![[Ca 2+ ]lys and lysosomal pH: resting levels and effects of variable extracellular calcium and bafilomycin A1. (A) [Ca 2+ ]lys. Black bars represent ratiometric measurements using a probe cocktail consisting of furaDx, FDx, and OGDx. The gray bar represents calcium measurements using fluorescence lifetime imaging with the calcium indicator OGBDx. The fluorescence ratio of furaDx was ≤Rmin for cells incubated in 10 mM EGTA, therefore [Ca 2+ ]lys was below the detection limit for furaDx at pH 4 (the bar represents the detection limit of furaDx at pH 4). Error bars represent s.e.m. (n≥10 cells). (B) Lysosomal pH. Error bars represent s.e.m. (n≥10 cells).](https://www.researchgate.net/profile/Kenneth-Christensen-3/publication/11499814/figure/fig2/AS:339538430971905@1457963678050/Ca-2-lys-and-lysosomal-pH-resting-levels-and-effects-of-variable-extracellular_Q320.jpg)
![Data from a time-course experiment showing the effects of NH4Cl on macrophage [Ca 2+ ]cyt and [Ca 2+ ]lys. (A) [Ca 2+ ]cyt in activated macrophages () following addition of NH4Cl and control cells (; activated macrophages without addition of NH4Cl). (B) [Ca 2+ ]lys () and lysosomal pH () in activated macrophages following addition of NH4Cl. (C) [Ca 2+ ]lys () and lysosomal pH () in control cells (no NH4Cl added). To measure [Ca 2+ ]cyt, cells were loaded with FFP-18AM. To measure [Ca 2+ ]lys, cells were loaded with a probe cocktail consisting of furaDx, FDx, and OGDx. Arrows show times of addition of ammonium chloride. Error bars represent s.e.m. (n=30 cells for [Ca 2+ ]cyt measurements and n≥15 cells for [Ca 2+ ]lys measurements).](https://www.researchgate.net/profile/Kenneth-Christensen-3/publication/11499814/figure/fig3/AS:339538430971906@1457963678072/Data-from-a-time-course-experiment-showing-the-effects-of-NH4Cl-on-macrophage-Ca-2-cyt_Q320.jpg)
Content uploaded by Kenneth A Christensen
Author content
All content in this area was uploaded by Kenneth A Christensen
Content may be subject to copyright.
A preview of the PDF is not available
PH-dependent regulation of lysosomal calcium in macrophages
Abstract and Figures
Calcium measurements in acidic vacuolar compartments of living cells are few, primarily because calibration of fluorescent probes for calcium requires knowledge of pH and the pH-dependence of the probe calcium-binding affinities. Here we report pH-corrected measurements of free calcium concentrations in lysosomes of mouse macrophages, using both ratiometric and time-resolved fluorescence microscopy of probes for pH and calcium. Average free calcium concentration in macrophage lysosomes was 4-6x10(-4) M, less than half of the extracellular calcium concentration, but much higher than cytosolic calcium levels. Incubating cells in varying extracellular calcium concentrations did not alter lysosomal pH, and had only a modest effect on lysosomal calcium concentrations, indicating that endocytosis of extracellular fluid provided a small but measurable contribution to lysosomal calcium concentrations. By contrast, increases in lysosomal pH, mediated by either bafilomycin A(1) or ammonium chloride, decreased lysosomal calcium concentrations by several orders of magnitude. Re-acidification of the lysosomes allowed rapid recovery of lysosomal calcium concentrations to higher concentrations. pH-dependent reductions of lysosomal calcium concentrations appeared to result from calcium movement out of lysosomes into cytoplasm, since increases in cytosolic calcium levels could be detected upon lysosome alkalinization. These studies indicate that lysosomal calcium concentration is high and is maintained in part by the proton gradient across lysosomal membranes. Moreover, lysosomes could provide an intracellular source for physiological increases in cytosolic calcium levels.
Figures - uploaded by Kenneth A Christensen
Author content
All figure content in this area was uploaded by Kenneth A Christensen
Content may be subject to copyright.
... These findings suggest that NaW increases lysosomal pH and the glucose transport rate by changing TMEM175 expression. Because lysosomes are acidic Ca 2+ reservoirs (Christensen et al. 2002), an increase in lysosomal pH increases triggers the release of lysosomal Ca 2+ into the cytoplasm and activate macrophages (Chen et al. 2018;Li et al. 2018). NaW treatment increased the M2 macrophage Ca 2+ level by increasing the lysosomal pH (Fig. 4K). ...
