A sustained increase of cytosolic Ca in γδ T cells triggered by co-stimulation via TCR/CD3 and LFA-1

We previously reported that co-stimulation with LFA-1 triggered apoptosis in γδ T cells but not in αβ T cells after TCR engagement. We extended our earlier study on TCR/LFA-1 triggered apoptosis to two autoreactive TCR γδ and TCR αβ T cell clones, which were derived from syngeneic mixed lymphocyte culture of BALB/c mice. A γδ T cell clone, KM1, expressed the Vγ4 and Vδ5 genes and CD4-CD8-CD45RB⁺ phenotype; and an αβ T cell clone, BASL1.1, expressed Vβ6 and CD4⁺CD8-CD45RB⁺. Both clones produced Th-1-type cytokines in response to syngeneic BALB/c stimulator cells. KM1 underwent apoptosis upon stimulation with immobilized anti-CD3/LFA-1 mAbs, whereas BASL1.1 could proliferate successfully in response to stimulation with the immobilized mAbs. BASL1.1 was able to down-regulate the increased cytosolic Ca²⁺ after the simultaneous stimulation, but KM1 exhibited a sustained increase of cytosolic Ca²⁺ after stimulation via CD3 and LFA-1. Similar results with respect to the kinetics of cytosolic Ca²⁺ were obtained with normal heterogeneous γδ and αβ T cell populations after co-stimulation via CD3 and LFA-1. Our results suggested that persistently high levels of cytosolic Ca²⁺ might be related to apoptosis in γδ T cell clone triggered by costimulation via CD3 and LFA-1.     

Integrin Cross Talk: Activation of Lymphocyte Function-associated Antigen-1 on Human T Cells Alters α4β1- and α5β1-mediated Function

A regulated order of adhesion events directs leukocytes from the vascular compartment into injured tissues in response to inflammatory stimuli. We show that on human T cells, the interaction of the β2 integrin leucocyte function–associated antigen-1 (LFA-1) with its ligand intercellular adhesion molecule-1 (ICAM-1) will decrease adhesion mediated by α4β1 and, to a lesser extent, α5β1. Similar inhibition is also seen when T cells are exposed to mAb 24, which stabilizes LFA-1 in an active state after triggering integrin function through divalent cation Mg²⁺, PdBu, or T cell receptor/ CD3 complex (TCR/CD3) cross-linking. Such cross talk decreases α4β1 integrin–mediated binding of T cells to fibronectin and vascular cell adhesion molecule-1 (VCAM-1). In contrast, ligand occupancy or prolonged activation of β1 integrin has no effect on LFA-1 adhesion to ICAM-1. We also show that T cell migration across fibronectin, unlike adhesion, is mediated solely by α5β1, and is increased when the α4β1-mediated component of fibronectin adhesion is decreased either by cross talk or the use of α4-blocking mAb. The ability of mAb 24 Fab′ fragments to induce cross talk without cross-linking LFA-1 suggests signal transduction through the active integrin. These data provide the first direct evidence for cross talk between LFA-1 and β1 integrins on T cells. Together, these findings imply that activation of LFA-1 on the extravasating T cell will decrease the binding to VCAM-1 while enhancing the subsequent migration on fibronectin. This sequence of events provides a further level of complexity to the coordination of T cell integrins, whose sequential but overlapping roles are essential for transmigration.     

