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A central concept in the physiology of neurosecretion is that a rise in cytosolic [Ca(2+)] in the vicinity of plasmalemmal Ca(2+) channels due to Ca(2+) influx elicits exocytosis. Here, we examine the effect on spontaneous exocytosis of a rise in focal cytosolic [Ca(2+)] in the vicinity of ryanodine receptors (RYRs) due to release from internal stores in the form of Ca(2+) syntillas. Ca(2+) syntillas are focal cytosolic transients mediated by RYRs, which we first found in hypothalamic magnocellular neuronal terminals. (scintilla, Latin for spark; found in nerve terminals, normally synaptic structures.) We have also observed Ca(2+) syntillas in mouse adrenal chromaffin cells. Here, we examine the effect of Ca(2+) syntillas on exocytosis in chromaffin cells. In such a study on elicited exocytosis, there are two sources of Ca(2+): one due to influx from the cell exterior through voltage-gated Ca(2+) channels, and that due to release from intracellular stores. To eliminate complications arising from Ca(2+) influx, we have examined spontaneous exocytosis where influx is not activated. We report here that decreasing syntillas leads to an increase in spontaneous exocytosis measured amperometrically. Two independent lines of experimentation each lead to this conclusion. In one case, release from stores was blocked by ryanodine; in another, stores were partially emptied using thapsigargin plus caffeine, after which syntillas were decreased. We conclude that Ca(2+) syntillas act to inhibit spontaneous exocytosis, and we propose a simple model to account quantitatively for this action of syntillas.

Ca(2+) sparks are highly localized, transient releases of Ca(2+) from sarcoplasmic reticulum through ryanodine receptors (RyRs). In smooth muscle, Ca(2+) sparks trigger spontaneous transient outward currents (STOCs) by opening nearby clusters of large-conductance Ca(2+)-activated K(+) channels, and also gate Ca(2+)-activated Cl(-) (Cl((Ca))) channels to induce spontaneous transient inward currents (STICs). While the molecular mechanisms underlying the activation of STOCs by Ca(2+) sparks is well understood, little information is available on how Ca(2+) sparks activate STICs. In the present study, we investigated the spatial organization of RyRs and Cl((Ca)) channels in spark sites in airway myocytes from mouse. Ca(2+) sparks and STICs were simultaneously recorded, respectively, with high-speed, widefield digital microscopy and whole-cell patch-clamp. An image-based approach was applied to measure the Ca(2+) current underlying a Ca(2+) spark (I(Ca(spark))), with an appropriate correction for endogenous fixed Ca(2+) buffer, which was characterized by flash photolysis of NPEGTA. We found that I(Ca(spark)) rises to a peak in 9 ms and decays with a single exponential with a time constant of 12 ms, suggesting that Ca(2+) sparks result from the nonsimultaneous opening and closure of multiple RyRs. The onset of the STIC lags the onset of the I(Ca(spark)) by less than 3 ms, and its rising phase matches the duration of the I(Ca(spark)). We further determined that Cl((Ca)) channels on average are exposed to a [Ca(2+)] of 2.4 microM or greater during Ca(2+) sparks. The area of the plasma membrane reaching this level is <600 nm in radius, as revealed by the spatiotemporal profile of [Ca(2+)] produced by a reaction-diffusion simulation with measured I(Ca(spark)). Finally we estimated that the number of Cl((Ca)) channels localized in Ca(2+) spark sites could account for all the Cl((Ca)) channels in the entire cell. Taken together these results lead us to propose a model in which RyRs and Cl((Ca)) channels in Ca(2+) spark sites localize near to each other, and, moreover, Cl((Ca)) channels concentrate in an area with a radius of approximately 600 nm, where their density reaches as high as 300 channels/microm(2). This model reveals that Cl((Ca)) channels are tightly controlled by Ca(2+) sparks via local Ca(2+) signaling.

Caffeine-activated, large-conductance, nonselective cation channels (LCCs) have been found in the plasma membrane of isolated cardiac myocytes in several species. However, little is known about the effects of opening these channels. To examine such effects and to further understand the caffeine-activation mechanism, we carried out studies using whole-cell patch-clamp techniques with freshly isolated cardiac myocytes from rats and mice. Unlike previous studies, thapsigargin was used so that both the effect of opening LCCs and the action of caffeine were independent of Ca(2+) release from intracellular stores. These Ca(2+)-permeable LCCs were found in a majority of the cells from atria and ventricles, with a conductance of approximately 370 pS in rat atria. Caffeine and all its direct metabolic products (theophylline, theobromine, and paraxanthine) activated the channel, while isocaffeine did not. Although they share some similarities with ryanodine receptors (RyRs, the openings of which give rise to Ca(2+) sparks), LCCs also showed some different characteristics. With simultaneous Ca(2+) imaging and current recording, the localized fluorescence increase due to Ca(2+) entry through a single opening of an LCC (SCCaFT) was detected. When membrane potential, instead of current, was recorded, SCCaFT-like fluorescence transients (indicating single LCC openings) were found to accompany membrane depolarizations. To our knowledge, this is the first report directly linking membrane potential changes to a single opening of an ion channel. Moreover, these events in cardiac cells suggest a possible additional mechanism by which caffeine and theophylline contribute to the generation of cardiac arrhythmias.

This protocol describes a method for observing and measuring the movement of RNA molecules in the nucleus of living mammalian cells. Caged fluorescein-labeled DNA oligonucleotides are introduced into living mammalian cells, where they demonstrably hybridize to complementary RNA. After site-specific photoactivation at desired sites within the cell, the RNA movements away from those sites are followed and digitally recorded using a rapid acquisition microscopy system.

Eukaryotic cilia are assembled via intraflagellar transport (IFT) in which large protein particles are motored along ciliary microtubules. The IFT particles are composed of at least 17 polypeptides that are thought to contain binding sites for various cargos that need to be transported from their site of synthesis in the cell body to the site of assembly in the cilium. We show here that the IFT20 subunit of the particle is localized to the Golgi complex in addition to the basal body and cilia where all previous IFT particle proteins had been found. In living cells, fluorescently tagged IFT20 is highly dynamic and moves between the Golgi complex and the cilium as well as along ciliary microtubules. Strong knock down of IFT20 in mammalian cells blocks ciliary assembly but does not affect Golgi structure. Moderate knockdown does not block cilia assembly but reduces the amount of polycystin-2 that is localized to the cilia. This work suggests that IFT20 functions in the delivery of ciliary membrane proteins from the Golgi complex to the cilium.

Ca2+ stores were studied in a preparation of freshly dissociated terminals from hypothalamic magnocellular neurons. Depolarization from a holding level of -80 mV in the absence of extracellular Ca2+ elicited Ca2+ release from intraterminal stores, a ryanodine-sensitive process designated as voltage-induced Ca2+ release (VICaR). The release took one of two forms: an increase in the frequency but not the quantal size of Ca2+ syntillas, which are brief, focal Ca2+ transients, or an increase in global [Ca2+]. The present study provides evidence that the sensors of membrane potential for VICaR are dihydropyridine receptors (DHPRs). First, over the range of -80 to -60 mV, in which there was no detectable voltage-gated inward Ca2+ current, syntilla frequency was increased e-fold per 8.4 mV of depolarization, a value consistent with the voltage sensitivity of DHPR-mediated VICaR in skeletal muscle. Second, VICaR was blocked by the dihydropyridine antagonist nifedipine, which immobilizes the gating charge of DHPRs but not by Cd2+ or FPL 64176 (methyl 2,5 dimethyl-4[2-(phenylmethyl)benzoyl]-1H-pyrrole-3-carboxylate), a non-dihydropyridine agonist specific for L-type Ca2+ channels, having no effect on gating charge movement. At 0 mV, the IC50 for nifedipine blockade of VICaR in the form of syntillas was 214 nM in the absence of extracellular Ca2+. Third, type 1 ryanodine receptors, the type to which DHPRs are coupled in skeletal muscle, were detected immunohistochemically at the plasma membrane of the terminals. VICaR may constitute a new link between neuronal activity, as signaled by depolarization, and a rise in intraterminal Ca2+.