... Notably, this degradation process depends on the acidic pH of the lysosomal lumen. Previous reports have shown that the lysosome is an acidic reservoir of Ca 2+ (Christensen et al. 2002;Morgan et al. 2011). NaW elevates the lysosomal pH and is a key factor that releases Ca 2+ from these acidic reservoirs and promotes the translocation of TFEB, a cytoplasmic transcription factor. ...
Background
Macrophages play a crucial role in the development of cardiac fibrosis (CF). Although our previous studies have shown that glycogen metabolism plays an important role in macrophage inflammatory phenotype, the role and mechanism of modifying macrophage phenotype by regulating glycogen metabolism and thereby improving CF have not been reported.
Methods
Here, we took glycogen synthetase kinase 3β (GSK3β) as the target and used its inhibitor NaW to enhance macrophage glycogen metabolism, transform M2 phenotype into anti-fibrotic M1 phenotype, inhibit fibroblast activation into myofibroblasts, and ultimately achieve the purpose of CF treatment.
Results
NaW increases the pH of macrophage lysosome through transmembrane protein 175 (TMEM175) and caused the release of Ca ²⁺ through the lysosomal Ca ²⁺ channel mucolipin-2 (Mcoln2). At the same time, the released Ca ²⁺ activates TFEB, which promotes glucose uptake by M2 and further enhances glycogen metabolism. NaW transforms the M2 phenotype into the anti-fibrotic M1 phenotype, inhibits fibroblasts from activating myofibroblasts, and ultimately achieves the purpose of treating CF.
Conclusion
Our data indicate the possibility of modifying macrophage phenotype by regulating macrophage glycogen metabolism, suggesting a potential macrophage-based immunotherapy against CF.
Graphical Abstract
... Lysosomes are the second largest Ca 2+ store inside cells. The Ca 2+ concentration in the lysosomal lumen is approximately 0.5 mM [43], which is hundreds to thousands of times that of the cytoplasm. Since ChR2 permeates calcium [10], activation of lyso-ChR2 can mediate the release of lysosomal Ca 2+ . ...
... The addition of bafilomycin A1, a V-ATPase inhibitor, effectively prevents this pH reduction (Fig 2j), suggesting that lyso-ChR2 reduces lysosomal pH by activating V-ATPase. The H + gradient across the lysosomal membrane has long been recognized as a crucial driving force for lysosomal Ca 2+ uptake [43,44]. A recent study identified TMEM165 as the protein responsible for mediating this pHdependent lysosomal Ca 2+ uptake [45]. ...
Lysosomes are degradation centers of cells and intracellular hubs of signal transduction, nutrient sensing, and autophagy regulation. Dysfunction of lysosomes contributes to a variety of diseases, such as lysosomal storage diseases (LSDs) and neurodegeneration, but the mechanisms are not well understood. Altering lysosomal activity and examining its impact on the occurrence and development of disease is an important strategy for studying lysosome-related diseases. However, methods to dynamically regulate lysosomal function in living cells or animals are still lacking. Here, we constructed lysosome-localized optogenetic actuators, named lyso-NpHR3.0, lyso-ArchT, and lyso-ChR2, to achieve optogenetic manipulation of lysosomes. These new actuators enable light-dependent control of lysosomal membrane potential, pH, hydrolase activity, degradation, and Ca ²⁺ dynamics in living cells. Notably, lyso-ChR2 activation induces autophagy through the mTOR pathway, promotes Aβ clearance in an autophagy-dependent manner in cellular models, and alleviates Aβ-induced paralysis in the Caenorhabditis elegans model of Alzheimer’s disease. Our lysosomal optogenetic actuators supplement the optogenetic toolbox and provide a method to dynamically regulate lysosomal physiology and function in living cells and animals.
... Although the concentration of calcium reported to reside within an acidic store is high, 400-600 µM in macrophages [29], the proportion of acidic stores contributing to a given signal and the time course of this contribution is unknown. Immunostaining of lysosomes using LAMP2 antibody was conducted on fixed NRAMs to confirm the presence of lysosomes in neonatal cultured atrial cardiomyocytes used for all FRET experiments (Fig. 4a). ...
