Second-harmonic generation imaging of membrane potential with retinal analogues

Second-harmonic generation (SHG) by membrane-incorporated probes is a nonlinear optical signal that is voltage-sensitive and the basis of a sensitive method for imaging membrane potential. The voltage dependence of SHG by four different probes, three retinoids (all-trans retinal), and two new retinal analogs, 3-methyl-7-(4′-dimethylamino-phenyl)-2,4,6-heptatrienal (AR-3) and 3,7-dimethyl-9-(4′-dimethylamino-phenyl)-2,4,6,8-nonatetraenal (AR-4), and a styryl dye (FM4-64), were compared in HEK-293 cells. Results were analyzed by fitting data with an expression based on an electrooptic mechanism for SHG, which depends on the complex-valued first- and second-order nonlinear electric susceptibilities (χ₂ and χ₃) of the probe. This gave values for the voltage sensitivity at the cell's resting potential, the voltage where the SHG is minimal, and the amplitude of the signal at that voltage for each of the four compounds. These measures show that χ₂ and χ₃ are complex numbers for all compounds except all-trans retinal, consistent with the proximities of excitation and/or emission wavelengths to molecular resonances. Estimates of probe orientation and location in the membrane electric field show that, for the far-from-resonance case, the shot noise-limited signal/noise ratio depends on the location of the probe in the membrane, and on χ₃ but not on χ₂.     

Detecting Subtle Plasma Membrane Perturbation in Living Cells Using Second Harmonic Generation Imaging

The requirement of center asymmetry for the creation of second harmonic generation (SHG) signals makes it an attractive technique for visualizing changes in interfacial layers such as the plasma membrane of biological cells. In this article, we explore the use of lipophilic SHG probes to detect minute perturbations in the plasma membrane. Three candidate probes, Di-4-ANEPPDHQ (Di-4), FM4-64, and all-trans-retinol, were evaluated for SHG effectiveness in Jurkat cells. Di-4 proved superior with both strong SHG signal and limited bleaching artifacts. To test whether rapid changes in membrane symmetry could be detected using SHG, we exposed cells to nanosecond-pulsed electric fields, which are believed to cause formation of nanopores in the plasma membrane. Upon nanosecond-pulsed electric fields exposure, we observed an instantaneous drop of ∼50% in SHG signal from the anodic pole of the cell. When compared to the simultaneously acquired fluorescence signals, it appears that the signal change was not due to the probe diffusing out of the membrane or changes in membrane potential or fluidity. We hypothesize that this loss in SHG signal is due to disruption in the interfacial nature of the membrane. The results show that SHG imaging has great potential as a tool for measuring rapid and subtle plasma membrane disturbance in living cells.     

A new vital stain for visualizing vacuolar membrane dynamics and endocytosis in yeast

We have used a lipophilic styryl dye, N-(3-triethylammoniumpropyl)-4- (p-diethylaminophenyl-hexatrienyl) pyridinium dibromide (FM 4-64), as a vital stain to follow bulk membrane-internalization and transport to the vacuole in yeast. After treatment for 60 min at 30 degrees C, FM 4- 64 stained the vacuole membrane (ring staining pattern). FM 4-64 did not appear to reach the vacuole by passive diffusion because at 0 degree C it exclusively stained the plasma membrane (PM). The PM staining decreased after warming cells to 25 degrees C and small punctate structures became apparent in the cytoplasm within 5-10 min. After an additional 20-40 min, the PM and cytoplasmic punctate staining disappeared concomitant with staining of the vacuolar membrane. Under steady state conditions, FM 4-64 staining was specific for vacuolar membranes; other membrane structures were not stained. The dye served as a sensitive reporter of vacuolar dynamics, detecting such events as segregation structure formation during mitosis, vacuole fission/fusion events, and vacuolar morphology in different classes of vacuolar protein sorting (vps) mutants. A particularly striking pattern was observed in class E mutants (e.g., vps27) where 500-700 nm organelles (presumptive prevacuolar compartments) were intensely stained with FM 4- 64 while the vacuole membrane was weakly fluorescent. Internalization of FM 4-64 at 15 degrees C delayed vacuolar labeling and trapped FM 4- 64 in cytoplasmic intermediates between the PM and the vacuole. The intermediate structures in the cytoplasm are likely to be endosomes as their staining was temperature, time, and energy dependent. Interestingly, unlike Lucifer yellow uptake, vacuolar labeling by FM 4- 64 was not blocked in sec18, sec14, end3, and end4 mutants, but was blocked in sec1 mutant cells. Finally, using permeabilized yeast spheroplasts to reconstitute FM 4-64 transport, we found that delivery of FM 4-64 from the endosome-like intermediate compartment (labeled at 15 degrees C) to the vacuole was ATP and cytosol dependent. Thus, we show that FM 4-64 is a new vital stain for the vacuolar membrane, a marker for endocytic intermediates, and a fluor for detecting endosome to vacuole membrane transport in vitro.     