Clathrin assembly at the plasma membrane is a fundamental process required for endocytosis. In cultured cells, most of the clathrin is localized to large patches that display little lateral mobility. The functional role of these regions is not clear, and it has been thought that they may represent artifacts of cell adhesion of cultured cells. Here we have analyzed clathrin organization in primary adipose cells isolated from mice, which are nonadherent and fully differentiated. The majority of clathrin on the plasma membrane of these cells (>60%) was found in large clathrin patches that displayed virtually no lateral mobility and persisted for many minutes, and a smaller amount was found in small spots that appeared and disappeared rapidly. Direct visualization of transferrin revealed that it bound onto large arrays of clathrin, internalizing through vesicles that emerge from these domains. High resolution imaging (50 images/s) revealed fluorescence intensity fluctuations consistent with the formation and detachment of coated vesicles from within large patches. These results reveal that large clathrin assemblies are active regions of endocytosis in mammalian cells and highlight the importance of understanding the mechanistic basis for this organization.

Spontaneous, short-lived, focal cytosolic Ca2+ transients were found for the first time and characterized in freshly dissociated chromaffin cells from mouse. Produced by release of Ca2+ from intracellular stores and mediated by type 2 and perhaps type 3 ryanodine receptors (RyRs), these transients are quantitatively similar in magnitude and duration to Ca2+ syntillas in terminals of hypothalamic neurons, suggesting that Ca2+ syntillas are found in a variety of excitable, exocytotic cells. However, unlike hypothalamic nerve terminals, chromaffin cells do not display syntilla activation by depolarization of the plasma membrane, nor do they have type 1 RyRs. It is widely thought that focal Ca2+ transients cause "spontaneous" exocytosis, although there is no direct evidence for this view. Hence, we monitored catecholamine release amperometrically while simultaneously imaging Ca2+ syntillas, the first such simultaneous measurements. Syntillas failed to produce exocytotic events; and, conversely, spontaneous exocytotic events were not preceded by syntillas. Therefore, we suggest that a spontaneous syntilla, at least in chromaffin cells, releases Ca2+ into a cytosolic microdomain distinct from the microdomains containing docked, primed vesicles. Ryanodine (100 microM) reduced the frequency of Ca2+ syntillas by an order of magnitude but did not alter the frequency of spontaneous amperometric events, suggesting that syntillas are not involved in steps preparatory to spontaneous exocytosis. Surprisingly, ryanodine also increased the total charge of individual amperometric events by 27%, indicating that intracellular Ca2+ stores can regulate quantal size.

Speckles are nuclear bodies that contain pre-mRNA splicing factors and polyadenylated RNA. Because nuclear poly(A) RNA consists of both mRNA transcripts and nucleus-restricted RNAs, we tested whether poly(A) RNA in speckles is dynamic or rather an immobile, perhaps structural, component. Fluorescein-labeled oligo(dT) was introduced into HeLa cells stably expressing a red fluorescent protein chimera of the splicing factor SC35 and allowed to hybridize. Fluorescence correlation spectroscopy (FCS) showed that the mobility of the tagged poly(A) RNA was virtually identical in both speckles and at random nucleoplasmic sites. This same result was observed in photoactivation-tracking studies in which caged fluorescein-labeled oligo(dT) was used as hybridization probe, and the rate of movement away from either a speckle or nucleoplasmic site was monitored using digital imaging microscopy after photoactivation. Furthermore, the tagged poly(A) RNA was observed to rapidly distribute throughout the entire nucleoplasm and other speckles, regardless of whether the tracking observations were initiated in a speckle or the nucleoplasm. Finally, in both FCS and photoactivation-tracking studies, a temperature reduction from 37 to 22 degrees C had no discernible effect on the behavior of poly(A) RNA in either speckles or the nucleoplasm, strongly suggesting that its movement in and out of speckles does not require metabolic energy.

Ca(2+) sparks are highly localized Ca(2+) transients caused by Ca(2+) release from sarcoplasmic reticulum through ryanodine receptors (RyR). In smooth muscle, Ca(2+) sparks activate nearby large-conductance, Ca(2+)-sensitive K(+) (BK) channels to generate spontaneous transient outward currents (STOC). The properties of individual sites that give rise to Ca(2+) sparks have not been examined systematically. We have characterized individual sites in amphibian gastric smooth muscle cells with simultaneous high-speed imaging of Ca(2+) sparks using wide-field digital microscopy and patch-clamp recording of STOC in whole cell mode. We used a signal mass approach to measure the total Ca(2+) released at a site and to estimate the Ca(2+) current flowing through RyR [I(Ca(spark))]. The variance between spark sites was significantly greater than the intrasite variance for the following parameters: Ca(2+) signal mass, I(Ca(spark)), STOC amplitude, and 5-ms isochronic STOC amplitude. Sites that failed to generate STOC did so consistently, while those at the remaining sites generated STOC without failure, allowing the sites to be divided into STOC-generating and STOC-less sites. We also determined the average number of spark sites, which was 42/cell at a minimum and more likely on the order of at least 400/cell. We conclude that 1) spark sites differ in the number of RyR, BK channels, and coupling ratio of RyR-BK channels, and 2) there are numerous Ca(2+) spark-generating sites in smooth muscle cells. The implications of these findings for the organization of the spark microdomain are explored.

The feasibility of determining localized Ca(2+) influx using only wide-field fluorescence images was explored by imaging (using fluo-3) single channel Ca(2+) fluorescence transients (SCCaFTs), due to Ca(2+) entry through single openings of Ca(2+)-permeable ion channels, while recording unitary channel currents. Since the image obtained with wide-field optics is an integration of both in-focus and out-of-focus light, the total fluorescence increase (DeltaF(total) or "signal mass") associated with a SCCaFT can be measured directly from the image by adding together the fluorescence increase due to Ca(2+) influx in all of the pixels. The assumptions necessary for obtaining the signal mass from confocal linescan images are not required. Two- and three-dimensional imaging was used to show that DeltaF(total) is essentially independent of the position of the channel with respect to the focal plane of the microscope. The relationship between Ca(2+) influx and DeltaF(total) was obtained using SCCaFTs from plasma membrane caffeine-activated cation channels when Ca(2+) was the only charge carrier of the inward current. This relationship was found to be linear, with the value of the slope (or converting factor) affected by the particular imaging system set-up, the experimental conditions, and the properties of the fluorescent indicator, including its binding capacity with respect to other cellular buffers. The converting factor was used to estimate the Ca(2+) current passing through caffeine-activated channels in near physiological saline and to estimate the endogenous buffer binding capacity. In addition, it allowed a more accurate estimate of the Ca(2+) current underlying Ca(2+) sparks resulting from Ca(2+) release from intracellular stores via ryanodine receptors in the same preparation.

