Optical complexities of living cytoplasm--implications for live cell imaging and photo-micromanipulation techniques.

The ability to manipulate the intracellular environment within living cells and to monitor the cytosolic chemical changes which occur during cell stimulation has lead to major advances in our understanding of how cells read and respond to their environment. Perhaps the most powerful suite of techniques for achieving these dual objectives is based on the use of light (photons). Because cells are 'transparent', light has been used to both interrogate and manipulate the chemistry inside living cells, exploiting technical advances in both the physical and biochemical sciences. However, cells are neither transparent nor homogeneous with respect to their optical properties. The interface between light and the living cell cytoplasm thus represent an important, yet largely ignored, interface. There has been no review of the optical properties of cytoplasm and little discussion about how the optical properties of living cytoplasm influence the outcome of such measurements and manipulations. In this short review, we discuss the importance of understanding the optical properties of cytoplasm for such techniques and how imperfections in experimental interpretation can arise.

Exogenous glycosyl phosphatidylinositol-anchored CD59 associates with kinases in membrane clusters on U937 cells and becomes Ca(2+)-signaling competent.

CD59, an 18-20-kD complement inhibitor anchored to the membrane via glycosyl phosphatidylinositol (GPI), can induce activation of T cells and neutrophils upon cross-linking with antibody. GPI-anchored molecules cocluster in high mol wt detergent-resistant complexes containing tyrosine kinases that are implicated in the signaling pathway. Exogenous, incorporated GPI-anchored molecules are initially unable to induce activation, presumably because they are not associated with kinases. Here we demonstrate that erythrocyte-derived CD59 incorporated in a CD59-negative cell line acquires signaling capacity in a time-dependent manner. Confocal microscopy revealed an initial diffuse distribution of CD59 that became clustered within 2 h to give a pattern similar to endogenous GPI-anchored molecules. Gel filtration of detergent-solubilized cells immediately after incorporation revealed that CD59 was mainly monomeric, but after 3 h incubation all was in high mol wt complexes and had become associated with protein kinases. Newly incorporated CD59 did not deliver a Ca2+ signal upon cross-linking, but at a time when it had become clustered and associated with kinase activity, cross-linking induced a large calcium transient, indicating that CD59 had incorporated in a specialized microenvironment that allowed it to function fully as a signal-transducing molecule.

Regulation of the expression of E-cadherin on human cancer cells by gamma-linolenic acid (GLA).

E-cadherin is a cell to cell adhesion molecule which acts as a suppressor of metastasis. This study examined the effect of gamma-linolenic acid (GLA), a n-6 polyunsaturated fatty acid, on the expression of E-cadherin in human cancer cells. Western blotting studies demonstrated that treatment of cells with GLA for 24 h increased the expression of E-cadherin in lung, colon, breast, melanoma, and liver cancer cells, but not in endothelial cells and fibroblasts. The results were confirmed by immunocytochemistry. In contrast, two other n-6 fatty acids, linoleic acid and arachidonic acid, failed to induce these changes. The increased expression of E-cadherin was correlated with reduced in vitro invasion and increased aggregation, indicating that the increased E-cadherin expression induced by GLA was biologically active. These data add GLA to the short list of E-cadherin up-regulatory factors. The up-regulation of E-cadherin expression in human cancer cells may contribute to the anticancer properties of GLA.

Quantal secretion and response lag demonstrated in single rat neutrophils.

The release of 9-aminoacridine loaded into neutrophil granules has been monitored using quantitative fluorescence microscopy of individual rat neutrophils. Within the granule, the fluorescence of the dye was substantially quenched, but release into the surrounding medium restored fluorescence. From kinetic analysis of the increase in fluorescence it was shown that secretion from a single neutrophil in response to a low concentration of chemotactic peptide occurred in 'bursts'. Each 'burst' of secretion was of equal size and kinetics, which were equal to the size and kinetics of the smallest evoked response possible. A significant time-lag of 5-10 s between the arrival of the stimulus at the cell and the onset of secretion was recorded. It was therefore concluded that secretion from neutrophils was the result of release of quantal amounts of dye following a delay period.

Development of oxidase 'priming' in maturing HL60 cells: correlation with protein expression and tyrosine phosphorylation.