Concentration-dependent staining of lactotroph vesicles by FM 4-64

Hormones are released from neuroendocrine cells by passing through an exocytotic pore that forms after vesicle and plasma membrane fusion. An elegant way to study this process at the single-vesicle level is to use styryl dyes, which stain not only the membrane, but also the matrix of individual vesicles in some neuroendocrine cells. However, the mechanism by which the vesicle matrix is stained is not completely clear. One possibility is that molecules of the styryl dye in the bath solution dissolve first in the plasma membrane and are then transported into the vesicle by lateral diffusion in the plane of the membrane, and finally the vesicle matrix is stained from the vesicle membrane. On the other hand, these molecules may enter the vesicle lumen and reach the vesicle matrix by permeation through an open aqueous fusion pore. To address these questions, we exposed pituitary lactotrophs to different concentrations of FM 4-64 to monitor the fluorescence increase of single vesicles by confocal microscopy after the stimulation of cells by high K(+). The results show that the membrane and the vesicle matrix exhibit different concentration-dependent properties: the plasma membrane staining by FM 4-64 has a higher affinity in comparison to the vesicle matrix. Moreover, the kinetics of vesicle loading by FM 4-64 exhibited a concentration-dependent process, which indicates that FM 4-64 molecules stain the vesicle matrix by aqueous permeation through an open fusion pore.     

Detecting Subtle Plasma Membrane Perturbation in Living Cells Using Second Harmonic Generation Imaging