Recently, it has become possible to record the localized fluorescence transient associated with the opening of a single plasma membrane Ca(2+) permeable ion channel using Ca(2+) indicators like fluo-3. These Single Channel Ca(2+) Fluorescence Transients (SCCaFTs) share some of the characteristics of such elementary events as Ca(2+) sparks and Ca(2+) puffs caused by Ca(2+) release from intracellular stores (due to the opening of ryanodine receptors and IP(3) receptors, respectively). In contrast to intracellular Ca(2+) release events, SCCaFTs can be observed while simultaneously recording the unitary channel currents using patch-clamp techniques to verify the channel openings. Imaging SCCaFTs provides a way to examine localized Ca(2+) handling in the vicinity of a channel with a known Ca(2+) influx, to obtain the Ca(2+) current passing through plasma membrane cation channels in near physiological solutions, to localize Ca(2+) permeable ion channels on the plasma membrane, and to estimate the Ca(2+) currents underlying those elementary events where the Ca(2+) currents cannot be recorded. Here we review studies of these fluorescence transients associated with caffeine-activated channels, L-type Ca(2+) channels, and stretch-activated channels. For the L-type Ca(2+) channel, SCCaFTs have been termed sparklets. In addition, we discuss how SCCaFTs have been used to estimate Ca(2+) currents using the rate of rise of the fluorescence transient as well as the signal mass associated with the total fluorescence increase.

The mitochondrial membrane potential (DeltaPsi(m)) underlies many mitochondrial functions, including Ca(2+) influx into the mitochondria, which allows them to serve as buffers of intracellular Ca(2+). Spontaneous depolarizations of DeltaPsi(m), flickers, have been observed in isolated mitochondria and intact cells using the fluorescent cationic lipophile tetramethylrhodamine ethyl ester (TMRE), which distributes across the inner mitochondrial membrane in accordance with the Nernst equation. Flickers in cardiomyocytes have been attributed to uptake of Ca(2+) released from the sarcoplasmic reticulum (SR) via ryanodine receptors in focal transients called Ca(2+) sparks. We have shown previously that an increase in global Ca(2+) in smooth muscle cells causes an increase in mitochondrial Ca(2+) and depolarization of DeltaPsi(m). Here we sought to determine whether flickers in smooth muscle cells are caused by uptake of Ca(2+) released focally in Ca(2+) sparks. High-speed three-dimensional imaging was used to monitor DeltaPsi(m) in freshly dissociated myocytes from toad stomach that were simultaneously voltage clamped at 0 mV to ensure the cytosolic TMRE concentration was constant and equal to the low level in the bath (2.5 nM). This approach allows quantitative analysis of flickers as we have previously demonstrated. Depletion of SR Ca(2+) not only failed to eliminate flickers but rather increased their magnitude and frequency somewhat. Flickers were not altered in magnitude or frequency by ryanodine or xestospongin C, inhibitors of intracellular Ca(2+) release, or by cyclosporin A, an inhibitor of the permeability transition pore. Focal Ca(2+) release from the SR does not cause flickers in the cells employed here.

Localized, brief Ca2+ transients (Ca2+ syntillas) caused by release from intracellular stores were found in isolated nerve terminals from magnocellular hypothalamic neurons and examined quantitatively using a signal mass approach to Ca2+ imaging. Ca2+ syntillas (scintilla, L., spark, from a synaptic structure, a nerve terminal) are caused by release of approximately 250,000 Ca ions on average by a Ca2+ flux lasting on the order of tens of milliseconds and occur spontaneously at a membrane potential of -80 mV. Syntillas are unaffected by removal of extracellular Ca2+, are mediated by ryanodine receptors (RyRs) and are increased in frequency, in the absence of extracellular Ca2+, by physiological levels of depolarization. This represents the first direct demonstration of mobilization of Ca2+ from intracellular stores in neurons by depolarization without Ca2+ influx. The regulation of syntillas by depolarization provides a new link between neuronal activity and cytosolic [Ca2+] in nerve terminals.

Although the complex process of ribosome assembly in the nucleolus is beginning to be understood, little is known about how the ribosomal subunits move from the nucleolus to the nuclear membrane for transport to the cytoplasm. We show here that large ribosomal subunits move out from the nucleolus and into the nucleoplasm in all directions, with no evidence of concentrated movement along directed paths. Mobility was slowed compared with that expected in aqueous solution in a manner consistent with anomalous diffusion. Once nucleoplasmic, the subunits moved in the same random manner and also sometimes visited another nucleolus before leaving the nucleus.

Spontaneous transient depolarizations in mitochondrial membrane potential (DeltaPsi(m)), mitochondrial flickers, have been observed in isolated mitochondria and intact cells using the fluorescent probe, tetramethylrhodamine ethyl ester (TMRE). In theory, the ratio of [TMRE] in cytosol and mitochondrion allows DeltaPsi(m) to be calculated with the Nernst equation, but this has proven difficult in practice due to fluorescence quenching and binding of dye to mitochondrial membranes. We developed a new method to determine the amplitude of flickers in terms of millivolts of depolarization. TMRE fluorescence was monitored using high-speed, high-sensitivity three-dimensional imaging to track individual mitochondria in freshly dissociated smooth muscle cells. Resting mitochondrial fluorescence, an exponential function of resting DeltaPsi(m), varied among mitochondria and was approximately normally distributed. Spontaneous changes in mitochondrial fluorescence, indicating depolarizations and repolarizations in DeltaPsi(m), were observed. The depolarizations were reversible and did not result in permanent depolarization of the mitochondria. The magnitude of the flickers ranged from <10 mV to >100 mV with a mean of 17.6 +/- 1.0 mV (n = 360) and a distribution skewed to smaller values. Nearly all mitochondria flickered, and they did so independently of one another, indicating that mitochondria function as independent units in the myocytes employed here.

Myosin light chain phosphatase (PP1M) is composed of three subunits, i.e., M20, MBS, and a catalytic subunit. Whereas MBS is assigned as a myosin binding subunit, the function of M20 is unknown. In the present study, we found that M20 binds to microtubules. The binding activity was revealed by cosedimentation of M20 with microtubules and binding of tubulin to M20 affinity resin. Green fluorescent protein (GFP)-tagged M20 (M20-GFP) was expressed in chicken primary smooth muscle cells and COS-7 cells and was used as a probe for studying the association between M20 and microtubules in living cells. M20-GFP was localized on filamentous structures in both cell types. Colocalization analysis revealed that M20-GFP colocalized with tubulin. Treatment with nocodazole, but not cytochalasin B, abolished the filamentous structure of M20-GFP. These results indicate that M20-GFP associates with microtubules in cells. Microinjection of rhodamine-tubulin into the M20-expressing cells revealed that incorporation of rhodamine-tubulin into microtubules was significantly facilitated by microtubule-associated M20. Consistent with this result, M20 enhanced the rate of tubulin polymerization in vitro and produced elongated microtubules. These results suggest that M20 has a microtubule binding activity and plays a role in regulating microtubule dynamics.

