Intact Single Cells Research Articles

G Protein-independent Activation of an Inward Na+Current by Muscarinic Receptors in Mouse Pancreatic β-Cells

Depolarization of pancreatic beta-cells is critical for stimulation of insulin secretion by acetylcholine but remains unexplained. Using voltage-clamped beta-cells, we identified a small inward current produced by acetylcholine, which was suppressed by atropine or external Na(+) omission, but was not mimicked by nicotine, and was insensitive to nicotinic antagonists, tetrodotoxin, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DiDS), thapsigargin pretreatment, and external Ca(2+) and K(+) removal. This suggests that muscarinic receptor stimulation activates voltage-insensitive Na(+) channels distinct from store-operated channels. No outward Na(+) current was produced by acetylcholine when the electrochemical Na(+) gradient was reversed, indicating that the channels are inward rectifiers. No outward K(+) current occurred either, and the reversal potential of the current activated by acetylcholine in the presence of Na(+) and K(+) was close to that expected for a Na(+)-selective membrane, suggesting that the channels opened by acetylcholine are specific for Na(+). Overnight pretreatment with pertussis toxin or the addition of guanosine 5'-O-(3-thiotriphosphate) (GTP-gamma-S) or guanosine-5'-O-(2-thiodiphosphate) (GDP-beta-S) instead of GTP to the pipette solution did not alter this current, excluding involvement of G proteins. Injection of a current of a similar amplitude to that induced by acetylcholine elicited electrical activity in beta-cells perifused with a subthreshold glucose concentration. These results demonstrate that muscarinic receptor activation in pancreatic beta-cells triggers, by a G protein-independent mechanism, a selective Na(+) current that explains the plasma membrane depolarization.

Mitochondria are morphologically and functionally heterogeneous within cells.

We investigated whether mitochondria represent morphologically continuous and functionally homogenous entities within single intact cells. Physical continuity of mitochondria was determined by three-dimensional reconstruction of fluorescence from mitochondrially targeted DsRed1 or calcein. The mitochondria of HeLa, PAEC, COS-7, HUVEC, hepatocytes, cortical astrocytes and neuronal cells all displayed heterogeneous distributions and were of varying sizes. There was a denser aggregation of mitochondria in perinuclear positions than in the cell periphery, where individual isolated mitochondria could be seen clearly. Using fluorescence-recovery after photobleaching, we observed that DsRed1 and calcein were highly mobile within the matrix of individual mitochondria, and that mitochondria within a cell were not lumenally continuous. Mitochondria were not electrically coupled, since only individual mitochondria were observed to depolarize following irradiation of TMRE-loaded cells. Functional heterogeneity of mitochondria in single cells was observed with respect to membrane potential, sequestration of hormonally evoked cytosolic calcium signals and timing of permeability transition pore opening in response to tert-butyl hydroperoxide. Our data indicate that mitochondria within individual cells are morphologically heterogeneous and unconnected, allowing them to have distinct functional properties.

Characterization of Mitochondrial Function During Cell Injury

Mitochondria play an important role in apoptotic and necrotic cell death. Depending on the stimulus, a variety of mitochondrial dysfunctions can trigger onset of cell death. Assessment of mitochondrial dysfunction in living cells in situis important to understand the sequence of events producing cell injury and death. Laser scanning confocal microscopy, a technique that creates submicron thin optical slices through living cells and tissues, allows us to monitor several mitochondrial events simultaneously in intact single cells over time.During oxidative stress, we monitored mitochondrial redox status, Ca2+ increase, reactive oxygen species (ROS), the mitochondrial permeability transition, and mitochondrial depolarization in cultured hepatocytes. When an oxidant chemical, t-BuOOH, was added to cultured hepatocytes, oxidation of mitochondrial pyridine nucleotides occurred as visualized directly by ultraviolet confocal microscopy of NAD(P)H autofluorescence. This event was followed by an increase of mitochondrial Ca2+ measured by the Ca2+-indicating fluorophore, Rhod-2.