Acute Inhibition of Selected Membrane-Proximal Mouse T Cell Receptor Signaling by Mitochondrial Antagonists

T cells absorb nanometric membrane vesicles, prepared from plasma membrane of antigen presenting cells, via dual receptor/ligand interactions of T cell receptor (TCR) with cognate peptide/major histocompatibility complex (MHC) plus lymphocyte function-associated antigen 1 (LFA-1) with intercellular adhesion molecule 1. TCR-mediated signaling for LFA-1 activation is also required for the vesicle absorption. Exploiting those findings, we had established a high throughput screening (HTS) platform and screened a library for isolation of small molecules inhibiting the vesicle absorption. Follow-up studies confirmed that treatments (1 hour) with various mitochondrial antagonists, including a class of anti-diabetic drugs (i.e., Metformin and Phenformin), resulted in ubiquitous inhibition of the vesicle absorption without compromising viability of T cells. Further studies revealed that the mitochondrial drug treatments caused impairment of specific membrane-proximal TCR signaling event(s). Thus, activation of Akt and PLC-γ1 and entry of extracellular Ca²⁺ following TCR stimulation were attenuated while polymerization of monomeric actins upon TCR triggering progressed normally after the treatments. Dynamic F-actin rearrangement concurring with the vesicle absorption was also found to be impaired by the drug treatments, implying that the inhibition by the drug treatments of downstream signaling events (and the vesicle absorption) could result from lack of directional relocation of signaling and cell surface molecules. We also assessed the potential application of mitochondrial antagonists as immune modulators by probing effects of the long-term drug treatments (24 hours) on viability of resting primary T cells and cell cycle progression of antigen-stimulated T cells. This study unveils a novel regulatory mechanism for T cell immunity in response to environmental factors having effects on mitochondrial function.     

Vaccinia Virus Induces Ca2+-Independent Cell-Matrix Adhesion during the Motile Phase of Infection

Vaccinia virus (VV) induces two forms of cell motility: cell migration, which is dependent on the expression of early genes, and the formation of cellular projections, which requires the expression of late genes. The need for viral gene expression prior to cell motility suggests that VV proteins may affect how infected cells interact with the extracellular matrix. To address this, we have analyzed changes in cell-matrix adhesion after infection of BS-C-1 cells with VV. Whereas uninfected cells round up and detach from the culture flask in the presence of EGTA, infected cells remain attached to the culture flask with a stellate morphology. Ca²⁺-independent cell-matrix adhesion was evident by 10 h postinfection, after the onset of cell motility but before the formation of virus-induced cellular projections. Progression to Ca²⁺-independent adhesion required the expression of late viral genes but not the formation of intracellular enveloped virus particles or intracellular actin tails. Analyses of specific matrix proteins identified vitronectin and fibronectin as optimal ligands for Ca²⁺-independent adhesion and the formation of cellular projections. Adhesion to fibronectin was mediated via RGD motifs alone and was not inhibited by 500 μg of heparin/ml. Kistrin, a disintegrin which binds preferentially to the αvβ3 (vitronectin/fibronectin) receptor inhibited the formation of cellular projections without disrupting preformed matrix interactions. Finally, we show that Ca²⁺-independent cell-matrix adhesion is a dynamic process which mediates changes in the morphology of VV-infected cells and uninfected cells which exhibit a transformed phenotype.     

Cross Talk between Adhesion Molecules: Control of N-cadherin Activity by Intracellular Signals Elicited by β1 and β3 Integrins in Migrating Neural Crest Cells

During embryonic development, cell migration and cell differentiation are associated with dynamic modulations both in time and space of the repertoire and function of adhesion receptors, but the nature of the mechanisms responsible for their coordinated occurrence remains to be elucidated. Thus, migrating neural crest cells adhere to fibronectin in an integrin-dependent manner while maintaining reduced N-cadherin–mediated intercellular contacts. In the present study we provide evidence that, in these cells, the control of N-cadherin may rely directly on the activity of integrins involved in the process of cell motion. Prevention of neural crest cell migration using RGD peptides or antibodies to fibronectin and to β1 and β3 integrins caused rapid N-cadherin–mediated cell clustering. Restoration of stable intercellular contacts resulted essentially from the recruitment of an intracellular pool of N-cadherin molecules that accumulated into adherens junctions in tight association with the cytoskeleton and not from the redistribution of a preexisting pool of surface N-cadherin molecules. In addition, agents that cause elevation of intracellular Ca²⁺ after entry across the plasma membrane were potent inhibitors of cell aggregation and reduced the N-cadherin– mediated junctions in the cells. Finally, elevated serine/ threonine phosphorylation of catenins associated with N-cadherin accompanied the restoration of intercellular contacts. These results indicate that, in migrating neural crest cells, β1 and β3 integrins are at the origin of a cascade of signaling events that involve transmembrane Ca²⁺ fluxes, followed by activation of phosphatases and kinases, and that ultimately control the surface distribution and activity of N-cadherin. Such a direct coupling between adhesion receptors by means of intracellular signals may be significant for the coordinated interplay between cell–cell and cell–substratum adhesion that occurs during embryonic development, in wound healing, and during tumor invasion and metastasis.     