Although the physiological relevance of mitochondrial Ca2+ homeostasis is widely accepted, no information is yet available on the molecular identity of the proteins involved in this process. Here we analyzed the role of the voltage-dependent anion channel (VDAC) of the outer mitochondrial membrane in the transmission of Ca2+ signals between the ER and mitochondria by measuring cytosolic and organelle [Ca2+] with targeted aequorins and Ca2+-sensitive GFPs. In HeLa cells and skeletal myotubes, the transient expression of VDAC enhanced the amplitude of the agonist-dependent increases in mitochondrial matrix Ca2+ concentration by allowing the fast diffusion of Ca2+ from ER release sites to the inner mitochondrial membrane. Indeed, high speed imaging of mitochondrial and cytosolic [Ca2+] changes showed that the delay between the rises occurring in the two compartments is significantly shorter in VDAC-overexpressing cells. As to the functional consequences, VDAC-overexpressing cells are more susceptible to ceramide-induced cell death, thus confirming that mitochondrial Ca2+ uptake plays a key role in the process of apoptosis. These results reveal a novel function for the widely expressed VDAC channel, identifying it as a molecular component of the routes for Ca2+ transport across the mitochondrial membranes.

Telokin is an acidic protein with a sequence identical to the COOH-terminal domain of myosin light chain kinase (MLCK) produced by an alternate promoter of the MLCK gene. Although it is abundantly expressed in smooth muscle, its physiological function is not understood. In the present study, we attempted to clarify the function of telokin by analyzing its spatial and temporal localization in living single smooth muscle cells. Primary cultured smooth muscle cells were transfected with green fluorescent protein (GFP)-tagged telokin. The telokin-GFP localized mostly diffusely in cytosol. Stimulation with both sodium nitroprusside (SNP) and 8-bromo-cyclic GMP induced translocation of GFP-tagged telokin to near plasma membrane in living single smooth muscle cells. The translocation was slow, and it took more than 10 min at room temperature. Mutation of the phosphorylation sites of telokin (S13A, S19A, and S13A/S19A) significantly attenuated SNP-induced translocation. Both KT-5823 (cGMP-dependent protein kinase inhibitor) and PD-98059 (mitogen-activated protein kinase inhibitor) diminished the telokin-GFP translocation. These results suggest that telokin changes its intracellular localization because of phosphorylation at Ser13 and/or Ser19 via the cGMP-signaling pathway.

Ca(2+) sparks are small, localized cytosolic Ca(2+) transients due to Ca(2+) release from sarcoplasmic reticulum through ryanodine receptors. In smooth muscle, Ca(2+) sparks activate large conductance Ca(2+)-activated K(+) channels (BK channels) in the spark microdomain, thus generating spontaneous transient outward currents (STOCs). The purpose of the present study is to determine experimentally the level of Ca(2+) to which the BK channels are exposed during a spark. Using tight seal, whole-cell recording, we have analyzed the voltage-dependence of the STOC conductance (g((STOC))), and compared it to the voltage-dependence of BK channel activation in excised patches in the presence of different [Ca(2+)]s. The Ca(2+) sparks did not change in amplitude over the range of potentials of interest. In contrast, the magnitude of g((STOC)) remained roughly constant from 20 to -40 mV and then declined steeply at more negative potentials. From this and the voltage dependence of BK channel activation, we conclude that the BK channels underlying STOCs are exposed to a mean [Ca(2+)] on the order of 10 microM during a Ca(2+) spark. The membrane area over which a concentration > or =10 microM is reached has an estimated radius of 150-300 nm, corresponding to an area which is a fraction of one square micron. Moreover, given the constraints imposed by the estimated channel density and the Ca(2+) current during a spark, the BK channels do not appear to be uniformly distributed over the membrane but instead are found at higher density at the spark site.

Stretch-activated channels (SACs) have been found in smooth muscle and are thought to be involved in myogenic responses. Although SACs have been shown to be Ca(2+) permeable when Ca(2+) is the only charge carrier, it has not been clearly demonstrated that significant Ca(2+) passes through SACs in physiological solutions. By imaging at high temporal and spatial resolution the single-channel Ca(2+) fluorescence transient (SCCaFT) arising from Ca(2+) entry through a single SAC opening, we provide direct evidence that significant Ca(2+) can indeed pass through SACs and increase the local [Ca(2+)]. Results were obtained under conditions where the only source of Ca(2+) was the physiological salt solution in the patch pipette containing 2 mM Ca(2+). Single smooth muscle cells were loaded with fluo-3 acetoxymethyl ester, and the fluorescence was recorded by using a wide-field digital imaging microscope while SAC currents were simultaneously recorded from cell-attached patches. Fluorescence increases at the cell-attached patch were clearly visualized before the simultaneous global Ca(2+) increase that occurred because of Ca(2+) influx through voltage-gated Ca(2+) channels when the membrane was depolarized by inward SAC current. From measurements of total fluorescence ("signal mass") we determined that about 18% of the SAC current is carried by Ca(2+) at membrane potentials more negative than the resting level. This would translate into at least a 0.35-pA unitary Ca(2+) current at the resting potential. Such Ca(2+) currents passing through SACs are sufficient to activate large-conductance Ca(2+)-activated K(+) channels and, as shown previously, to trigger Ca(2+) release from intracellular stores.

Agonist-induced translocation of RhoA and the spatio-temporal change in myosin regulatory light chain (MLC20) phosphorylation in smooth muscle was clarified at the single cell level. We expressed green fluorescent protein-tagged RhoA in the differentiated tracheal smooth muscle cells and visualized the translocation of RhoA in a living cell with three-dimensional digital imaging analysis. The stimulation of the cells by carbachol initiated the translocation of green fluorescent protein-tagged wild type RhoA to the plasma membrane within a minute. The change in MLC20 phosphorylation level after carbachol stimulation was monitored by using phospho-Ser-19-specific antibody recognizing the phosphorylated MLC20 in single cells. Cells expressing the dominant negative form (T19N) of RhoA significantly suppressed sustained MLC20 phosphorylation during the prolonged phase (>300 s), whereas the maximum phosphorylation level (reached at 10 s after stimulation) of these cells was not significantly different from the control cells. The kinetics of RhoA translocation was consistent with that of sustained myosin phosphorylation, suggesting the involvement of a RhoA pathway. Carbachol stimulation increased myosin phosphorylation within a minute both at the cortical and the central region. On the other hand, during prolonged phase, myosin phosphorylation was sustained at the cortical region of the cells but not at the central fibers. A myosin light chain kinase-specific inhibitor, ML-9, diminished myosin phosphorylation at the central region of the cells after the stimulation but not at the cortical area. On the other hand, Y-27632, a Rho kinase-specific inhibitor, diminished myosin phosphorylation at the cortical region but not the central region. The results clearly show that the myosin light chain kinase pathway and the Rho pathway distinctly change myosin phosphorylation in smooth muscle cells in both a temporal and spatial manner.

Recycling endosomes in astrocytes show hormone-regulated, actin fiber-dependent delivery to the endosomal sorting pool. Recycling vesicle trafficking was followed in real time using a fusion protein composed of green fluorescent protein coupled to the 29-kDa subunit of the short-lived, membrane-bound enzyme type 2 deiodinase. Primary endosomes budded from the plasma membrane and oscillated near the cell periphery for 1-4 min. The addition of thyroid hormone triggered the processive, centripetal movement of the recycling vesicle in linear bursts at velocities of up to 200 nm/s. Vesicle migration was hormone-specific and blocked by inhibitors of actin polymerization and myosin ATPase. Domain mapping confirmed that the hormone-dependent vesicle-binding domain was located at the C terminus of the motor. In addition, the interruption of normal dimerization of native myosin 5a monomers inactivated vesicle transport, indicating that single-headed myosin 5a motors do not transport cargo in situ. This is the first demonstration of processive hormone-dependent myosin 5a movement in living cells.