Femtosecond Dynamics of Intracellular Water Probed with Nonlinear Optical Kerr Effect Microspectroscopy

A nonlinear optical Kerr effect (OKE) microscope was developed and used to elucidate the ultra-fast diffusive motions of intracellular water molecules. In the OKE microscope, a pump-induced birefringence is sensed by a delayed probe pulse within a spatially confined volume that measures 0.5 μm in the lateral direction and 4.0 μm along the axial coordinate. This microscope allows the recording of time-resolved Kerr signals, which reflect the ultra-fast structural relaxation of the liquid, exclusively from intracellular aqueous domains. Because relaxation occurs on a picosecond time scale, only local diffusive motions are probed. The microscopic OKE signal is therefore insensitive to long-time-scale hindered translational motions enforced by intracellular mechanical barriers but probes the intrinsic orientational mobility of water molecules in cells instead. The Kerr response as determined from single intact mammalian cells under physiological conditions shows a structural relaxation time of 1.35 ps, which is 1.7 times slower than the Kerr decay observed in pure water. The data indicate that the mobility of water molecules in cellular domains is moderately restricted due to the high intracellular content of proteins and solutes.

Subcellular distribution of chelatable iron: a laser scanning microscopic study in isolated hepatocytes and liver endothelial cells

The pool of cellular chelatable iron (‘free iron’, ‘low-molecular-weight iron’, the ‘labile iron pool’) is usually considered to reside mainly within the cytosol. For the present study we adapted our previously established Phen Green method, based on quantitative laser scanning microscopy, to examine the subcellular distribution of chelatable iron in single intact cells for the first time. These measurements, performed in isolated rat hepatocytes and rat liver endothelial cells, showed considerable concentrations of chelatable iron, not only in the cytosol but also in several other subcellular compartments. In isolated rat hepatocytes we determined a chelatable iron concentration of 5.8±2.6μM within the cytosol and of at least 4.8μM in mitochondria. The hepatocellular nucleus contained chelatable iron at the surprisingly high concentration of 6.6±2.9μM. In rat liver endothelial cells, the concentration of chelatable iron within all these compartments was even higher (cytosol, 7.3±2.6μM; nucleus, 11.8±3.9μM; mitochondria, 9.2±2.7μM); in addition, chelatable iron (approx. 16±4μM) was detected in a small subpopulation of the endosomal/lysosomal apparatus. Hence there is an uneven distribution of subcellular chelatable iron, a fact that is important to consider for (patho)physiological processes and that also has implications for the use of iron chelators to inhibit oxidative stress.

Subcellular distribution of chelatable iron: a laser scanning microscopic study in isolated hepatocytes and liver endothelial cells.

The pool of cellular chelatable iron ('free iron', 'low-molecular-weight iron', the 'labile iron pool') is usually considered to reside mainly within the cytosol. For the present study we adapted our previously established Phen Green method, based on quantitative laser scanning microscopy, to examine the subcellular distribution of chelatable iron in single intact cells for the first time. These measurements, performed in isolated rat hepatocytes and rat liver endothelial cells, showed considerable concentrations of chelatable iron, not only in the cytosol but also in several other subcellular compartments. In isolated rat hepatocytes we determined a chelatable iron concentration of 5.8+/-2.6 microM within the cytosol and of at least 4.8 microM in mitochondria. The hepatocellular nucleus contained chelatable iron at the surprisingly high concentration of 6.6+/-2.9 microM. In rat liver endothelial cells, the concentration of chelatable iron within all these compartments was even higher (cytosol, 7.3+/-2.6 microM; nucleus, 11.8+/-3.9 microM; mitochondria, 9.2+/-2.7 microM); in addition, chelatable iron (approx. 16+/-4 microM) was detected in a small subpopulation of the endosomal/lysosomal apparatus. Hence there is an uneven distribution of subcellular chelatable iron, a fact that is important to consider for (patho)physiological processes and that also has implications for the use of iron chelators to inhibit oxidative stress.

Detection of biotin in individual sea urchin oocytes using a bioluminescence binding assay.

The ability to detect biomolecules in single cells is important in order to fully understand the processes by which many biochemical events occur. To that end, we have developed a bioluminescence binding assay capable of measuring the intracellular biotin content of individual cells. The assay depends on competition between an aequorin-biotin conjugate (AEQ-biotin) and free biotin within the oocytes for binding sites on the protein avidin. The assay is performed by microinjecting each component into the oocytes and following the resulting bioluminescence within the oocyte upon triggering of aequorin. Results obtained using sea urchin oocytes show that the assay performed within the cells behaves in a manner consistent with assay theory. Using the assay, the individual biotin content of the oocytes is an average of approximately 20 amol. To our knowledge, this is the first reported multicomponent binding assay to be performed inside an intact single cell.