Integrin α2β1 Is a Receptor for the Cartilage Matrix Protein Chondroadherin

Chondroadherin (the 36-kD protein) is a leucine-rich, cartilage matrix protein known to mediate adhesion of isolated chondrocytes. In the present study we investigated cell surface proteins involved in the interaction of cells with chondroadherin in cell adhesion and by affinity purification. Adhesion of bovine articular chondrocytes to chondroadherin-coated dishes was dependent on Mg²⁺ or Mn²⁺ but not Ca²⁺. Adhesion was partially inhibited by an antibody recognizing β1 integrin subunit. Chondroadherin-binding proteins from chondrocyte lysates were affinity purified on chondroadherin-Sepharose. The β1 integrin antibody immunoprecipitated two proteins with molecular mass ~110 and 140 kD (nonreduced) from the EDTA-eluted material. These results indicate that a β1 integrin on chondrocytes interacts with chondroadherin. To identify the α integrin subunit(s) involved in interaction of cells with the protein, we affinity purified chondroadherin-binding membrane proteins from human fibroblasts. Immunoprecipitation of the EDTA-eluted material from the affinity column identified α2β1 as a chondroadherin-binding integrin. These results are in agreement with cell adhesion experiments where antibodies against the integrin subunit α2 partially inhibited adhesion of human fibroblast and human chondrocytes to chondroadherin. Since α2β1 also is a receptor for collagen type II, we tested the ability of different antibodies against the α2 subunit to inhibit adhesion of T47D cells to collagen type II and chondroadherin. The results suggested that adhesion to collagen type II and chondroadherin involves similar or nearby sites on the α2β1 integrin. Although α2β1 is a receptor for both collagen type II and chondroadherin, only adhesion of cells to collagen type II was found to mediate spreading.     

Guard Cells Possess a Calcium-Dependent Protein Kinase That Phosphorylates the KAT1 Potassium Channel

Increasing evidence suggests that changes in cytosolic Ca²⁺ levels and phosphorylation play important roles in the regulation of stomatal aperture and as ion transporters of guard cells. However, protein kinases responsible for Ca²⁺ signaling in guard cells remain to be identified. Using biochemical approaches, we have identified a Ca²⁺-dependent protein kinase with a calmodulin-like domain (CDPK) in guard cell protoplasts of Vicia faba. Both autophosphorylation and catalytic activity of CDPK are Ca²⁺ dependent. CDPK exhibits a Ca²⁺-induced electrophoretic mobility shift and its Ca²⁺-dependent catalytic activity can be inhibited by the calmodulin antagonists trifluoperazine and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide. Antibodies to soybean CDPKα cross-react with CDPK. Micromolar Ca²⁺ concentrations stimulate phosphorylation of several proteins from guard cells; cyclosporin A, a specific inhibitor of the Ca²⁺-dependent protein phosphatase calcineurin enhances the Ca²⁺-dependent phosphorylation of several soluble proteins. CDPK from guard cells phosphorylates the K⁺ channel KAT1 protein in a Ca²⁺-dependent manner. These results suggest that CDPK may be an important component of Ca²⁺ signaling in guard cells.     