Haematopoietic stem cells (HSCs) have been extensively characterized regarding in vivo engraftment, surface epitopes and genetic regulation. However, little is known about the homing of these rare cells, and their intrinsic motility and membrane deformation capacity. We used high-speed optical-sectioning microscopy and inverted fluorescent videomicroscopy to study highly purified murine lineage-negative, rhodamine-low, Hoechst-low HSCs over time under various in vitro conditions. We discovered extremely rapid motility, directed migration to stromal cells and marked membrane modulation. High resolution images with three-dimensional reconstruction showed the general presence of microspikes. Further, pseudopodia (proteopodia) were observed that were induced by stromal-derived factor-1 and steel factor. Proteopodia were directed towards and were quenched by stromal cells, at times bridged HSCs, and could rapidly retract or detach from cells. Proteopodia were also observed in vivo with homed HSCs in frozen sections of murine spleen, lung and heart. This is the first demonstration that HSCs are both fast and highly malleable in phenotype.

We recorded Ca2+ sparks and spontaneous transient outward currents (STOCs) simultaneously in smooth muscle cells using whole-cell patch recording and a unique, high-speed widefield digital imaging system to monitor fluo-3 fluorescence in both two and three dimensions (2D and 3D). In 2D imaging, the correlation between the amplitude of a spark and its corresponding STOC was a weak one, and 27 % of the sparks failed to cause STOCs. The STOCless sparks were not significantly different in amplitude from those that caused STOCs. Three-dimensional imaging disclosed that STOCless sparks were located close to the cell surface, and on average their apparent distance from the cell surface was not significantly different from the sparks that cause STOCs. Statistical evaluation of spark clusters disclosed that there were regions of the cell where the probability of spark occurrence was high and others where it was quite low.

Ca(2+) sparks are highly localized cytosolic Ca(2+) transients caused by a release of Ca(2+) from the sarcoplasmic reticulum via ryanodine receptors (RyRs); they are the elementary events underlying global changes in Ca(2+) in skeletal and cardiac muscle. In smooth muscle and some neurons, Ca(2+) sparks activate large conductance Ca(2+)-activated K(+) channels (BK channels) in the spark microdomain, causing spontaneous transient outward currents (STOCs) that regulate membrane potential and, hence, voltage-gated channels. Using the fluorescent Ca(2+) indicator fluo-3 and a high speed widefield digital imaging system, it was possible to capture the total increase in fluorescence (i.e., the signal mass) during a spark in smooth muscle cells, which is the first time such a direct approach has been used in any system. The signal mass is proportional to the total quantity of Ca(2+) released into the cytosol, and its rate of rise is proportional to the Ca(2+) current flowing through the RyRs during a spark (I(Ca(spark))). Thus, Ca(2+) currents through RyRs can be monitored inside the cell under physiological conditions. Since the magnitude of I(Ca(spark)) in different sparks varies more than fivefold, Ca(2+) sparks appear to be caused by the concerted opening of a number of RyRs. Sparks with the same underlying Ca(2+) current cause STOCs, whose amplitudes vary more than threefold, a finding that is best explained by variability in coupling ratio (i.e., the ratio of RyRs to BK channels in the spark microdomain). The time course of STOC decay is approximated by a single exponential that is independent of the magnitude of signal mass and has a time constant close to the value of the mean open time of the BK channels, suggesting that STOC decay reflects BK channel kinetics, rather than the time course of [Ca(2+)] decline at the membrane. Computer simulations were carried out to determine the spatiotemporal distribution of the Ca(2+) concentration resulting from the measured range of I(Ca(spark)). At the onset of a spark, the Ca(2+) concentration within 200 nm of the release site reaches a plateau or exceeds the [Ca(2+)](EC50) for the BK channels rapidly in comparison to the rate of rise of STOCs. These findings suggest a model in which the BK channels lie close to the release site and are exposed to a saturating [Ca(2+)] with the rise and fall of the STOCs determined by BK channel kinetics. The mechanism of signaling between RyRs and BK channels may provide a model for Ca(2+) action on a variety of molecular targets within cellular microdomains.

Myosin plays an important role in mitosis, especially during cytokinesis. Although it has been assumed that phosphorylation of regulatory light chain of myosin (RLC) controls motility of mammalian non-muscle cells, the functional significance of RLC phosphorylation remains uninvestigated. To address this problem, we have produced unphosphorylatable RLC (T18A/S19A RLC) and overexpressed it in COS-7 cells and normal rat kidney cells. Overexpression of T18A/S19A RLC but not wild type RLC almost completely abolished concanavalin A-induced receptor cap formation. The results indicate that myosin phosphorylation is critical for concanavalin A-induced gathering of surface receptors. T18A/S19A RLC overexpression resulted in the production of multinucleated cells, suggesting the failure of proper cell division in these cells. Video microscopic observation revealed that cells expressing T18A/S19A RLC showed abnormalities during mitosis in two respects. One is that the cells produced abnormal cleavage furrows, resulting in incomplete cytokinesis, which suggests that myosin phosphorylation is important for the normal recruitment of myosin molecules into the contractile ring structure. The other is that separation of chromosomes from the metaphase plate is disrupted in T18A/S19A RLC expressing cells, thus preventing proper transition from metaphase to anaphase. These results suggest that, in addition to cytokinesis, myosin and myosin phosphorylation play a role in the karyokinetic process.

InsP(3)-evoked elementary Ca(2+) release events have been postulated to play a role in providing the building blocks of larger Ca(2+) signals. In pancreatic acinar cells, low concentrations of acetylcholine or the injection of low concentrations of InsP(3) elicit a train of spatially localized Ca(2+) spikes. In this study we have quantified these responses and compared the Ca(2+) signals to the elementary events shown in Xenopus oocytes. The results demonstrate, at the same concentrations of InsP(3), Ca(2+) signals consisting of one population of small transient Ca(2+) release events and a second distinct population of larger Ca(2+) spikes. The signal mass amplitudes of both types of events are within the range of amplitudes for the elementary events in Xenopus oocytes. However, the bimodal Ca(2+) distribution of Ca(2+) responses we observe is not consistent with the continuum of event sizes seen in Xenopus. We conclude that the two types of InsP(3)-dependent events in acinar cells are both elementary Ca(2+) signals, which are independent of one another. Our data indicate a complexity to the organization of the Ca(2+) release apparatus in acinar cells, which might result from the presence of multiple InsP(3) receptor isoforms, and is likely to be important in the physiology of these cells.

This study was undertaken to obtain direct evidence for the involvement of gap junctions in the propagation of intercellular Ca(2+) waves. Gap junction-deficient HeLa cells were transfected with plasmids encoding for green fluorescent protein (GFP) fused to the cytoplasmic carboxyl termini of connexin 43 (Cx43), 32 (Cx32), or 26 (Cx26). The subsequently expressed GFP-labeled gap junctions rendered the cells dye- and electrically coupled and were detected at the plasma membranes at points of contact between adjacent cells. To correlate the distribution of gap junctions with the changes in [Ca(2+)](i) associated with Ca(2+) waves and the distribution of the endoplasmic reticulum (ER), cells were loaded with fluorescent Ca(2+)-sensitive (fluo-3 and fura-2) and ER membrane (ER-Tracker) dyes. Digital high-speed microscopy was used to collect a series of image slices from which the three-dimensional distribution of the gap junctions and ER were reconstructed. Subsequently, intercellular Ca(2+) waves were induced in these cells by mechanical stimulation with or without extracellular apyrase, an ATP-degrading enzyme. In untransfected HeLa cells and in the absence of apyrase, cell-to-cell propagating [Ca(2+)](i) changes were characterized by initiating Ca(2+) puffs associated with the perinuclear ER. By contrast, in Cx-GFP-transfected cells and in the presence of apyrase, [Ca(2+)](i) changes were propagated without initiating perinuclear Ca(2+) puffs and were communicated between cells at the sites of the Cx-GFP gap junctions. The efficiency of Cx expression determined the extent of Ca(2+) wave propagation. These results demonstrate that intercellular Ca(2+) waves may be propagated simultaneously via an extracellular pathway and an intracellular pathway through gap junctions and that one form of communication may mask the other.