Microscope laser light scattering spectroscopy and its applications to non-invasivein situ real time measurements on single intact cells

The principle of the technique of microscope laser light scattering spectroscopy, its development and applications to the studies on single intact cells, including our work on non-invasivein situ real time measurements on single intact erythrocyte simultaneously at the levels of cellular, sub-cellular and intracellular molecules, are introduced.

Fluorescence Lifetime Imaging (FLI) in Real-Time - a New Technique in Photosynthesis Research

We describe an instrument that allows the rapid measurement of fluorescence lifetime-resolved images of leaves as well as sub-cellular structures of intact plants or single cells of algae. Lifetime and intensity fluorescence images can be acquired and displayed in real time (up to 55 lifetime-resolved images per s). Our imaging technique therefore allows rapid measurements that are necessary to determine the fluorescence lifetimes at the maximum (P level) fluorescence following initial illumination during the chlorophyll (Chl) a fluorescence transient (induction) in photosynthetic organisms. We demonstrate the application of this new instrument and methodology to measurements of: (1) Arabidopsis thaliana leaves showing the effect of dehydration on the fluorescence lifetime images; (2) Zea mays leaves showing differences in the fluorescence lifetimes due to differences in the bundle sheath cells (having a higher amount of low yield photosystem 1) and the mesophyll cells (having a higher amount of high yield photosystem 2); and (3) single cells of wild type Chlamydomonas reinhardtii and its non-photochemical quenching mutant NPQ2 (where the conversion of zeaxanthin to violaxanthin is blocked), with NPQ2 showing lowered lifetime of Chl a fluorescence. In addition to the lifetime differences referred to in (1) and (2), structural dependent heterogeneities in the fluorescence lifetimes were generally observed when imaging mesophyll cells in leaves.

Dual Video Microscopic Imaging of Membrane Potential and Cytosolic Calcium of Immunoidentified Embryonic Rat Cortical Cells

Membrane potential (MP) and cytosolic Ca2+ (Ca2+c) constitute important components involved in the physiological regulation of a myriad of cell functions in eukaryotic organisms. In particular, during development of the central nervous system, both properties are thought to be important in the regulation of cell cycle, cell migration, cell differentiation, cell–cell communication, and naturally occurring cell death. However, obtaining insight into the precise relationship between these two parameters of cell function is relatively limited either by technical difficulties inherent in using electrical recordings of membrane properties in conjunction with optical imaging of single cells or by employing optical imaging of either one or another property alone. Here, we describe in detail a novel strategy to record changes in both MP and Ca2+c from many intact single cells in a noninvasive manner using digital video microscopy. This method involves double-loading the cells with voltage- and calcium-sensitive fluorescent indicator dyes, green oxonol, and fura-2, which can be sequentially excited with a mercury arc lamp filtered at appropriate wavelengths and their resulting emissions can be captured with an intensified charged-coupled device camera at 1-s intervals. As an example of the utility of dual-recording strategy, we present data on a distinct functional expression of excitable membrane and cytoplasmic calcium properties in proliferating and differentiating embryonic rat cerebral cortical cells.

Determination of the Chelatable Iron Pool of Single Intact Cells by Laser Scanning Microscopy

We have previously established a method of detecting intracellular chelatable iron in viable cells based on digital fluorescence microscopy. To quantify cellular chelatable iron, it was crucial to determine the intracellular indicator concentration. In the present study, we therefore adapted the method to confocal laser scanning microscopy, which should allow the determination of the indicator concentration on the single-cell level. The fluorescent heavy-metal indicator phen green SK (PG SK), the fluorescence of which is quenched by iron, was loaded into cultured rat hepatocytes. The hepatocellular fluorescence increased when cellular chelatable iron available to PG SK was removed from the probe by an excess of the membrane-permeable transition metal chelator 2,2′-dipyridyl (2,2′-DPD, 5 mM). We optimized the scanning parameters for quantitatively recording changes in fluorescence and determined individual intracellular PG SK concentrations from the unquenched cellular fluorescence (after 2,2′-DPD) compared with PG SK standards in a “cytosolic” medium. An ex situ calibration method based on laser scanning microscopy was set up to determine the concentration of cellular chelatable iron from the increase of PG SK fluorescence after addition of 2,2′-DPD (5 mM). As the stoichiometry of the PG SK:Fe2+ complex was 3:1 as long as PG SK was not limiting, cellular chelatable iron was calculated directly from absolute changes in cellular fluorescence. Using this method, we found 2.5 ± 2.2 μM chelatable iron in hepatocytes. This method makes it possible to determine the pool of chelatable iron in single vital cells independently of cellular differences (e.g., dye loading, cell volume) in heterogeneous cell populations.

