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1.
J Gen Physiol ; 134(4): 267-80, 2009 Oct.
Article in English | MEDLINE | ID: mdl-19786582

ABSTRACT

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.


Subject(s)
Adrenal Glands/metabolism , Calcium/metabolism , Chromaffin Cells/metabolism , Exocytosis/physiology , Animals , Calcium Signaling , Cells, Cultured , Cytosol/metabolism , Male , Mice , Neurons/metabolism , Ryanodine Receptor Calcium Release Channel/metabolism , Synaptic Membranes/metabolism
2.
J Gen Physiol ; 132(1): 145-60, 2008 Jul.
Article in English | MEDLINE | ID: mdl-18591421

ABSTRACT

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.


Subject(s)
Calcium Signaling/physiology , Chloride Channels/physiology , Myocytes, Smooth Muscle/physiology , Ryanodine Receptor Calcium Release Channel/physiology , Aniline Compounds/chemistry , Animals , Calcium/analysis , Calcium/metabolism , Cells, Cultured , Computer Simulation , Egtazic Acid/analogs & derivatives , Egtazic Acid/chemistry , Electrophysiology , Kinetics , Male , Membrane Potentials/physiology , Mice , Models, Biological , Muscle, Smooth/cytology , Muscle, Smooth/physiology , Photolysis , Trachea/cytology , Xanthenes/chemistry
3.
Am J Physiol Heart Circ Physiol ; 293(4): H2448-61, 2007 Oct.
Article in English | MEDLINE | ID: mdl-17483243

ABSTRACT

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.


Subject(s)
Caffeine/pharmacology , Calcium Channel Agonists/pharmacology , Calcium Channels/drug effects , Calcium Signaling/drug effects , Cell Membrane/drug effects , Ion Channel Gating/drug effects , Myocytes, Cardiac/drug effects , Animals , Arrhythmias, Cardiac/chemically induced , Arrhythmias, Cardiac/metabolism , Caffeine/adverse effects , Calcium Channel Agonists/adverse effects , Calcium Channel Blockers/pharmacology , Calcium Channels/metabolism , Cell Membrane/metabolism , Cresols/pharmacology , Enzyme Inhibitors/pharmacology , Heart Atria/cytology , Heart Atria/drug effects , Heart Atria/metabolism , Heart Ventricles/cytology , Heart Ventricles/drug effects , Heart Ventricles/metabolism , In Vitro Techniques , Membrane Potentials/drug effects , Mice , Myocytes, Cardiac/enzymology , Myocytes, Cardiac/metabolism , Patch-Clamp Techniques , Rats , Ruthenium Red , Ryanodine/pharmacology , Ryanodine Receptor Calcium Release Channel/drug effects , Ryanodine Receptor Calcium Release Channel/metabolism , Sarcoplasmic Reticulum/drug effects , Sarcoplasmic Reticulum/metabolism , Sarcoplasmic Reticulum Calcium-Transporting ATPases/antagonists & inhibitors , Sarcoplasmic Reticulum Calcium-Transporting ATPases/metabolism , Tetracaine/pharmacology , Thapsigargin/pharmacology
4.
J Neurosci ; 26(29): 7565-74, 2006 Jul 19.
Article in English | MEDLINE | ID: mdl-16855084

ABSTRACT

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+.


Subject(s)
Calcium Channels, L-Type/physiology , Calcium/metabolism , Nerve Endings/metabolism , Ryanodine Receptor Calcium Release Channel/physiology , Animals , Calcium Channel Agonists/pharmacology , Calcium Channel Blockers/pharmacology , Calcium Channels, L-Type/drug effects , Cell Membrane/metabolism , Electric Stimulation , Electrophysiology , Hypothalamus/cytology , Hypothalamus/metabolism , Immunohistochemistry , In Vitro Techniques , Mice , Neurons/metabolism , Nifedipine/pharmacology , Pyrroles/pharmacology
5.
Mol Biol Cell ; 17(9): 3781-92, 2006 Sep.
Article in English | MEDLINE | ID: mdl-16775004

ABSTRACT

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.


Subject(s)
Carrier Proteins/metabolism , Cilia/metabolism , Golgi Apparatus/metabolism , Animals , Cell Cycle , Cells, Cultured , Centrosome/metabolism , Epithelial Cells/cytology , Humans , Mice , Protein Binding , Protein Transport , Rats , TRPP Cation Channels/metabolism
6.
J Biol Chem ; 281(23): 16139-46, 2006 Jun 09.
Article in English | MEDLINE | ID: mdl-16537543

ABSTRACT

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.


Subject(s)
Clathrin/physiology , Endocytosis/physiology , 3T3-L1 Cells , Animals , Cells, Cultured , Fluorescent Dyes , Male , Mice , Mice, Inbred C57BL
7.
Biophys J ; 90(6): 2027-37, 2006 Mar 15.
Article in English | MEDLINE | ID: mdl-16387759

ABSTRACT

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.