1. A digital imaging microscope with fura-2 as the Ca2+ indicator was used to determine the sources for the rise in intracellular calcium concentration ([Ca2+]i) that occurs when the membrane in a cell-attached patch is stretched. Unitary ionic currents from stretch-activated channels and [Ca2+]i images were recorded simultaneously. 2. When suction was applied to the patch pipette to stretch a patch of membrane, Ca2+-permeable cation channels (stretch-activated channels) opened and a global increase in [Ca2+]i occurred, as well as a greater focal increase in the vicinity of the patch pipette. The global changes in [Ca2+]i occurred only when stretch-activated currents were sufficient to cause membrane depolarization, as indicated by the reduction in amplitude of the unitary currents. 3. When Ca2+ was present only in the pipette solution, just the focal change in [Ca2+]i was obtained. This focal change was not seen when the contribution from Ca2+ stores was eliminated using caffeine and ryanodine. 4. These results suggest that the opening of stretch-activated channels allows ions, including Ca2+, to enter the cell. The entry of positive charge triggers the influx of Ca2+ into the cell by causing membrane depolarization, which presumably activates voltage-gated Ca2+ channels. The entry of Ca2+ through stretch-activated channels is also amplified by Ca2+ release from internal stores. This amplification appears to be greater than that obtained by activation of whole-cell Ca2+ currents. These multiple pathways whereby membrane stretch causes a rise in [Ca2+]i may play a role in stretch-induced contraction, which is a characteristic of many smooth muscle tissues.

1. The Ca(2+)-sensitive fluorescent indicator rhod-2 was used to monitor mitochondrial Ca2+ concentration ([Ca2+]m) in gastric smooth muscle cells from Bufo marinus. In some studies, fura-2 was used in combination with rhod-2, allowing simultaneous measurement of cytoplasmic Ca2+ concentration ([Ca2+]i) and [Ca2+]m, respectively. 2. During a short train of depolarizations, which causes Ca2+ influx from the extracellular medium, there was an increase in both [Ca2+]i and [Ca2+]m. The half-time (t1/2) to peak for the increase in [Ca2+]m was considerably longer than the t1/2 to peak for the increase in [Ca2+]i. [Ca2+]m remained elevated for tens of seconds after [Ca2+]i had returned to its resting value. 3. Stimulation with caffeine, which causes release of Ca2+ from the sarcoplasmic reticulum (SR), also produced increases in both [Ca2+]i and [Ca2+]m. The values of t1/2 to peak for the increase in [Ca2+] in both cytoplasm and mitochondria were similar; however, [Ca2+]i returned to baseline values much faster than [Ca2+]m. 4. Using a wide-field digital imaging microscope, changes in [Ca2+]m were monitored within individual mitochondria in situ, during stimulation of Ca2+ influx or Ca2+ release from the SR. 5. Mitochondrial Ca2+ uptake during depolarizing stimulation caused depolarization of the mitochondrial membrane potential. The mitochondrial membrane potential recovered considerably faster than the recovery of [Ca2+]m. 6. This study shows that Ca2+ influx from the extracellular medium and Ca2+ release from the SR are capable of increasing [Ca2+]m in smooth muscle cells. The efflux of Ca2+ from the mitochondria is a slow process and appears to be dependent upon the amount of Ca2+ in the SR.

Discrete localized fluorescence transients due to openings of a single plasma membrane Ca(2+) permeable cation channel were recorded using wide-field digital imaging microscopy with fluo-3 as the Ca(2+) indicator. These transients were obtained while simultaneously recording the unitary channel currents using the whole-cell current-recording configuration of the patch-clamp technique. This cation channel in smooth muscle cells is opened by caffeine (Guerrero, A., F.S. Fay, and J.J. Singer. 1994. J. Gen. Physiol. 104:375-394). The localized fluorescence transients appeared to occur at random locations on the cell membrane, with the duration of the rising phase matching the duration of the channel opening. Moreover, these transients were only observed in the presence of sufficient extracellular Ca(2+), suggesting that they are due to Ca(2+) influx from the bathing solution. The fluorescence transient is characterized by an initial fast rising phase when the channel opens, followed by a slower rising phase during prolonged openings. When the channel closes there is an immediate fast falling phase followed by a slower falling phase. Computer simulations of the underlying events were used to interpret the time course of the transients. The rapid phases are mainly due to the establishment or removal of Ca(2+) and Ca(2+)-bound fluo-3 gradients near the channel when the channel opens or closes, while the slow phases are due to the diffusion of Ca(2+) and Ca(2+)-bound fluo-3 into the cytoplasm. Transients due to short channel openings have a "Ca(2+) spark-like" appearance, suggesting that the rising and early falling components of sparks (due to openings of ryanodine receptors) reflect the fast phases of the fluorescence change. The results presented here suggest methods to determine the relationship between the fluorescence transient and the underlying Ca(2+) current, to study intracellular localized Ca(2+) handling as might occur from single Ca(2+) channel openings, and to localize Ca(2+) permeable ion channels on the plasma membrane.

1. Cytosolic Ca2+ has been proposed to act as both a positive and a negative feedback signal on the inositol trisphosphate (InsP3) receptor. However, it is unclear how this might affect the Ca2+ response in vivo. 2. Mouse pancreatic acinar cells were whole-cell patch clamped to record the Ca2+-dependent chloride (Cl(Ca)) current spikes and imaged to record the cytosolic Ca2+ spikes elicited by the injection of Ins(2,4,5)P3. Increasing concentrations of Ca2+ buffer (up to 200 microM EGTA or BAPTA) were associated with the appearance of steps in the current activation phase and a prevalence of smaller-amplitude Cl(Ca) spikes. Imaging experiments showed that with increased buffer the secretory pole cytosolic Ca2+ signal became fragmented and spatially discrete Ca2+ release events were observed. 3. At higher buffer concentrations (200-500 microM), increasing concentrations of EGTA increased spike frequency and reduced spike amplitude. In contrast, BAPTA decreased spike frequency and maintained large spike amplitudes. 4. We conclude that, during InsP3-evoked spiking, long-range Ca2+ feedback ( approximately 2-4 microm) shapes the rising phase of the Ca2+ signal by acting to co-ordinate discrete Ca2+ release events and short-range ( approximately 40 nm) Ca2+ feedback acts to inhibit further Ca2+ release.