Protein-protein interaction of FHL3 with FHL2 and visualization of their interaction by green fluorescent proteins (GFP) two-fusion fluorescence resonance energy transfer (FRET)

LIM domain proteins are found to be important regulators in cell growth, cell fate determination, cell differentiation and remodeling of the cell cytoskeleton. Human Four-and-a-half LIM-only protein 3 (FHL3) is a type of LIM-only protein that contains four tandemly repeated LIM motifs with an N-terminal single zinc finger (half LIM motif). FHL3 expresses predominantly in human skeletal muscle. In this report, FHL3 was shown to be a novel interacting partner of FHL2 using the yeast two-hybrid assay. Furthermore, site-directed mutagenesis of FHL3 indicated that the LIM2 of FHL3 is the essential LIM domain for interaction with FHL2. Green fluorescent protein (GFP) was used to tag FHL3 in order to study its distribution during myogenesis. Our result shows that FHL3 was localized in the focal adhesions and nucleus of the cells. FHL3 mainly stayed in the focal adhesion during myogenesis. Moreover, using site-directed mutagenesis, the LIM1 of FHL3 was identified as an essential LIM domain for its subcellular localization. Mutants of GFP have given rise to a novel technique, two-fusion fluorescence resonance energy transfer (FRET), in the determination of protein-protein interaction at particular subcellular locations of eukaryotic cells. To determine whether FHL2 and FHL3 can interact with one another and to locate the site of this interaction in a single intact mammalian cell, we fused FHL2 and FHL3 to different mutants of GFP and studied their interactions using FRET. BFP/GFP fusion constructs were cotransfected into muscle myoblast C2C12 to verify the colocalization and subcellular localization of FRET. We found that FHL2 and FHL3 were colocalized in the mitochondria of the C2C12 cells and FRET was observed by using an epi-fluorescent microscope equipped with an FRET specific filter set.

The effect of cyclopiazonic acid on excitation-contraction coupling in guinea-pig ureteric smooth muscle: role of the sarcoplasmic reticulum.

1. We have investigated the effect of cyclopiazonic acid (CPA), an inhibitor of the sarcoplasmic reticulum (SR) Ca2+-ATPase on excitation-contraction (EC) coupling in guinea-pig ureter, by measuring membrane currents, action potentials, intracellular [Ca2+] and force. 2. CPA (20 micrometers) significantly enhanced the amplitude and duration of phasic contractions of ureteric smooth muscle associated with action potentials. This was accompanied by an increase in the duration of the intracellular Ca2+ transient in intact tissue and single cells but not their amplitude. However, CPA also slowed the rate of rise, and fall, of the force 1|1|Phiand1Phi Ca2+ transients. 3. Membrane potential recordings showed that CPA produced a small depolarization and a large increase in the duration of the plateau phase of the action potential. 4. Patch-clamp studies showed marked inhibition of outward potassium current in the presence of CPA and an inhibition of spontaneous transient outward currents (STOCs). CPA had no effect on inward Ca2+ current. 5. These data suggest that the SR plays a major role in modulating the excitability of the ureter, particularly via curtailing the action potential duration. This in turn will shorten the Ca2+ transient and decrease force. This negative action on developed force predominates over any small role it may play in initiating force in the guinea-pig ureter.