In the heart, endogenous nicotinic acid adenine dinucleotide phosphate (NAADP) triggers lysosomal calcium release to augment sarcoplasmic reticulum (SR) calcium sequestration, producing larger calcium transients. However, the role of lysosomal calcium signals in pacemaker activity, a distinct calcium-operated function of the sino-atrial node (SAN) or atria, a distinct calcium-operated function, has not been investigated. Pharmacological or genetic ablation of the NAADP pathway inhibits spontaneous beating rate response to β-adrenergic stimulation in intact SAN. We found intracellular signalling microdomains between lysosomes and neighbouring SR or mitochondria in mouse, rabbit, goat, and human atrial tissue. The spatial relationship between lysosomes and other calcium-handling organelles are altered in goat and human atrial fibrillation. Furthermore, we demonstrate atrial myocytes produce 3′–5′-cyclic adenosine monophosphate in response to lysosomal signalling, adding a novel trigger for cyclic nucleotide signalling. Our findings support the hypothesis that lysosomal calcium signaling directly increases cardiomyocyte cAMP and modulates pacemaker activity.
... Channels and transporters delicately manage the influx and efflux of Ca 2+ , finely tuning essential cellular activities such as autophagy, apoptosis, and gene regulation [57]. The lysosomal Ca 2+ concentration ([Ca 2+ ] lumen ) is around 500 µM, roughly 5000 times higher than the cytosolic concentration [51], making it the second-largest store of Ca 2+ within the mammalian cell. However, the mechanisms of Ca 2+ influx and maintenance of high Ca 2+ concentration in lysosomes remain unclear. ...
Lysosomes are highly dynamic organelles that maintain cellular homeostasis and regulate fundamental cellular processes by integrating multiple metabolic pathways. Lysosomal ion channels such as TRPML1-3, TPC1/2, ClC6/7, CLN7, and TMEM175 mediate the flux of Ca2+, Cl−, Na+, H+, and K+ across lysosomal membranes in response to osmotic stimulus, nutrient-dependent signals, and cellular stresses. These ion channels serve as the crucial transducers of cell signals and are essential for the regulation of lysosomal biogenesis, motility, membrane contact site formation, and lysosomal homeostasis. In terms of pathophysiology, genetic variations in these channel genes have been associated with the development of lysosomal storage diseases, neurodegenerative diseases, inflammation, and cancer. This review aims to discuss the current understanding of the role of these ion channels in the central nervous system and to assess their potential as drug targets.
... Although the concentration of calcium reported to reside within an acidic store is high, 400-600 µM in macrophages [29], the proportion of acidic stores contributing to a given signal and the time course of this contribution is unknown. Immunostaining of lysosomes using LAMP2 antibody was conducted on fixed NRAMs to confirm the presence of lysosomes in neonatal cultured atrial cardiomyocytes used for all FRET experiments (Fig. 4a). ...
In the heart, endogenous nicotinic acid adenine dinucleotide phosphate (NAADP) triggers lysosomal calcium release to augment sarcoplasmic reticulum (SR) calcium sequestration, producing larger calcium transients. However, the role of lysosomal calcium signals in pacemaker activity, a distinct calcium-operated function of the sino-atrial node (SAN) or atria, a distinct calcium-operated function, has not been investigated. Pharmacological or genetic ablation of the NAADP pathway inhibits spontaneous beating rate response to beta-adrenergic stimulation in intact SAN. We found intracellular signalling microdomains between lysosomes and neighbouring SR or mitochondria in mouse, rabbit, goat, and human atrial tissue. The spatial relationship between lysosomes and other calcium-handling organelles are altered in goat and human atrial fibrillation. Furthermore, we demonstrate atrial myocytes produce 3'-5'-cyclic adenosine monophosphate in response to lysosomal signalling, adding a novel trigger for cyclic nucleotide signalling. Our findings support the hypothesis that lysosomal calcium signaling directly increases cardiomyocyte cAMP and modulates pacemaker activity.
... This article contains supporting information (70)(71)(72)(73)(74)(75)(76)(77)(78). ...