1. The Ca2+-sensitive fluorescent indicator rhod-2 was used to measure mitochondrial [Ca2+] ([Ca2+]m) in single smooth muscle cells from the rat pulmonary artery, while simultaneously monitoring cytosolic [Ca2+] ([Ca2+]i) with fura-2. 2. Application of caffeine produced an increase in [Ca2+]i and also increased [Ca2+]m. The increase in [Ca2+]m occurred after the increase in [Ca2+]i, and remained elevated for a considerable time after [Ca2+]i had returned to resting values. 3. The protonophore carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP), which causes the mitochondrial membrane potential to collapse, markedly attenuated the increase in [Ca2+]m following caffeine application and also increased the half-time for recovery of [Ca2+]i to resting values. 4. Activation of purinoceptors with ATP also produced increases in both [Ca2+]i and [Ca2+]m in these smooth muscle cells. In some cells, oscillations in [Ca2+]i were observed during ATP application, which produced corresponding oscillations in [Ca2+]m and membrane currents. 5. This study provides direct evidence that Ca2+ release from the sarcoplasmic reticulum, either through ryanodine or inositol 1,4, 5-trisphosphate (InsP3) receptors, increases both cytosolic and mitochondrial [Ca2+] in smooth muscle cells. These results have potential implications both for the role of mitochondria in Ca2+ regulation in smooth muscle, and for understanding how cellular metabolism is regulated.

Background: Messenger RNA (mRNA) is transcribed and processed in the nucleus of eucaryotic cells and then exported to the cytoplasm through nuclear pores. It is not known whether the movement of mRNA from its site of synthesis to the nuclear pore is directed or random. Directed movement would suggest that there is an energy-requiring step in addition to the step required for active transport through the pore, whereas random movement would indicate that mRNAs can make their way to the nuclear envelope by diffusion., Results: We devised a method to visualize movement of endogenous polymerase II transcripts in the nuclei of living cells. Oligo(dT) labeled with chemically masked (caged) fluorescein was allowed to penetrate cells and hybridize to nuclear poly(A) RNA. Laser spot photolysis then uncaged the oligo(dT) at a given intranuclear site and the resultant fluorescent, hybridized oligo(dT) was tracked using high-speed imaging microscopy. Poly(A) RNA moved away from the uncaging spot in all directions with a mean square displacement that varied linearly with time, and the same apparent diffusion coefficient was measured for the movement at both 37 degrees C and 23 degrees C. These properties are characteristic of a random diffusive process. High resolution three-dimensional imaging of live cells containing both Hoechst-labeled chromosomes and uncaged oligo(dT) showed that, excluding nucleoli, the poly(A) RNA could access most, if not all, of the non-chromosomal space in the nucleus., Conclusions: Poly(A) RNA can move freely throughout the interchromatin space of the nucleus with properties characteristic of diffusion.

Localized, transient elevations in cytosolic Ca2+, known as Ca2+ sparks, caused by Ca2+ release from sarcoplasmic reticulum, are thought to trigger the opening of large conductance Ca2+-activated potassium channels in the plasma membrane resulting in spontaneous transient outward currents (STOCs) in smooth muscle cells. But the precise relationships between Ca2+ concentration within the sarcoplasmic reticulum and a Ca2+ spark and that between a Ca2+ spark and a STOC are not well defined or fully understood. To address these problems, we have employed two approaches using single patch-clamped smooth muscle cells freshly dissociated from toad stomach: a high speed, wide-field imaging system to simultaneously record Ca2+ sparks and STOCs, and a method to simultaneously measure free global Ca2+ concentration in the sarcoplasmic reticulum ([Ca2+]SR) and in the cytosol ([Ca2+]CYTO) along with STOCs. At a holding potential of 0 mV, cells displayed Ca2+ sparks and STOCs. Ca2+ sparks were associated with STOCs; the onset of the sparks coincided with the upstroke of STOCs, and both had approximately the same decay time. The mean increase in [Ca2+]CYTO at the time and location of the spark peak was approximately 100 nM above a resting concentration of approximately 100 nM. The frequency and amplitude of spontaneous Ca2+ sparks recorded at -80 mV were unchanged for a period of 10 min after removal of extracellular Ca2+ (nominally Ca2+-free solution with 50 microM EGTA), indicating that Ca2+ influx is not necessary for Ca2+sparks. A brief pulse of caffeine (20 mM) elicited a rapid decrease in [Ca2+]SR in association with a surge in [Ca2+]CYTO and a fusion of STOCs, followed by a fast restoration of [Ca2+]CYTO and a gradual recovery of [Ca2+]SR and STOCs. The return of global [Ca2+]CYTO to rest was an order of magnitude faster than the refilling of the sarcoplasmic reticulum with Ca2+. After the global [Ca2+]CYTO was fully restored, recovery of STOC frequency and amplitude were correlated with the level of [Ca2+]SR, even though the time for refilling varied greatly. STOC frequency did not recover substantially until the [Ca2+]SR was restored to 60% or more of resting levels. At [Ca2+]SR levels above 80% of rest, there was a steep relationship between [Ca2+]SR and STOC frequency. In contrast, the relationship between [Ca2+]SR and STOC amplitude was linear. The relationship between [Ca2+]SR and the frequency and amplitude was the same for Ca2+ sparks as it was for STOCs. The results of this study suggest that the regulation of [Ca2+]SR might provide one mechanism whereby agents could govern Ca2+ sparks and STOCs. The relationship between Ca2+ sparks and STOCs also implies a close association between a sarcoplasmic reticulum Ca2+ release site and the Ca2+-activated potassium channels responsible for a STOC.

1. Local changes in cytosolic [Ca2+] were imaged with a wide-field, high-speed, digital imaging system while membrane currents were simultaneously recorded using whole-cell, perforated patch recording in freshly dissociated guinea-pig tracheal myocytes. 2. Depending on membrane potential, Ca2+ sparks triggered 'spontaneous' transient inward currents (STICs), 'spontaneous' transient outward currents (STOCs) and biphasic currents in which the outward phase always preceded the inward (STOICs). The outward currents resulted from the opening of large-conductance Ca2+-activated K+ (BK) channels and the inward currents from Ca2+-activated Cl- (ClCa) channels. 3. A single Ca2+ spark elicited both phases of a STOIC, and sparks originating from the same site triggered STOCs, STICs and STOICs, depending on membrane potential. 4. STOCs had a shorter time to peak (TTP) than Ca2+ sparks and a much shorter half-time of decay. In contrast, STICs had a somewhat longer TTP than sparks but the same half-time of decay. Thus, the STIC, not the STOC, more closely reflected the time course of cytosolic Ca2+ elevation during a Ca2+ spark. 5. These findings suggest that ClCa channels and BK channels may be organized spatially in quite different ways in relation to points of Ca2+ release from intracellular Ca2+ stores. The results also suggest that Ca2+ sparks may have functions in smooth muscle not previously suggested, such as a stabilizing effect on membrane potential and hence on the contractile state of the cell, or as activators of voltage-gated Ca2+ channels due to depolarization mediated by STICs.

Fluorescence microscopy has undergone a resurgence in interest following the discovery of green-fluorescent protein (GFP) and its increasing use in live-cell imaging. This article describes an enhanced form of epifluorescence microscopy, digital imaging microscopy, that can be used to produce high-resolution three-dimensional images of samples labelled with GFP, or other fluorochromes, using simple instrumentation and image-restoration software.

The spatial relation between mitochondria and endoplasmic reticulum (ER) in living HeLa cells was analyzed at high resolution in three dimensions with two differently colored, specifically targeted green fluorescent proteins. Numerous close contacts were observed between these organelles, and mitochondria in situ formed a largely interconnected, dynamic network. A Ca2+-sensitive photoprotein targeted to the outer face of the inner mitochondrial membrane showed that, upon opening of the inositol 1,4,5-triphosphate (IP3)-gated channels of the ER, the mitochondrial surface was exposed to a higher concentration of Ca2+ than was the bulk cytosol. These results emphasize the importance of cell architecture and the distribution of organelles in regulation of Ca2+ signaling.