A KATP Channel-Dependent Pathway within α Cells Regulates Glucagon Release from Both Rodent and Human Islets of Langerhans

Glucagon, secreted from pancreatic islet α cells, stimulates gluconeogenesis and liver glycogen breakdown. The mechanism regulating glucagon release is debated, and variously attributed to neuronal control, paracrine control by neighbouring β cells, or to an intrinsic glucose sensing by the α cells themselves. We examined hormone secretion and Ca²⁺ responses of α and β cells within intact rodent and human islets. Glucose-dependent suppression of glucagon release persisted when paracrine GABA or Zn²⁺ signalling was blocked, but was reversed by low concentrations (1–20 μM) of the ATP-sensitive K⁺ (K_ATP) channel opener diazoxide, which had no effect on insulin release or β cell responses. This effect was prevented by the K_ATP channel blocker tolbutamide (100 μM). Higher diazoxide concentrations (≥30 μM) decreased glucagon and insulin secretion, and α- and β-cell Ca²⁺ responses, in parallel. In the absence of glucose, tolbutamide at low concentrations (<1 μM) stimulated glucagon secretion, whereas high concentrations (>10 μM) were inhibitory. In the presence of a maximally inhibitory concentration of tolbutamide (0.5 mM), glucose had no additional suppressive effect. Downstream of the K_ATP channel, inhibition of voltage-gated Na⁺ (TTX) and N-type Ca²⁺ channels (ω-conotoxin), but not L-type Ca²⁺ channels (nifedipine), prevented glucagon secretion. Both the N-type Ca²⁺ channels and α-cell exocytosis were inactivated at depolarised membrane potentials. Rodent and human glucagon secretion is regulated by an α-cell K_ATP channel-dependent mechanism. We propose that elevated glucose reduces electrical activity and exocytosis via depolarisation-induced inactivation of ion channels involved in action potential firing and secretion.     

Modulation of the Local SR Ca2+ Release by Intracellular Mg2+ in Cardiac Myocytes

In cardiac muscle, Ca²⁺-induced Ca²⁺ release (CICR) from the sarcoplasmic reticulum (SR) defines the amplitude and time course of the Ca²⁺ transient. The global elevation of the intracellular Ca²⁺ concentration arises from the spatial and temporal summation of elementary Ca²⁺ release events, Ca²⁺ sparks. Ca²⁺ sparks represent the concerted opening of a group of ryanodine receptors (RYRs), which are under the control of several modulatory proteins and diffusible cytoplasmic factors (e.g., Ca²⁺, Mg²⁺, and ATP). Here, we examined by which mechanism the free intracellular Mg²⁺ ([Mg²⁺]_free) affects various Ca²⁺ spark parameters in permeabilized mouse ventricular myocytes, such as spark frequency, duration, rise time, and full width, at half magnitude and half maximal duration. Varying the levels of free ATP and Mg²⁺ in specifically designed solutions allowed us to separate the inhibition of RYRs by Mg²⁺ from the possible activation by ATP and Mg²⁺-ATP via the adenine binding site of the channel. Changes in [Mg²⁺]_free generally led to biphasic alterations of the Ca²⁺ spark frequency. For example, lowering [Mg²⁺]_free resulted in an abrupt increase of spark frequency, which slowly recovered toward the initial level, presumably as a result of SR Ca²⁺ depletion. Fitting the Ca²⁺ spark inhibition by [Mg²⁺]_free with a Hill equation revealed a K_i of 0.1 mM. In conclusion, our results support the notion that local Ca²⁺ release and Ca²⁺ sparks are modulated by Mg²⁺ in the intracellular environment. This seems to occur predominantly by hindering Ca²⁺-dependent activation of the RYRs through competitive Mg²⁺ occupancy of the high-affinity activation site of the channels. These findings help to characterize CICR in cardiac muscle under normal and pathological conditions, where the levels of Mg²⁺ and ATP can change.     