The mechanisms involved in 'priming' of the oxidase response of neutrophils are unknown. Two major problems are encountered in using circulating neutrophils; firstly, prior exposure to circulating 'priming' cytokines cannot be controlled and secondly, non-intentional 'priming' during cell separation can occur. In this study, these problems were avoided by differentiating the promyeloid leukaemic cell line, HL60, towards granulocytes using dibutyrl cyclic AMP, to produce a 'virgin cell' model system. We have demonstrated that the ability of substance P to both prime the oxidase response and induce tyrosine phosphorylation increased during differentiation. The major tyrosine-phosphorylated protein, with molecular weight of 74 kDa, was not recognised by anti-c-raf1 antibodies. Furthermore, c-raf1 expression rapidly declined during HL60 cell granulocytic differentiation. This data shows that although there was no simple relationship between c-raf quantity and priming, the data were consistent with tyrosine phosphorylation of a 74 kDa protein being important for oxidase 'priming'.

Activation and priming of the human neutrophil oxidase response by substance P: distinct signal transduction pathways.

The undecapeptide substance P (SP), both activates and 'primes' the neutrophil NADPH-oxidase response. In this paper we investigate the roles of Ca2+ and actin polymerisation in both the activation and 'priming' of the neutrophil oxidase response by the non-oxidisable SP-analog norleucine-SP (n-SP). We demonstrate that by binding to receptors which were distinct from the formylated peptide receptor, n-SP (100 nM-400 microM) directly triggered the NADPH-oxidase response, elevated cytosolic free Ca2+, and caused the polymerisation of G-actin. However at lower concentrations (1-100 nM), in the absence of these phenomena, n-SP primed the oxidase response to the peptide f-Met-Leu-Phe. We propose that occupancy of SP-activatable receptors on neutrophils triggers two different signal transduction pathways, one being responsible for the generation of signals for oxidase activation and the second, which is independent of Ca2+ and actin signalling, being responsible for priming.

Actin polymerization modifies stimulus-oxidase coupling in rat neutrophils.

Oxidase activity in rat neutrophils was monitored by oxygen consumption rate and luminol-dependent chemiluminescence. Two agents which inhibit actin polymerization, cytochalasin B and dihydrocytochalasin B, produced a marked enhancement (up to 10-fold) of oxidase activation induced by two Ca2+-dependent stimuli, chemotactic peptide and ionophore A23187. In contrast, activation by the calcium-independent stimulus, phorbol myristate acetate, was unaffected by these agents. Other agents that interact with the cytoskeleton, phalloidin and colchicine have no effect on activation by any stimulus tested. The effect of cytochalasin B, when added after stimulation by chemotactic peptide, was transient with t0.5 approx. 10 s. Similarly, the degree of actin polymerization following stimulation by chemotactic peptide was transient, decaying with a t0.5 of approx. 10 s. The half-maximal concentration of cytochalasin B for inhibition of actin polymerization was similar to that for enhancement of oxidase activation. It was concluded, therefore, that the intracellular Ca2+ rise in rat neutrophils that accompanies stimulation by chemotactic peptide affects actin polymerization in a manner that modifies oxidase activation.

Osmotically induced cytosolic free Ca(2+) changes in human neutrophils.

Cytosolic free Ca(2+) concentration in neutrophils was measured by ratiometric fluorometry of intracellular fura2. Increasing the extracellular osmolarity, by either NaCl (300-600 mM) or sucrose (600-1200 mM), caused a rise in cytosolic free Ca(2+) (Delta(max) approximately equal to 600 nM). This was not due to cell lysis as the cytosolic free Ca(2+) concentration was reversed by restoration of isotonicity and a second rise in cytosolic free Ca(2+) could be provoked by repeating the change in extracellular osmolarity. Furthermore, the rise in cytosolic free Ca(2+) concentration occurred in the absence of extracellular Ca(2+), demonstrating that release of intracellular fura2 into the external medium did not occur. The osmotically-induced rise in cytosolic free Ca(2+) was not inhibited by either the phospholipase C-inhibitor U73122, or the microfilament inhibitor cytochalasin B, suggesting that neither signalling via inositol tris-phosphate or the cytoskeletal system were involved. However, the rise in cytosolic free Ca(2+) may have resulted from a reduction in neutrophil water volume in hyperosmotic conditions. As these rises in cytosolic Ca(2+) (Delta(max) approximately equal to 600 nM) were large enough to provoke changes in neutrophil activity, we propose that conditions which removes cell water may similarly elevate cytosolic free Ca(2+) to physiologically important levels.

Ca2+ signalling delays in neutrophils: effects of prior exposure to platelet activating factor or formyl-met-leu-phe.

