drug vinblastine, chronically or acutely, rescues ER morphology, SOCE and flight ability, indicating that MT dynamics impairment is responsible for all the phenotypes observed., (Copyright © 2019 Vajente, Norante, Redolfi, Daga, Pizzo and Pendin.)

Ca 2+ handling by mitochondria is crucial for cell life and the direct measure of mitochondrial Ca 2+ concentration in living cells is of pivotal interest. Genetically-encoded indicators greatly facilitated this task, however they require demanding delivery procedures. On the other hand, existing mitochondria-targeted synthetic Ca 2+ indicators are plagued by several drawbacks, for example, non-specific localization, leakage, toxicity. Here we report the synthesis and characterization of a new fluorescent Ca 2+ sensor, named mt-fura-2, obtained by coupling two triphenylphosphonium cations to the molecular backbone of the ratiometric Ca 2+ indicator fura-2. Mt-fura-2 binds Ca 2+ with a dissociation constant of ≈1.5 μm in vitro. When loaded in different cell types as acetoxymethyl ester, the probe shows proper mitochondrial localization and accurately measures matrix [Ca 2+ ] variations, proving its superiority over available dyes. We describe the synthesis, characterization and application of mt-fura-2 to cell types where the delivery of genetically-encoded indicators is troublesome., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)