Adhesion molecule interactions facilitate human immunodeficiency virus type 1-induced virological synapse formation between T cells

Human immunodeficiency virus type 1 (HIV-1) can spread between CD4⁺ T cells by using a virological synapse (VS). The VS assembly is a cytoskeleton-driven process dependent on HIV-1 envelope glycoprotein (Env)-receptor engagement and is hypothesized to require adhesion molecule interactions. Here we demonstrate that leukocyte function-associated antigen 1 (LFA-1), intercellular adhesion molecule 1 (ICAM-1), and ICAM-3 are enriched at the VS and that inhibition of these interactions influences conjugate formation and reduces VS assembly. Moreover, CD4⁺ T cells deficient in LFA-1 or with modified LFA-1 function were less able to support VS assembly and cell-cell transfer of HIV-1. Thus, cognate adhesion molecule interactions at the VS are important for HIV-1 spread between T cells.     

Peripheral Hot Spots for Local Ca2+ Release after Single Action Potentials in Sympathetic Ganglion Neurons

Ca²⁺ release from the endoplasmic reticulum (ER) contributes to Ca²⁺ transients in frog sympathetic ganglion neurons. Here we use video-rate confocal fluo-4 fluorescence imaging to show that single action potentials reproducibly trigger rapidly rising Ca²⁺ transients at 1–3 local hot spots within the peripheral ER-rich layer in intact neurons in fresh ganglia and in the majority (74%) of cultured neurons. Hot spots were located near the nucleus or the axon hillock region. Other regions exhibited either slower and smaller signals or no response. Ca²⁺ signals spread into the cell at constant velocity across the ER in nonnuclear regions, indicating active propagation, but spread with a (time)^1/2 dependence within the nucleus, consistent with diffusion. 26% of cultured cells exhibited uniform Ca²⁺ signals around the periphery, but hot spots were produced by loading the cytosol with EGTA or by bathing such cells in low-Ca²⁺ Ringer's solution. Peripheral hot spots for Ca²⁺ release within the perinuclear and axon hillock regions provide a mechanism for preferential initiation of nuclear and axonal Ca²⁺ signals by single action potentials in sympathetic ganglion neurons.     

Microdomain Ca2+ Activation during Exocytosis in Paramecium Cells. Superposition of Local Subplasmalemmal Calcium Store Activation by Local Ca2+ Influx

In Paramecium tetraurelia, polyamine-triggered exocytosis is accompanied by the activation of Ca²⁺-activated currents across the cell membrane (Erxleben, C., and H. Plattner. 1994. J. Cell Biol. 127:935– 945). We now show by voltage clamp and extracellular recordings that the product of current × time (As) closely parallels the number of exocytotic events. We suggest that Ca²⁺ mobilization from subplasmalemmal storage compartments, covering almost the entire cell surface, is a key event. In fact, after local stimulation, Ca²⁺ imaging with high time resolution reveals rapid, transient, local signals even when extracellular Ca²⁺ is quenched to or below resting intracellular Ca²⁺ concentration ([Ca²⁺]_e [Ca²⁺]_i). Under these conditions, quenched-flow/freeze-fracture analysis shows that membrane fusion is only partially inhibited. Increasing [Ca²⁺]_e alone, i.e., without secretagogue, causes rapid, strong cortical increase of [Ca²⁺]_i but no exocytosis. In various cells, the ratio of maximal vs. minimal currents registered during maximal stimulation or single exocytotic events, respectively, correlate nicely with the number of Ca stores available. Since no quantal current steps could be observed, this is again compatible with the combined occurrence of Ca²⁺ mobilization from stores (providing close to threshold Ca²⁺ levels) and Ca²⁺ influx from the medium (which per se does not cause exocytosis). This implies that only the combination of Ca²⁺ flushes, primarily from internal and secondarily from external sources, can produce a signal triggering rapid, local exocytotic responses, as requested for Paramecium defense.     