Rapid-time confocal scanning of fluo3-loaded neutrophils revealed that in individual cells there were grossly heterogeneous time intervals between stimulation with either f-met-leu-phe or platelet activating factor (PAF) and the initiation of Ca2+ influx, ranging from 75 msec to several seconds. The distribution of lag times after stimulation with f-met-leu-phe (100 nM) was influenced by prior stimulation with either f-met-leu-phe or PAF. However, whereas prior stimulation with f-met-leu-phe (50 nM) caused the subsequent cytosolic free Ca2+ response to second challenge with f-met-leu-phe to be delayed, prior stimulation with PAF (100 nM) caused an increase in the rapidity of the onset of the second response to f-met-leu-phe. With both stimuli, the cytosolic free Ca2+ in some neutrophils (non-Ca2+ responders) in the population did not increase significantly. However, some of these cells responded to the subsequent challenge. However, with both pre-treatment stimuli, those cells in which a significant Ca2+ response was provoked by the first stimulus, responded significantly faster than the initial 'non-Ca2+ responders.' However, the reduced lag time provoked by pre-stimulation was not inhibited in neutrophils in which cytosolic free Ca2+ changes were dampened by intracellular BAPTA. These data point to post-receptor events, other than prior cytosolic free Ca2+ elevation, being important in determining the response Ca2+ delay to subsequent stimulation.

Spatial and temporal separation of calcium signals in myeloid cells stimulated by immune complexes.

Stimulation of large (100 microns) human myeloid cells with immune complexes resulted in Ca2+ spiking. Both global and regional changes in the intracellular cytosolic free Ca2+ concentration were detected in response to immune complex stimulation. The regional changes were mediated by release of Ca2+ from stores, whereas global changes were mediated by Ca2+ influx. They occurred independently of each other, with release of Ca2+ from intracellular Ca2+ stores being separated from transmembrane influx of Ca2+. Bromophenacyl bromide, an 1-plastin binding agent, inhibited store release without preventing transmembrane influx of Ca2+. The large size of the myeloid cells used here allowed the visualisation of the spatial and temporal separation of store release from transmembrane influx of Ca2+, providing further evidence for the existence of independent Ca2+ store release and Ca2+ influx mechanisms in these cells.

Two distinct Ca2+ storage and release sites in human neutrophils.

It is well established that changes in cytosolic free Ca2+ play a role in a number of neutrophil responses such as cell shape change and oxidase activation. The release of intracellular stored Ca2+ occurs with stimuli that either act by occupancy of seven membrane-spanning domain receptors or those which act by receptor cross-linking. Here, two distinct Ca2+ storage and release sites have been identified in neutrophils. Using chlortetracycline fluorescence as an indicator of Ca2+ storage, two separate Ca2+ storage sites have been identified. One site was located peripherally under the plasma membrane and the other was in the juxtanuclear space. Confocal imaging of Fluo3-loaded neutrophils demonstrated that the central Ca2+ storage site released Ca2+ in response to N-formyl-methionyl-leucyl-phenylalanine, whereas engagement and clustering of CD11b/CD18 integrins causes Ca2+ release from the peripheral stores. The release sites also correlated with organelles that stained with DiOC6(3). Localized phototoxicity generated by DiOC6(3) excitation resulted in inhibition of the release of stored Ca2+, which was selective for the stimulus used. The presence of two distinct cellular locations for these Ca2+ stores and their independent release raises the possibility that separate intracellular messengers for their release are generated.

Slow calcium waves imaged in myeloid cells derived from neonatal cord blood.

Myeloid cells were derived from neonatal cord blood by culture with granulocyte-macrophage colony-stimulating factor for approximately 8 days. The resultant cell population contained large adherent cells (diameter > or = microns), expressing formylated peptide receptors that were functionally coupled to cytosolic free Ca2+ signaling. Imaging of the cytosolic free Ca2+ changes in these cells revealed initial focal release of Ca2+ from a site from within the cell, with elevated Ca2+ also near the cell edge. Increased cytosolic free Ca2+ moved as a slow oscillating wave across the cell (velocity 1 microns/s). As similar events may occur in mature neutrophils and monocytes but be difficult to resolve because of the small size of these cells, it was concluded that neonatal myeloid cells may provide a useful model system for the investigation of Ca2+ signaling in myeloid cells.

Slow Ca2+ waves in large myeloid cells as a result of a diffusible cytosolic factor.

In the work reported here evidence is provided that shows the slow wave of Ca2+ large neonatal myeloid cells provoked by formyl-Met-Leu-Phe was generated by spatially delayed Ca2+ influx. Evidence is provided that the delay in Ca2+ influx was the result of diffusion of a factor from the Ca2+ storage site, which is responsible for Ca2+ channel opening. The location of the Ca2+ release site was correlated with a region near the nucleus, probably a specialized region of endoplasmic reticulum. It is proposed that similar mechanisms of Ca2+ signaling occur in mature myeloid cells, such as neutrophils, but on a shorter time scale as a consequence of their smaller size.