Separation and detection of the α- and β-chains of hemoglobin of a single intact red blood cell using capillary electrophoresis/electrospray ionization time-of-flight mass spectrometry

A single intact red blood cell (erythrocyte) was injected into a capillary electrophoresis column, and following in-capillary lysing the α- and β-chains of the hemoglobin (∼450 amol) were separated and detected using capillary electrophoresis/electrospray ionization time-of-flight mass spectrometry. The mass spectra of the electropherogram peaks of the α and β chains showed identifiable peaks corresponding to multiply protonated and sodiated α- and β-chains of hemoglobin.

Ca2+ homeostasis in the agonist-sensitive internal store: functional interactions between mitochondria and the ER measured In situ in intact cells.

Mitochondria have a well-established capacity to detect cytoplasmic Ca2+ signals resulting from the discharge of ER Ca2+ stores. Conversely, both the buffering of released Ca2+ and ATP production by mitochondria are predicted to influence ER Ca2+ handling, but this complex exchange has been difficult to assess in situ using conventional measurement techniques. Here we have examined this interaction in single intact BHK-21 cells by monitoring intraluminal ER [Ca2+] directly using trapped fluorescent low-affinity Ca2+ indicators. Treatment with mitochondrial inhibitors (FCCP, antimycin A, oligomycin, and rotenone) dramatically prolonged the refilling of stores after release with bradykinin. This effect was largely due to inhibition of Ca2+ entry pathways at the plasma membrane, but a significant component appears to arise from reduction of SERCA-mediated Ca2+ uptake, possibly as a consequence of ATP depletions in a localized subcellular domain. The rate of bradykinin-induced Ca2+ release was reduced to 51% of control by FCCP. This effect was largely overcome by loading cells with BAPTA-AM, highlighting the importance of mitochondrial Ca2+ buffering in shaping the release kinetics. However, mitochondria-specific ATP production was also a significant determinant of the release dynamic. Our data emphasize the localized nature of the interaction between these organelles, and show that competent mitochondria are essential for generating explosive Ca2+ signals.

Bcl-2 and Bax interactions in mitochondria probed with green fluorescent protein and fluorescence resonance energy transfer.

It has been hypothesized that interaction of Bcl-2 and Bax may regulate apoptosis. The spatial and temporal interaction of Bcl-2 and Bax at the single cell level has not, however, been demonstrated. To achieve this goal, we have developed two-fusion FRET (fluorescence resonance energy transfer). Using green fluorescent protein (GFP)-Bax and blue fluorescent protein (BFP)-Bcl-2 fusion proteins coexpressed in the same cell, we demonstrate a direct interaction between Bcl-2 and Bax in individual mitochondria. Mitochondrially localized cytochrome c-GFP and BFP-Bcl-2 showed little or no FRET, while nuclear-localized GFP-human papillomavirus E6 and BFP-Bcl-2 did not interact when coexpressed in the same cell. These findings indicate that two-fusion FRET provides an opportunity to examine the interaction between two different proteins coexpressed in single intact mammalian cells.

Free [Ca2+] dynamics measured in agonist-sensitive stores of single living intact cells: a new look at the refilling process.

Free [Ca2+] in agonist-sensitive internal stores of single intact cells was measured in situ in order to examine the role of [Ca2+] in modulating the store refilling process. BHK-21 fibroblasts were loaded with the low-affinity fluorescent calcium indicator mag-fura-2-AM such that >80% of the dye was trapped in organelles, where it reported [Ca2+] changes solely in an agonist- and thapsigargin-sensitive internal store. The rates of store reloading following stimulation by 100 nM bradykinin were essentially unchanged when cytosolic [Ca2+] was clamped to resting values with BAPTA-AM. In control cells, recharging of stores totally depended on the presence of external Ca2+, but pre-loading the cells with BAPTA-AM permitted efficient refilling in Ca2+-free, EGTA-containing external medium. Our results show: (i) Ca2+ stores normally are recharged by Ca2+ which must first transit the cytoplasm; (ii) an elevation in cytoplasmic [Ca2+] is not required to replenish Ca2+ stores; (iii) the activation of the plasma membrane Ca2+ pump during the Ca2+ spike ordinarily results in complete extrusion of released Ca2+; and (iv) the buffering capacity of the cytoplasm is an essential component of the store refilling process. An interesting finding was that acute treatment of cells with BAPTA-AM activated capacitative Ca2+ entry at the plasma membrane, due to its efficient hydrolysis in the stores, and the ensuing decrease in the endoplasmic reticulum [Ca2+].