Mitochondrial Participation in the Intracellular Ca2+ Network

Calcium can activate mitochondrial metabolism, and the possibility that mitochondrial Ca²⁺ uptake and extrusion modulate free cytosolic [Ca²⁺] (Ca_c) now has renewed interest. We use whole-cell and perforated patch clamp methods together with rapid local perfusion to introduce probes and inhibitors to rat chromaffin cells, to evoke Ca²⁺ entry, and to monitor Ca²⁺-activated currents that report near-surface [Ca²⁺]. We show that rapid recovery from elevations of Ca_c requires both the mitochondrial Ca²⁺ uniporter and the mitochondrial energization that drives Ca²⁺ uptake through it. Applying imaging and single-cell photometric methods, we find that the probe rhod-2 selectively localizes to mitochondria and uses its responses to quantify mitochondrial free [Ca²⁺] (Ca_m). The indicated resting Ca_m of 100–200 nM is similar to the resting Ca_c reported by the probes indo-1 and Calcium Green, or its dextran conjugate in the cytoplasm. Simultaneous monitoring of Ca_m and Ca_c at high temporal resolution shows that, although Ca_m increases less than Ca_c, mitochondrial sequestration of Ca²⁺ is fast and has high capacity. We find that mitochondrial Ca²⁺ uptake limits the rise and underlies the rapid decay of Ca_c excursions produced by Ca²⁺ entry or by mobilization of reticular stores. We also find that subsequent export of Ca²⁺ from mitochondria, seen as declining Ca_m, prolongs complete Ca_c recovery and that suppressing export of Ca²⁺, by inhibition of the mitochondrial Na⁺/ Ca²⁺ exchanger, reversibly hastens final recovery of Ca_c. We conclude that mitochondria are active participants in cellular Ca²⁺ signaling, whose unique role is determined by their ability to rapidly accumulate and then release large quantities of Ca²⁺.     

A function for tyrosine phosphorylation of type 1 inositol 1,4,5-trisphosphate receptor in lymphocyte activation

Sustained elevation of intracellular calcium by Ca²⁺ release–activated Ca²⁺ channels is required for lymphocyte activation. Sustained Ca²⁺ entry requires endoplasmic reticulum (ER) Ca²⁺ depletion and prolonged activation of inositol 1,4,5-trisphosphate receptor (IP₃R)/Ca²⁺ release channels. However, a major isoform in lymphocyte ER, IP3R1, is inhibited by elevated levels of cytosolic Ca²⁺, and the mechanism that enables the prolonged activation of IP₃R1 required for lymphocyte activation is unclear. We show that IP₃R1 binds to the scaffolding protein linker of activated T cells and colocalizes with the T cell receptor during activation, resulting in persistent phosphorylation of IP₃R1 at Tyr353. This phosphorylation increases the sensitivity of the channel to activation by IP₃ and renders the channel less sensitive to Ca²⁺-induced inactivation. Expression of a mutant IP3R1-Y353F channel in lymphocytes causes defective Ca²⁺ signaling and decreased nuclear factor of activated T cells activation. Thus, tyrosine phosphorylation of IP3R1-Y353 may have an important function in maintaining elevated cytosolic Ca²⁺ levels during lymphocyte activation.     