Together with its β-subunit OSTM1, ClC-7 performs 2Cl−/H+ exchange across lysosomal membranes. Pathogenic variants in either gene cause lysosome-related pathologies, including osteopetrosis, lysosomal storage, and pigmentation defects. CLCN7 variants can cause recessive or dominant disease. Different variants entail different sets of symptoms. Loss of ClC-7 causes osteopetrosis and mostly neuronal lysosomal storage. A recently reported de novo CLCN7 mutation (p.Tyr715Cys) causes widespread severe lysosome pathology and hypopigmentation (‘HOD syndrome’), but no osteopetrosis. We now describe two additional HOD individuals with the previously described p.Tyr715Cys and a novel p.Lys285Thr mutation, respectively. Both mutations decreased ClC-7 inhibition by PI(3,5)P2 and affected residues lining its binding pocket, and shifted voltage-dependent gating to less positive potentials, an effect partially conferred to WT subunits in WT/mutant heteromers. This shift predicts augmented pH gradient-driven Cl− uptake into vesicles. Overexpressing either mutant induced large lysosome-related vacuoles. This effect depended on Cl−/H+-exchange, as shown using mutants carrying uncoupling mutations. Fibroblasts from the p.Y715C patient also displayed giant vacuoles. This was not observed with p.K285T fibroblasts probably due to some ClC-7K285T-retained PI(3,5)P2 sensitivity. The gain of function caused by the shifted voltage-dependence of either mutant likely is the main cause of their pathogenicity. Their loss of PI(3,5)P2 inhibition will further increase currents, but may not be a general feature of HOD. Overactivity of ClC-7 induces pathologically enlarged vacuoles in many tissues, which is distinct from lysosomal storage observed with the loss of ClC-7 function. Osteopetrosis results from a loss of ClC-7, but osteoclasts remain resilient to increased ClC-7 activity.
... An increase in calcium ion levels prompts the release of calcium from lysosomes, subsequently triggering lysosome biogenesis and activating autophagyrelated genes 38 . The precise mechanisms governing the regulation of calcium levels and pH within lysosomes have been challenging to confirm 39 . Nevertheless, when TASL was knocked out, proper acidification within lysosomes did not occur despite the increases in extracellular calcium levels. ...
Maintaining epidermal homeostasis relies on a tightly organized process of proliferation and differentiation of keratinocytes. While past studies have primarily focused on calcium regulation in keratinocyte differentiation, recent research has shed light on the crucial role of lysosome dysfunction in this process. TLR adaptor interacting with SLC15A4 on the lysosome (TASL) plays a role in regulating pH within the endo-lysosome. However, the specific role of TASL in keratinocyte differentiation and its potential impact on proliferation remains elusive. In our study, we discovered that TASL deficiency hinders the proliferation and migration of keratinocytes by inducing G1/S cell cycle arrest. Also, TASL deficiency disrupts proper differentiation process in TASL knockout human keratinocyte cell line (HaCaT) by affecting lysosomal function. Additionally, our research into calcium-induced differentiation showed that TASL deficiency affects calcium modulation, which is essential for keratinocyte regulation. These findings unveil a novel role of TASL in the proliferation and differentiation of keratinocytes, providing new insights into the intricate regulatory mechanisms of keratinocyte biology.
The proper function of lysosomes depends on their ability to store and release calcium. While several lysosomal calcium release channels have been described, how mammalian lysosomes replenish their calcium stores has not been determined. Using genetic depletion and overexpression techniques combined with electrophysiology and visualization of subcellular ion concentrations and their fluxes across the lysosomal membrane, we show here that TMEM165 imports calcium to the lysosomal lumen and mediates calcium-induced lysosomal proton leakage. Accordingly, TMEM165 accelerates the recovery of cells from cytosolic calcium overload thereby enhancing cell survival while causing a significant acidification of the perilysosomal area and the entire cytosol. These data indicate that in addition to its essential role in the glycosylation in the Golgi, a small but significant fraction of TMEM165 localizes on the lysosomal limiting membrane, where it protects cells against cytosolic calcium overload, preserves lysosomal function by refilling lysosomal calcium stores and regulates perilysosomal and lysosomal pH.
Airway neuroendocrine (NE) cells have been proposed to serve as specialized sensory epithelial cells that modulate respiratory behavior by communicating with nearby nerve endings. However, their functional properties and physiological roles in the healthy lung, trachea, and larynx remain largely unknown. In this work, we show that murine NE cells in these compartments have distinct biophysical properties but share sensitivity to two commonly aspirated noxious stimuli, water and acid. Moreover, we found that tracheal and laryngeal NE cells protect the airways by releasing adenosine 5′-triphosphate (ATP) to activate purinoreceptive sensory neurons that initiate swallowing and expiratory reflexes. Our work uncovers the broad molecular and biophysical diversity of NE cells across the airways and reveals mechanisms by which these specialized excitable cells serve as sentinels for activating protective responses.