Conformationally restricted calpain inhibitors.

The cysteine protease calpain-I is linked to several diseases and is therefore a valuable target for inhibition. Selective inhibition of calpain-I has proved difficult as most compounds target the active site and inhibit a broad spectrum of cysteine proteases as well as other calpain isoforms. Selective inhibitors might not only be potential drugs but should act as tools to explore the physiological and pathophysiological roles of calpain-I. α-Mercaptoacrylic acid based calpain inhibitors are potent, cell permeable and selective inhibitors of calpain-I and calpain-II. These inhibitors target the calcium binding domain PEF(S) of calpain-I and -II. Here X-ray diffraction analysis of co-crystals of PEF(S) revealed that the disulfide form of an α-mercaptoacrylic acid bound within a hydrophobic groove that is also targeted by a calpastatin inhibitory region and made a greater number of favourable interactions with the protein than the reduced sulfhydryl form. Measurement of the inhibitory potency of the α-mercaptoacrylic acids and X-ray crystallography revealed that the IC50 values decreased significantly on oxidation as a consequence of the stereo-electronic properties of disulfide bonds that restrict rotation around the S-S bond. Consequently, thioether analogues inhibited calpain-I with potencies similar to those of the free sulfhydryl forms of α-mercaptoacrylic acids.

Near membrane Ca2+ changes resulting from store release in neutrophils: detection by FFP-18.

FFP-18 was incorporated into the inner face of the plasma membrane of human neutrophils by incubation with its acetoxymethyl ester. Conversion to the Ca2+ sensitive intracellular indicator was monitored by the change in excitation spectra. The fluorescence from extracellularly facing FFP-18 was quenched by the membrane impermeant ion Ni2+. Ratio fluorescence measurement of FFP-18 under these conditions permitted the detection of near membrane Ca2+ changes resulting from the release of Ca2+ from intracellular stores. Near membrane and cytosolic Ca2+ changes were measured under conditions in which store release and Ca2+ influx were triggered by FMLP, thapsigargin or immune complexes. There were significant differences in the timing and magnitude of Ca2+ changes near the plasma membrane and bulk cytosolic Ca2+ concentration, which were consistent with a Ca2+ storage site deep within the neutrophil released by thapsigargin and FMLP, but Ca2+ release sites with immune complex stimulation being close to the membrane. The use of FFP-18 to monitor Ca2+ near the inner face of the plasma membrane thus provides evidence for the existence of two distinct Ca2+ storage locations in neutrophils.

Is intracellular Ca2+ the trigger for oxygen radical production by polymorphonuclear leucocytes?

The aim of this paper is critically to evaluate the existing evidence for the role of intracellular Ca2+ in polymorphonuclear leucocyte (PMN) activation and in particular in oxygen radical production. Indirect experiments are based on the manipulation of extracellular Ca2+, measurement of 45Ca fluxes, employing pharmacological agents such as Ca2+-ionophores and intracellular Ca2+ antagonists and monitoring chlortetracycline fluorescence. Experiments of this type do not provide the necessary definitive evidence that an increase in intracellular Ca2+ is the trigger for PMN activation. Recent direct measurements of intracellular free Ca2+ using the Ca2+-activated photoprotein, obelin, and the Ca2+-sensitive fluorescent indicator, quin 2, have provided evidence for the existence of two distinct mechanisms of activation, one triggered by a rise in intracellular Ca2+ and the other independent of a rise in intracellular Ca2+. The source of the Ca2+ for the former mechanism is mainly extracellular but can also come from an intracellular Ca2+ store.

The use of fura-2 to determine the relationship between cytoplasmic free Ca2+ and oxidase activation in rat neutrophils.