Mitochondrial Regulation of Store-operated Calcium Signaling in T Lymphocytes

Mitochondria act as potent buffers of intracellular Ca²⁺ in many cells, but a more active role in modulating the generation of Ca²⁺ signals is not well established. We have investigated the ability of mitochondria to modulate store-operated or “capacitative” Ca²⁺ entry in Jurkat leukemic T cells and human T lymphocytes using fluorescence imaging techniques. Depletion of the ER Ca²⁺ store with thapsigargin (TG) activates Ca²⁺ release-activated Ca²⁺ (CRAC) channels in T cells, and the ensuing influx of Ca²⁺ loads a TG- insensitive intracellular store that by several criteria appears to be mitochondria. Loading of this store is prevented by carbonyl cyanide m-chlorophenylhydrazone or by antimycin A1 + oligomycin, agents that are known to inhibit mitochondrial Ca²⁺ import by dissipating the mitochondrial membrane potential. Conversely, intracellular Na⁺ depletion, which inhibits Na⁺-dependent Ca²⁺ export from mitochondria, enhances store loading. In addition, we find that rhod-2 labels mitochondria in T cells, and it reports changes in Ca²⁺ levels that are consistent with its localization in the TG-insensitive store. Ca²⁺ uptake by the mitochondrial store is sensitive (threshold is <400 nM cytosolic Ca²⁺), rapid (detectable within 8 s), and does not readily saturate. The rate of mitochondrial Ca²⁺ uptake is sensitive to extracellular [Ca²⁺], indicating that mitochondria sense Ca²⁺ gradients near CRAC channels. Remarkably, mitochondrial uncouplers or Na⁺ depletion prevent the ability of T cells to maintain a high rate of capacitative Ca²⁺ entry over prolonged periods of >10 min. Under these conditions, the rate of Ca²⁺ influx in single cells undergoes abrupt transitions from a high influx to a low influx state. These results demonstrate that mitochondria not only buffer the Ca²⁺ that enters T cells via store-operated Ca²⁺ channels, but also play an active role in modulating the rate of capacitative Ca²⁺ entry.     

Cysteine String Protein Functions Directly in Regulated Exocytosis

Cysteine string protein (Csp) is essential for neurotransmitter release in Drosophila. It has been suggested that Csp functions by regulating the activity of presynaptic Ca²⁺ channels, thus controlling exocytosis. We have examined the effect of overexpressing Csp1 in PC12 cells, a neuroendocrine cell line. PC12 cell clones overexpressing Csp1 did not show any changes in morphology, granule number or distribution, or in the levels of other key exocytotic proteins. This overexpression did not affect intracellular Ca²⁺ signals after depolarization, suggesting that Csp1 has no gross effect on Ca²⁺ channel activity in PC12 cells. In contrast, we show that Csp1 overexpression enhances the extent of exocytosis from permeabilized cells in response to Ca²⁺ or GTPγS in the absence of Ca²⁺. Because secretion from permeabilized cells is not influenced by Ca²⁺ channel activity, this represents the first demonstration that Csp has a direct role in regulated exocytosis.     

Polymorphism of Ca2+ Sparks Evoked from In-Focus Ca2+ Release Units in Cardiac Myocytes

Ca²⁺ sparks are the elementary release events in many types of cells. Here we present a morphometric analysis of Ca²⁺ sparks (i.e., amplitude and kinetic parameters) using an approach that minimizes the confounding factor of the detection of out-of-focus events. By activation and visualization of Ca²⁺ sparks from Ca²⁺ release units under loose-seal patch-clamp conditions, we found that the amplitude and rising rate of in-focus sparks exhibited a broad modal distribution, whereas spark rise time and spatial width appeared to be stereotyped. Spark morphometrics were constant irrespective of the latency of spark production and the time-dependent L-type Ca²⁺ channel activation. Polymorphism of Ca²⁺ sparks in terms of variable amplitude and rising rate was evident for events from the same release units, and intra- and interrelease unit variability contributed equally to the overall variability. The rising rate, a reporter of the underlying Ca²⁺ release flux, displayed a strong positive correlation with spark amplitude, but a negative correlation with spark rise time, an index of Ca²⁺ release duration. On the basis of Ca²⁺ spark morphometrics measured here, we suggested a model in which cohorts of variable number of ryanodine receptors are activated in the genesis of Ca²⁺ sparks, and the ensuing negative feedback overrides the regenerative Ca²⁺-induced Ca²⁺ release to extinguish the ongoing Ca²⁺ spark.     