The possibility of specifically addressing recombinant probes to mitochondria is a novel, powerful way to study these organelles within living cells. We first showed that the Ca(2+)-sensitive photoprotein aequorin, modified by the addition of a mitochondrial targeting sequence, allows to monitor specifically the Ca2+ concentration in the mitochondrial matrix ([Ca2+]m) of living cells. With this tool, we could show that, upon physiological stimulation, mitochondria undergo a major rise in [Ca2+]m, well in the range of the Ca2+ sensitivity of the matrix dehydrogenases, in a wide variety of cell types, ranging from non excitable, e.g., HeLa and CHO, and excitable, e.g., cell lines to primary cultures of various embryological origin, such as myocytes and neurons. This phenomenon, while providing an obvious mechanism for tuning mitochondrial activity to cell needs, appeared at first in striking contrast with the low affinity of mitochondrial Ca2+ uptake mechanisms. Based on indirect evidence, we proposed that the mitochondria might be close to the source of the Ca2+ signal and thus exposed to microdomains of high [Ca2+], hence allowing the rapid accumulation of Ca2+ into the organelle. In order to verify this intriguing possibility, we followed two approaches. In the first, we constructed a novel aequorin chimera, targeted to the mitochondrial intermembrane space (MIMS), i.e., the region sensed by the low-affinity Ca2+ uptake systems of the inner mitochondrial membrane. With this probe, we observed that, upon agonist stimulation, a portion of the MIMS is exposed to saturating Ca2+ concentrations, thus confirming the occurrence of microdomains of high [Ca2+] next to mitochondria. In the second approach, we directly investigated the spatial relationship of the mitochondria and the ER, the source of agonist-releasable Ca2+ in non-excitable cells. For this purpose, we constructed GFP-based probes of organelle structure; namely, by targeting to these organelles GFP mutants with different spectral properties, we could label them simultaneously in living cells. By using an imaging system endowed with high speed and sensitivity, which allows to obtain high-resolution 3D images, we could demonstrate that close contacts (< 80 nm) occur in vivo between mitochondria and the ER.

Cardiac muscle contraction is triggered by a small and brief Ca2+ entry across the t-tubular membranes, which is believed to be locally amplified by release of Ca2+ from the adjacent junctional sarcoplasmic reticulum (SR). As Ca2+ diffusion is thought to be markedly attenuated in cells, it has been predicted that significant intrasarcomeric [Ca2+] gradients should exist during activation. To directly test for this, we measured [Ca2+] distribution in single cardiac myocytes using fluorescent [Ca2+] indicators and high speed, three-dimensional digital imaging microscopy and image deconvolution techniques. Steep cytosolic [Ca2+] gradients from the t-tubule region to the center of the sarcomere developed during the first 15 ms of systole. The steepness of these [Ca2+] gradients varied with treatments that altered Ca2+ release from internal stores. Electron probe microanalysis revealed a loss of Ca2+ from the junctional SR and an accumulation, principally in the A-band during activation. We propose that the prolonged existence of [Ca2+] gradients within the sarcomere reflects the relatively long period of Ca2+ release from the SR, the localization of Ca2+ binding sites and Ca2+ sinks remote from sites of release, and diffusion limitations within the sarcomere. The large [Ca2+] transient near the t-tubular/ junctional SR membranes is postulated to explain numerous features of excitation-contraction coupling in cardiac muscle.

Calcium, a ubiquitous second messenger, stimulates the activity of several mitochondrial dehydrogenases. This has led to the suggestion that the same messenger that signals cell activation could also activate mitochondrial electron/proton transport, thereby meeting demands for increased cellular energy. To test this in live cells, quantitative three-dimensional microscopy and ratio imaging were used to measure membrane potential of individual mitochondria and cytosolic calcium distribution. Mitochondria reversibly depolarized as cytosolic calcium rose and then fell following physiological stimulation. Thus, the dominant response of the mitochondrion to a rise in cytosolic [Ca2+] is to draw on the electrochemical potential, possibly to accelerate processes directly involved in ATP synthesis and calcium homeostasis.

Local chemical events underlying chemotaxis were characterized in a new model cell, the newt eosinophil. These cells exhibit a chemotactic response to a trypsin-sensitive component of newt serum. Ca2+ plays a role in this process, since treatments expected to diminish Ca2+ availability from the medium [ethylene glycol-bis (beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, Co2+, and verapamil], to break down transmembrane Ca2+ gradients (ionomycin), or to interfere with the function of intracellular Ca2+ stores (caffeine and neomycin) inhibited cell polarization and movement. Using imaging techniques we found that cytosolic Ca2+ concentration ([Ca2+]i) increased in response to newt serum. Migrating newt eosinophils exhibited a dynamic heterogeneous distribution of [Ca2+]i. [Ca2+]i was elevated in cells undergoing a change of direction relative to cells migrating persistently in one direction. Migrating cells contained gradients of [Ca2+]i along their long axis, with the front of the cell having consistently lower [Ca2+]i than the rear. When cells were loaded with the cell-permeant form of fura 2, fura 2 acetoxymethyl ester, a caffeine-sensitive membrane-delimited region of elevated [Ca2+]i was seen associated with the microtubule organizing center. A model is proposed relating the distribution of [Ca2+]i and the location of the external stimulus to the generation and interaction of substances within the cell that both simulate and inhibit increases in [Ca2+]i.

Because of its importance in the chemiosmotic theory, mitochondrial membrane potential has been the object of many investigations. Significantly, however, quantitative data on how energy transduction might be regulated or perturbed by the physiological state of the cell has only been gathered via indirect studies on isolated mitochondrial suspensions; quantitative studies on individual mitochondria in situ have not been possible because of their small size, their intrinsic motility, and the absence of appropriate analytical reagents. In this article, we combine techniques for rapid, high resolution, quantitative three-dimensional imaging microscopy and mathematical modeling to determine accurate distributions of a potentiometric fluorescent probe between the cytosol and individual mitochondria inside a living cell. Analysis of this distribution via the Nernst equation permits assignment of potentials to each of the imaged mitochondrial membranes. The mitochondrial membrane potentials are distributed over a narrow range centered at -150 mV within the neurites of differentiated neuroblastoma cells. We find that the membrane potential of a single mitochondrion is generally remarkably stable over times of 40-80 s, but significant fluctuations can occasionally be seen. The motility of individual mitochondria is not directly correlated to membrane potential, but mitochondria do become immobile after prolonged treatment with respiratory inhibitors or uncouplers. Thus, three spatial dimensions, a key physiological parameter, and their changes over time are all quantitated for objects at the resolution limit of light microscopy. The methods described may be readily extended to permit investigations of how mitochondrial function is integrated with other processes in the intact cell.

The concentration of intracellular free calcium ([Ca2+]i) in polarized eosinophils was imaged during chemotaxis by monitoring fluorescence of the calcium-sensitive dye Fura-2 with a modified digital imaging microscope. Chemotactic stimuli caused [Ca2+]i to increase in a nonuniform manner that was related to cell activity. In cells moving persistently in one direction, [Ca2+]i was highest at the rear and lowest at the front of the cell. Before cells turned, [Ca2+]i transiently increased. The region of the cell that became the new leading edge had the lowest [Ca2+]i. These changes in [Ca2+]i provide a basis for understanding the organization and local activity of cytoskeletal proteins thought to underlie the directed migration of many cells.