Flow cytometric analysis and confocal imaging of anticancer alkylaminoanthraquinones and their n-oxides in intact human cells using 647-nm krypton laser excitation

Flow cytometry and laser-scanning confocal fluorescence microscopy have been used in the study of the pharmacodynamics, in single intact cells, of two novel alkylaminoanthraquinones (AQ4 and AQ6), structurally based upon the mid-red excitable but very weakly fluorescent anticancer agent mitoxantrone, together with their respective N-oxide derivatives (AQ4NO and AQ6NO). The drug design rationale was that N-oxide modifications generates prodrug forms suitable for selective bioreductive-activation in hypoxic tumor cells. DNA-binding ranked in the order of mitoxantrone > AQ6 > AQ4 > AQ6NO >> AQ4NO. Using both cytometric methods a similar ranking was found for whole cell and nuclear location in human transformed fibroblasts. However, AQ6 showed enhanced nuclear uptake compared with mitoxantrone, in keeping with its greater capacity to inhibit DNA synthesis. Partial charge neutralisation by N-oxide derivatization resulted in loss of DNA synthesis inhibition but retention of the ability to accumulate in the cytosol, an important property for prodrug development. We conclude that both flow cytometry and confocal imaging revealed biologically significant differences between analogues for subcellular distribution and retention properties. The study demonstrates the potential for these complementary 647-nm krypton laser line-based fluorometric methods to provide relevant structure-activity information in anthraquinone drug-design programmes.

Modulation of Ca2+ channels, charge movement and Ca2+ transients by heparin in frog skeletal muscle fibres.

This study is an investigation into the modulatory effects of heparin, a component of the extracellular matrix that binds to dihydropyridine receptors, on contraction and Ca2+ channels in frog skeletal muscle. Using tension and Ca2+ signal measurements in single intact skeletal muscle cells we have found that heparin (100-200 micrograms ml-1) substantially potentiates twitch and tetanic tension (55% and 28%, respectively). In contrast, heparin reduces the amplitude of K+ contractures. Heparin most likely potentiates twitch tension by prolonging action potentials. The ionic basis of this effect was investigated in voltage-clamp experiments. Membrane currents were monitored in voltage-clamped segments of single fibres using the triple Vaseline gap technique. We found that heparin partially blocks delayed rectifier potassium channels. The depressive effects of heparin on K+ contractures prompted us to investigate the effects of heparin on charge movement and Ca2+ currents (ICa) under voltage-clamp. Charge movement was measured using a subtraction procedure that employed a -20 mV control pulse from a holding potential of -100 mV. Heparin depresses the total charge by 25%. We propose that the reduction in the amplitude of potassium contractures is related to a partial blockade of charge movement. Extracellular heparin shifts the ICa-V relation toward more negative voltages and delays the deactivation of tail currents. Double pulse experiments revealed that conditioning depolarizations speed the activation of ICa during test depolarizations. Heparin does not affect this process. The primary action of heparin is to accelerate the activation of ICa during pulses not preceded by conditioning depolarizations. Overall, the kinetic effects of heparin on ICa would increase the Ca2+ influx associated with action potentials. However, mechanical and optical experiments performed in Ca(2+) -free solutions and in the presence of Ca2+ channel blockers revealed that twitch and tetanic potentiation occur even in the absence of Ca(2+) -influx.

Mechanical Changes after Histamine Activation of Intact Single Vascular Smooth Muscle Cells

Although several different models have been proposed to explain force maintenance in vascular smooth muscle, the molecular mechanism responsible for this phase of contraction has yet to be elucidated. To investigate the molecular mechanism for force maintenance, force and stiffness were measured during (1 μMhistamine) activation of single intact arterial smooth muscle cells. After histamine stimulation, the rise in quadrature stiffness preceded force (P< 0.05) and reached a steady state plateau before force (P< 0.05). These data suggest that the number of cycling cross-bridges increases during force activation and then remains constant during force maintenance. In-phase stiffness, on the other hand, continued to increase in amplitude after force had reached a steady state. The increase in the in-phase stiffness during force maintenance suggests that during force maintenance either a population of cross-bridges in an attached nonforce producing state develop or noncross-bridge force bearing structures are formed.