Subject(s)
Calcium Signaling/physiology , Calcium/metabolism , Chromaffin Cells/physiology , Exocytosis/physiology , Membrane Microdomains/physiology , Ryanodine Receptor Calcium Release Channel/metabolism , Synaptic Vesicles/physiology , Animals , Cells, Cultured , Chromaffin Cells/cytology , Membrane Microdomains/ultrastructure , Mice , Synaptic Vesicles/ultrastructure
8.
Mol Biol Cell ; 17(3): 1239-49, 2006 Mar.
Article in English | MEDLINE | ID: mdl-16371503

ABSTRACT

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.


Subject(s)
Cell Nucleus Structures/metabolism , RNA Transport , RNA, Messenger/metabolism , Animals , Diffusion , HeLa Cells , Humans , Light , Luminescent Proteins/metabolism , Nuclear Proteins/metabolism , Oligodeoxyribonucleotides/metabolism , RNA, Messenger/radiation effects , Rats , Ribonucleoproteins/metabolism , Serine-Arginine Splicing Factors , Spectrometry, Fluorescence , Time Factors , Red Fluorescent Protein
9.
CSH Protoc ; 2006(6)2006 Nov 01.
Article in English | MEDLINE | ID: mdl-22485987

ABSTRACT

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.

10.
J Gen Physiol ; 124(3): 259-72, 2004 Sep.
Article in English | MEDLINE | ID: mdl-15337821

ABSTRACT

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.


Subject(s)
Calcium Channels/metabolism , Calcium/metabolism , Fluorescence , Aniline Compounds , Animals , Bufo marinus , Caffeine/pharmacology , Electric Conductivity , Fluorescent Dyes , In Vitro Techniques , Ion Channel Gating/drug effects , Microscopy, Fluorescence , Myocytes, Smooth Muscle/drug effects , Myocytes, Smooth Muscle/metabolism , Patch-Clamp Techniques , Xanthenes
11.
Am J Physiol Cell Physiol ; 287(6): C1577-88, 2004 Dec.
Article in English | MEDLINE | ID: mdl-15306542

ABSTRACT

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.


Subject(s)
Calcium Signaling/physiology , Microscopy, Fluorescence/methods , Myocytes, Smooth Muscle/physiology , Potassium Channels, Calcium-Activated/physiology , Ryanodine Receptor Calcium Release Channel/physiology , Animals , Bufo marinus , Calcium/metabolism , Cytoplasm/metabolism , Large-Conductance Calcium-Activated Potassium Channels , Patch-Clamp Techniques , Sarcoplasmic Reticulum/physiology
12.
Am J Physiol Cell Physiol ; 286(5): C1139-51, 2004 May.
Article in English | MEDLINE | ID: mdl-15075213

ABSTRACT

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.


Subject(s)
Calcium/metabolism , Mitochondria, Muscle/physiology , Sarcoplasmic Reticulum/metabolism , Animals , Bufo marinus , Calcium Channels , Electrophysiology , Inositol 1,4,5-Trisphosphate Receptors , Ion Channels/physiology , Ion Channels/radiation effects , Light , Macrocyclic Compounds , Mitochondrial Membrane Transport Proteins , Mitochondrial Permeability Transition Pore , Muscle, Smooth/cytology , Muscle, Smooth/physiology , Oxazoles/pharmacology , Receptors, Cytoplasmic and Nuclear/antagonists & inhibitors , Ryanodine/pharmacology
13.
Cell Calcium ; 35(6): 523-33, 2004 Jun.
Article in English | MEDLINE | ID: mdl-15110142

ABSTRACT

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.


Subject(s)
Calcium Channels/metabolism , Calcium/analysis , Cell Membrane/metabolism , Cytosol/metabolism , Animals , Calcium/metabolism , Electric Conductivity , Fluorescence
14.
J Neurosci ; 24(5): 1226-35, 2004 Feb 04.
Article in English | MEDLINE | ID: mdl-14762141

ABSTRACT

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.


Subject(s)
Calcium Signaling/physiology , Calcium/metabolism , Hypothalamus/cytology , Neurons/metabolism , Presynaptic Terminals/metabolism , Animals , Caffeine/pharmacology , Calcium Signaling/drug effects , Hypothalamus/chemistry , Membrane Potentials/physiology , Mice , Neurons/ultrastructure , Patch-Clamp Techniques , Presynaptic Terminals/chemistry , Presynaptic Terminals/drug effects , Ryanodine Receptor Calcium Release Channel/metabolism , Subcellular Fractions/chemistry , Subcellular Fractions/drug effects , Subcellular Fractions/metabolism
15.
Biophys J ; 85(5): 3350-7, 2003 Nov.
Article in English | MEDLINE | ID: mdl-14581236

ABSTRACT

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.