Ca2+ Homeostasis in the Endoplasmic Reticulum: Coexistence of High and Low [Ca2+] Subcompartments in Intact HeLa Cells

Two recombinant aequorin isoforms with different Ca²⁺ affinities, specifically targeted to the endoplasmic reticulum (ER), were used in parallel to investigate free Ca²⁺ homeostasis in the lumen of this organelle. Here we show that, although identically and homogeneously distributed in the ER system, as revealed by both immunocytochemical and functional evidence, the two aequorins measured apparently very different concentrations of divalent cations ([Ca²⁺]_er or [Sr²⁺]_er). Our data demonstrate that this contradiction is due to the heterogeneity of the [Ca²⁺] of the aequorin-enclosing endomembrane system. Because of the characteristics of the calibration procedure used to convert aequorin luminescence into Ca²⁺ concentration, the [Ca²⁺]_er values obtained at steady state tend, in fact, to reflect not the average ER values, but those of one or more subcompartments with lower [Ca²⁺]. These subcompartments are not generated artefactually during the experiments, as revealed by the dynamic analysis of the ER structure in living cells carried out by means of an ER-targeted green fluorescent protein. When the problem of ER heterogeneity was taken into account (and when Sr²⁺ was used as a Ca²⁺ surrogate), the bulk of the organelle was shown to accumulate free [cation²⁺]_er up to a steady state in the millimolar range. A theoretical model, based on the existence of multiple ER subcompartments of high and low [Ca²⁺], that closely mimics the experimental data obtained in HeLa cells during accumulation of either Ca²⁺ or Sr²⁺, is presented. Moreover, a few other key problems concerning the ER Ca²⁺ homeostasis have been addressed with the following conclusions: (a) the changes induced in the ER subcompartments by receptor generation of InsP₃ vary depending on their initial [Ca²⁺]. In the bulk of the system there is a rapid release whereas in the small subcompartments with low [Ca²⁺] the cation is simultaneously accumulated; (b) stimulation of Ca²⁺ release by receptor-generated InsP₃ is inhibited when the lumenal level is below a threshold, suggesting a regulation by [cation²⁺]_er of the InsP₃ receptor activity (such a phenomenon had already been reported, however, but only in subcellular fractions analyzed in vitro); and (c) the maintenance of a relatively constant level of cytosolic [Ca²⁺], observed when the cells are incubated in Ca²⁺-free medium, depends on the continuous release of the cation from the ER, with ensuing activation in the plasma membrane of the channels thereby regulated (capacitative influx).     

Evidence that Distinct States of the Integrin α6β1 Interact with Laminin and an ADAM

Integrins can exist in different functional states with low or high binding capacity for particular ligands. We previously provided evidence that the integrin α6β1, on mouse eggs and on α6-transfected cells, interacted with the disintegrin domain of the sperm surface protein ADAM 2 (fertilin β). In the present study we tested the hypothesis that different states of α6β1 interact with fertilin and laminin, an extracellular matrix ligand for α6β1. Using α6-transfected cells we found that treatments (e.g., with phorbol myristate acetate or MnCl₂) that increased adhesion to laminin inhibited sperm binding. Conversely, treatments that inhibited laminin adhesion increased sperm binding. Next, we compared the ability of fluorescent beads coated with either fertilin β or with the laminin E8 fragment to bind to eggs. In Ca²⁺-containing media, fertilin β beads bound to eggs via an interaction mediated by the disintegrin loop of fertilin β and by the α6 integrin subunit. In Ca²⁺-containing media, laminin E8 beads did not bind to eggs. Treatment of eggs with phorbol myristate acetate or with the actin disrupting agent, latrunculin A, inhibited fertilin bead binding, but did not induce laminin E8 bead binding. Treatment of eggs with Mn²⁺ dramatically increased laminin E8 bead binding, and inhibited fertilin bead binding. Our results provide the first evidence that different states of an integrin (α6β1) can interact with an extracellular matrix ligand (laminin) or a membrane-anchored cell surface ligand (ADAM 2).