Incubation of rat neutrophils with fura-2-acetoxy-methyl ester (fura-2/AM) resulted in the loading of fura-2 almost exclusively into the cytoplasm. Despite the additional presence of fura-2/AM esterase activity in the granules, only 1.5% of cell-associated fura-2 was located within these organelles. Fura-2 leaked from neutrophils at an acceptably low rate 0.16 +/- 0.05% min-1 at 37 degrees C. At intracellular concentrations of fura-2 up to 500 microM, there was no effect on oxidase activation; although the cellular ATP content was reduced to approximately 50%. The peptide, f-met-leu-phe (fmlp), 1 microM, produced intensity changes of fluorescence excited at 340nm and 380nm which were consistent with a cytoplasmic Ca2+ rise from the resting level of 94 +/- 13nM to 768 +/- 173nM (n = 6). Intracellular concentrations of fura-2 greater than 1mM were required to buffer effectively this rise, and it was estimated that an intracellular fura-2 concentration required for a high signal:autofluorescence ratio (100 microM) the cytoplasmic Ca2+ buffering capacity of the cells was increased by only 10%. The rise in cytoplasmic free Ca2+ induced by the peptide preceded activation of the oxidase by several seconds, and the magnitude of the response was dependent on the extent of the Ca2+ rise, half-maximal activation being achieved at approx. 600nM. These data were therefore consistent with a secondary messenger role for cytoplasmic Ca2+ in triggering neutrophil oxidase activation.

Complement component C9-dependent cytosolic free Ca2+ rise and recovery in neutrophils.

The cytosolic free Ca2+ concentration in rat neutrophils was determined by ratiometric fluorometry and imaging of Fura-2. Transient elevations of cytosolic free Ca2+ concentration were provoked by addition of C9 to neutrophils pre-coated with C5b-8. The rate of rise of the cytosolic free Ca2+ concentration was dependent upon the concentration of C9. These changes in cytosolic free Ca2+ concentration occurred in the absence of cell lysis, since there was no release of Fura-2, and were the result of increased permeability to extracellular Ca2+. More than 96% of the rise in cytosolic free Ca2+ generation by C9 was dependent upon the presence of extracellular Ca2+, but did not occur via channels which were inhibited by the Ca2+ channel blocker SKF96365. The decrease in the permeability of the membrane to Ca2+ after C9 was not triggered by the rise in cytosolic free Ca2+. After attack by C9, individual neutrophils remained responsive to f-met-leu-phe, or further attack by C9, both producing Ca2+ transients. The recovery of the Ca2+ signal was consistent with the complement membrane attack complex generating a series of permeability thresholds in the plasma membrane. These data have implications for the understanding of the mechanisms underlying the inappropriate responsiveness of neutrophils at inflammatory sites.

Coelenterate photoproteins as indicators of cytoplasmic free Ca2+ in small cells.

The Ca2+-activated photoproteins aequorin and obelin are capable of detecting rapid changes in free Ca2+ over the range 10nM-100uM. Whilst they have been used to quantify free Ca transients in giant cells for some time, their use in small mammalian cells has been restricted because of the difficulty of incorporating them into live cells without impairment of cell function. We have developed three methods for incorporating photoproteins into small cells (a) reversible cell swelling (b) membrane fusion and (c) intracellular release from pinocytotic vesicles. Formation of the membrane attack complex of complement (C5b6789), via a specific cell surface antibody to activate complement, causes a rapid increase in cytoplasmic Ca2+ detectable within 5-10 s. It provides a specific method for quantifying cytoplasmic photoprotein. As a result new insights into the role of intracellular Ca2+ in cell physiology and pathology have been established.

Oxidase activation in individual neutrophils is dependent on the onset and magnitude of the Ca2+ signal.

Using single-cell ratio imaging of Fura-2-loaded neutrophils, we demonstrate that the heterogeneity and asynchrony of the oxidase response originates from variability in the timing and magnitude of the cytosolic free Ca2+ signal. The Ca2+ signals from individual cells could be classified into four types: (a) type 1, a transient rise in Ca2+ occurring within 6 s; (b) type 2, an oscillating cytosolic free Ca2+; (c) type 3, a latent Ca2+ transient significantly delayed (21-56 s); and (d) type 4, no significant Ca2+ rise. These response types accounted for approximately 41%, 15%, 26% and 18% of the population respectively for stimulation with 1 microM f-met-leu-phe peptide (n = 27) and 52.5%, 15%, 11.5% and 21% respectively for 0.1 microM f-met-leu-phe peptide (n = 52). The oxidase in neutrophils in which the cytosolic free Ca2+ concentration rose to greater than 250 nM always became activated. In the presence of extracellular Ca2+, cytosolic Ca2+ rose uniformly throughout the cell, whereas in the absence of extracellular Ca2+, a localised Ca2+ 'cloud' was observed in approximately 30% of cells. A localised activation of the oxidase accompanied the presence of the Ca2+ 'cloud' when the 250 nM Ca2+ threshold was exceeded. The data presented here therefore demonstrate a tight coupling in individual neutrophils between an elevation in cytosolic free Ca2+ above a threshold of 250 nM and activation of the oxidase.