A new family of highly fluorescent indicators has been synthesized for biochemical studies of the physiological role of cytosolic free Ca2+. The compounds combine an 8-coordinate tetracarboxylate chelating site with stilbene chromophores. Incorporation of the ethylenic linkage of the stilbene into a heterocyclic ring enhances the quantum efficiency and photochemical stability of the fluorophore. Compared to their widely used predecessor, “quin2”, the new dyes offer up to 30-fold brighter fluorescence, major changes in wavelength not just intensity upon Ca2+ binding, slightly lower affinities for Ca2+, slightly longer wavelengths of excitation, and considerably improved selectivity for Ca2+ over other divalent cations. These properties, particularly the wavelength sensitivity to Ca2+, should make these dyes the preferred fluorescent indicators for many intracellular applications, especially in single cells, adherent cell layers, or bulk tissues.
We have measured changes of pH in a protein's microenvironment consequent on its binding to the cell surface and incorporation into pinosomes. Changes of pH were measured from single, living cells and selected regions of cells by the fluorescence ratio technique using a photon-counting microspectrofluorimeter. The chemotactic agent and pinocytosis inducer, ribonuclease, labeled with fluorescein (FTC-RNase), adsorbed to the surface of Amoeba proteus, and was pinocytosed by cells in culture media at pH 7.0. The FTC-RNase entered an apparently acidic microenvironment, pH approximately 6.1, upon binding to the surface of amoebae. Once enclosed within pinosomes, this protein's microenvironment became steadily more acidic, reaching a minimum of pH approximately 5.6 in less than 10 min. FTC-RNase pinocytosed by the giant amoeba, Chaos carolinensis, entered pinosomes whose pH was correlated with their cytoplasmic location during the initial 30-40 min after pinocytosis. The majority of pinosomes containing FTC-RNase clustered in the tail ectoplasm of C. carolinensis during this interval and had a pH of approximately 6.5; those released into endoplasm and carried into the tip of cells had a pH below 5.0. As pinosomes became distributed at random in C. carolinensis (1-2 h after initial pinocytosis), differences in pH between tip and tail pinosomes vanished. We have also measured the pH within single phagosomes of A. proteus. Phagosomal pH dropped steadily to approximately 5.4 within 5 min after particle ingestion in 70% of the cells measured, and reached this level of acidity within 10 min in 90% of the cells measured. By contrast, stain for the lysosomal enzyme, acid phosphatase, was evident within only 20% of 5-min-old phagosomes visualized by light microscopy, and within only 40% of 10-min-old phagosomes. A microfluorimetric assay was used to simultaneously record changes in pH, and the initial deposition of lysosomal esterases, within phagosomes of single, living Amoeba proteus. Near complete acidification of the phagosome was recorded from some cells before phagosomal fusion was evident by this microfluorimetric assay. From other cells, however, continued acidification of phagosomes was recorded after lysosomal fusion was initiated. We conclude that acidification of phagosomes by A. proteus is initiated but not necessarily completed prior to phagosome-lysosome formation, and that the two events are closely linked in time. Initial acidification of endosomes is a property intrinsic to the plasma membrane which envelops particles at the cell surface, rather than the result of lysosomal fusion with phagosomes.
This chapter discusses the mechanisms and functional significance of phagosomal acidification. To function as an effective microbicidal organelle, the early phagosome must mature by fusing with endosomes and lysosomes. In concert with phagosomal maturation there is a progressive acidification of the interior of the phagosome, which confers and/or enhances its microbicidal capabilities by several mechanisms. First, the acidification can have a direct bactericidal or bacteriostatic effect, inasmuch as various microorganisms are unable to grow at low pH. In addition, the acidic milieu can activate microbicidal enzymes, such as pH-sensitive proteases or lipases that are delivered to the phagosome during fusion with lysosomes. Lastly, the increased acidification will promote the dismutation of superoxide to hydrogen peroxide that in turn functions as the substrate for myeloperoxidase, another potent microbicidal agent delivered by phago-lysosomal fusion. The critical role of phagosomal acidification in bacterial killing is illustrated by the failure of macrophages to eliminate many species of mycobacteria. These bacteria survive and replicate within phagosomes, at least in part by impairing the process of phagosomal acidification. An understanding of the mechanisms responsible for phagosomal acidification is therefore essential in the analysis of the antimicrobial features of phagocytes.