Subject(s)
Image Interpretation, Computer-Assisted/methods , Imaging, Three-Dimensional/methods , Membrane Potentials/physiology , Microscopy, Fluorescence/methods , Mitochondria/physiology , Mitochondria/ultrastructure , Myocytes, Smooth Muscle/cytology , Myocytes, Smooth Muscle/physiology , Organometallic Compounds , Animals , Bufo marinus , Cells, Cultured
16.
Mol Biol Cell ; 14(12): 4805-12, 2003 Dec.
Article in English | MEDLINE | ID: mdl-12960421

ABSTRACT

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.


Subject(s)
Cell Nucleolus/physiology , Cytoplasm/physiology , Myoblasts/physiology , RNA, Ribosomal/physiology , Ribosomes/physiology , Animals , Biological Transport/physiology , Cell Nucleolus/metabolism , Cells, Cultured , Cytoplasm/metabolism , Image Processing, Computer-Assisted , In Situ Hybridization , Microscopy, Video , Myoblasts/metabolism , RNA, Ribosomal/metabolism , Rats , Ribosomes/metabolism
17.
Am J Physiol Cell Physiol ; 284(2): C250-62, 2003 Feb.
Article in English | MEDLINE | ID: mdl-12388116

ABSTRACT

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.


Subject(s)
Eukaryotic Cells/metabolism , Microtubules/metabolism , Phosphoprotein Phosphatases/metabolism , Animals , Binding Sites/physiology , COS Cells , Chick Embryo , Eukaryotic Cells/ultrastructure , Green Fluorescent Proteins , Holoenzymes/metabolism , Luminescent Proteins , Microscopy, Electron , Microtubule Proteins/chemistry , Microtubules/ultrastructure , Myosin-Light-Chain Phosphatase , Peptide Fragments/metabolism , Peptide Fragments/pharmacology , Protein Binding/physiology , Protein Structure, Tertiary/physiology , Recombinant Fusion Proteins
18.
J Cell Biol ; 159(4): 613-24, 2002 Nov 25.
Article in English | MEDLINE | ID: mdl-12438411

ABSTRACT

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.


Subject(s)
Calcium/metabolism , Fura-2/analogs & derivatives , Ion Channels/metabolism , Mitochondria/metabolism , Porins/metabolism , Proto-Oncogene Proteins c-bcl-2 , Sphingosine/analogs & derivatives , Animals , Apoptosis/physiology , Cells, Cultured , Endoplasmic Reticulum/metabolism , Enzyme Inhibitors/metabolism , Fluorescent Dyes/metabolism , Fura-2/metabolism , HeLa Cells , Homeostasis , Humans , Ion Channels/genetics , Ion Transport , Muscle Fibers, Skeletal/cytology , Muscle Fibers, Skeletal/metabolism , Porins/genetics , Proto-Oncogene Proteins/metabolism , Rats , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Sphingosine/metabolism , Voltage-Dependent Anion Channels , bcl-2-Associated X Protein
19.
Am J Physiol Cell Physiol ; 283(3): C752-61, 2002 Sep.
Article in English | MEDLINE | ID: mdl-12176732

ABSTRACT

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.


Subject(s)
Cyclic GMP/analogs & derivatives , Cyclic GMP/metabolism , Muscle Proteins/metabolism , Muscle, Smooth/metabolism , Signal Transduction/physiology , Animals , Binding Sites/physiology , COS Cells , Cell Membrane/metabolism , Cells, Cultured , Cyclic GMP/pharmacology , Cyclic GMP-Dependent Protein Kinases/metabolism , Enzyme Inhibitors/pharmacology , Green Fluorescent Proteins , Luminescent Proteins/genetics , Muscle Proteins/genetics , Muscle, Smooth/cytology , Muscle, Smooth/drug effects , Mutagenesis, Site-Directed , Myosin-Light-Chain Kinase , Myosins/metabolism , Nitric Oxide Donors/pharmacology , Peptide Fragments , Peptides , Phosphorylation , Protein Binding/physiology , Protein Transport/drug effects , Protein Transport/physiology , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Signal Transduction/drug effects , Swine , Trachea , Transfection
20.
J Gen Physiol ; 120(1): 15-27, 2002 Jul.
Article in English | MEDLINE | ID: mdl-12084772

ABSTRACT

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.


Subject(s)
Potassium Channels, Calcium-Activated/physiology , Animals , Bufo marinus , Calcium/pharmacology , Calcium/physiology , Electric Conductivity , Large-Conductance Calcium-Activated Potassium Channels , Membrane Potentials/physiology , Muscle, Smooth/cytology , Muscle, Smooth/metabolism , Osmolar Concentration
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