This chapter discusses the various pathways through the vacuolar compartment. The essential route through the vacuolar compartment consists of three component activities: entry, transit and recycling. Entry that includes the processes of phagocytosis, pinocytosis and receptor-mediated endocytosis, occurs when plasma membrane invaginates to enclose extracellular molecules or particles into intracellular vesicles. Subsequent membrane fusion and fission reactions typically deliver the contents of these vesicles to endosomes and then to lysosomes, where most macromolecules are degraded by acid hydrolases. The exchange of membrane and content that occurs en route to lysosomes, and the mechanisms that reestablish compartment identities, allow some internalized membrane and solutes to recycle to the cell surface. The net effect is that extracellular solutes and particles are delivered efficiently to lysosomes through a series of compartments whose distinct identities are maintained even as the exogenous passengers move through them. The chapter also describes a model to explain pathways dynamics. The aim is to describe the general flow of membrane, solutes, water, and macromolecules into and through the macrophage.
Activation revisited. Since the time of Metchinkoff (1) and his contemporaries it has been clear that tissue macrophages exist in various states of activity. One reads about the resting-wandering cells of the connective tissue, arising from the mesenchyme and stimulated by the products of local injury. Such observations performed with the light microscope, vital dyes, and keen insight have in the past two decades been expanded with our increasing knowledge of the mononuclear phagocyte system (2).
In the recent past the term “activated macrophage” has largely been relegated to the bactericidal process and to the role of these cells in cell-mediated immunity. Unraveled largely through the imaginative in vivo studies of Mackaness and North and their colleagues (3, 4), the two cell nature of the process, the effector role of the macrophage, and the unique sensitizing qualities of viable organisms were elucidated. Yet, it is apparent when one scans the varied roles of tissue macrophages in the physiology and pathology of mammals that this is a much restricted definition.
Certain bacteria secrete protein toxins that catalytically modify and disrupt essential processes in mammalian cells, often leading to cell death. As the substrates modified by these toxins are located in the mammalian cell cytosol, a catalytically active toxin polypeptide must reach this compartment in order to act. The toxins bind to receptors on the surface of susceptible cells and enter them by endocytic uptake. Endocytosed toxins initially accumulate in endosomes, where some of these proteins take advantage of the acidic environment within these organelles to form, or contribute to the formation of, protein-conducting channels through which the catalytic polypeptide is able to translocate into the cytosol. Other toxins are unable to respond to low pH in this way and must undergo intracellular vesicular transport to reach a compartment where pre-existing protein-conducting channels occur and can be exploited for membrane translocation — the endoplasmic reticulum. In this way, cell entry by this second group of toxins demonstrates that the secretory pathway of mammalian cells is completely reversible.
Survival of Salmonella typhimurium within macrophage phagosomes requires the coordinate expression of bacterial gene products. This report examines the contribution of phagosomal pH as a signal for expression of genes positively regulated by the S. typhimurium virulence regulators PhoP and PhoQ. Several hours after bacterial phagocytosis by murine bone marrow-derived macrophages, PhoP-activated gene transcription increased 50- to 77-fold. In contrast, no difference in PhoP-activated gene expression was observed after infection of cultured epithelial cells, suggesting that the membrane sensor PhoQ recognized signals unique to macrophage phagosomes. The increase in PhoP-regulated gene expression was abolished when macrophage culture medium contained NH4Cl or chloroquine, weak bases that raise the pH of acidic compartments. Measurements of pH documented that S. typhimurium delayed and attenuated acidification of its intracellular compartment. Phagosomes containing S. typhimurium required 4-5 hr to reach pH < 5.0. In contrast, within 1 hr vacuoles containing heat-killed bacteria were measured at pH < 4.5. The eventual acidification of phagosomes to pH < 5.0 correlated with the period of maximal PhoP-dependent gene expression. These observations implicate phagosome acidification as an intracellular inducer of PhoP-regulated gene expression and suggest that Salmonella survival is dependent on its ability to attenuate phagosome acidification.
Fluorescent calcium indicators fluo-3, fura-2 and indo-1, and fluorescent magnesium indicators mag-fura-2 (FURAPTRA) and mag-indo-1 were evaluated for the effects of pH on their association and dissociation rates, ion selectivity and thermodynamic properties. Calcium indicator affinities for Ca and Mg were reduced and the discrimination between Ca and Mg decreased in fura-2 and indo-1 at acidic pH. Alterations in apparent dissociation constants were caused primarily by reduced association rates. Magnesium indicators did not show these changes. The enthalphies of the calcium indicators' Ca complex were 1-3 kcal/mole and magnesium indicators' Mg complex were 7-9 kcal/mole. The potential effects of a biexponential dissociation rate of fluo-3 and of Ca interactions with magnesium indicators were examined.