The requirement of center asymmetry for the creation of second harmonic generation (SHG) signals makes it an attractive technique for visualizing changes in interfacial layers such as the plasma membrane of biological cells. In this article, we explore the use of lipophilic SHG probes to detect minute perturbations in the plasma membrane. Three candidate probes, Di-4-ANEPPDHQ (Di-4), FM4-64, and all-trans-retinol, were evaluated for SHG effectiveness in Jurkat cells. Di-4 proved superior with both strong SHG signal and limited bleaching artifacts. To test whether rapid changes in membrane symmetry could be detected using SHG, we exposed cells to nanosecond-pulsed electric fields, which are believed to cause formation of nanopores in the plasma membrane. Upon nanosecond-pulsed electric fields exposure, we observed an instantaneous drop of ∼50% in SHG signal from the anodic pole of the cell. When compared to the simultaneously acquired fluorescence signals, it appears that the signal change was not due to the probe diffusing out of the membrane or changes in membrane potential or fluidity. We hypothesize that this loss in SHG signal is due to disruption in the interfacial nature of the membrane. The results show that SHG imaging has great potential as a tool for measuring rapid and subtle plasma membrane disturbance in living cells.As the epicenter for many cellular functions, understanding the dynamics of the plasma membrane is important to monitoring biological phenomena. External forces acting upon the plasma membrane (e.g., electric, mechanical) have been shown to cause rapid disturbances, often resulting in dramatic changes in cell physiology (1–3). To understand this interaction, a minimally invasive, highly sensitive imaging technique that enables monitoring the structure of the plasma membrane is needed. Lipophilic dyes, which embed themselves into lipid membranes, are sensitive to the surrounding electric field and, therefore, report changes in membrane fluidity as well as voltage due to the capacitive nature of the membranes (4,5). This sensitivity is typically detected as a shift in the fluorescence emission spectrum. Localization of the fluorescence signal to only the plasma membrane is difficult because the probes also label internal membrane structures. Thus, to overcome this lack of spatial selectivity, second harmonic generation (SHG) has been used as an alternative to fluorescence for membrane imaging (6,7).In SHG, a second-order nonlinear polarization is induced by electronic disruption of a probe molecule from the electromagnetic field of the incident laser beam. This polarization generates oscillating dipole moments that reradiate light at twice the energy of the excitation beam. The induction of this dipole is sensitive to the static electric field surrounding the probe and the steady-state molecular polarization of the probe molecule. These properties make SHG probes useful for monitoring changes in biological membranes.First, as the voltage potential across the membrane changes, the static electric field around the probe also shifts, making the probe sensitive to these variations (7). Several SHG probes have, therefore, been employed to monitor plasma membrane potential (7,8).Second, because the dipole is affected by the steady-state molecular polarization of the probe itself, a SHG signal is only produced in materials that lack a center of inversion symmetry. In the centrosymmetric case, any emitted radiation is cancelled out by destructive interference. The properties of an interfacial environment, such as a cellular plasma membrane, not only provide the necessary asymmetry, but cause the polarized lipophilic dyes to be aligned in respect to the interface, instead of being randomly distributed as they would in a bulk environment. This alignment allows the generation of a coherent SHG signal from the plasma membrane while the rest of the cell remains nearly signal-free (6,7).We investigated whether the alignment sensitivity of the SHG response could be used to detect minute changes in the organization of the plasma membrane. Jurkat clone E6-1 human T-lymphocytes with a spherical morphology were selected for optimum signal clarity and cultured as directed by American Type Culture Collection (ATCC, Manassas, VA) with 1 I.U./mL penicillin and 0.1 μg/mL streptomycin. Cells were added to 35-mm poly-L-lysine-coated glass-bottomed dishes (MatTek, Ashland, MA) and incubated for 1 h in growth media to allow adherence. Before loading, the cells were rinsed with a buffer consisting of 135 mM NaCl, 5 mM KCl, 2 mM MgCl2, 10 mM HEPES, 10 mM glucose, 2 mM CaCl2, pH 7.4, 290–310 mOsm. SHG probes, Di-4-ANEPPDHQ (Di-4) (5 μM final concentration), FM4-64 (15 μM) or ATR (100 μM, 1 mg/mL BSA) were added to the buffer solution and incubated for 1 h. Cellular imaging was performed in the labeling buffer to limit diffusion of the probe molecules out of the cell membranes.A Ti:sapphire oscillator at 980 nm (Coherent Chameleon, 130 fs, 80 MHz, ∼15 mW at the sample; Coherent Laser, Santa Clara, CA) was coupled through the scan head of a modified model No. TCS SP5 II (Leica Geosystems, Norcross, GA) for SHG and multiphoton-excited fluorescence imaging (40×, water, 1.1 NA) using resonant scanning. SHG signal was collected in transmission by a photomultiplier tube after 680-nm shortpass and 485/25-nm bandpass filters; simultaneous fluorescence signal was collected in the epi-direction by two non-descanned photomultiplier tubes with 540/60-nm and 650/60-nm bandpass filters.Three SHG probes previously used to monitor voltage or membrane order in living cells were tested (8–10). Although ATR is reported to be effective in monitoring membrane voltage, we obtained nearly no SHG signal, despite successful loading as indicated by the fluorescence signal (Fig. 1). When FM4-64 and Di-4 were loaded to similar fluorescence intensities, nearly equivalent SHG signal was collected. Di-4 did appear to have a greater internalization of the dye. However, after the first frame, the FM4-64 signal dropped considerably (Fig. 1 b), an observation reported as a membrane voltage-independent bleaching effect (8). This drop in signal recovered after excitation was blocked for several seconds, but quantification of the response was difficult. Di-4 did not suffer as dramatic a drop in signal upon excitation, and still had sufficient SHG signal/noise after several seconds, so it was used in all further experiments.Open in a separate windowFigure 1(a) Fluorescence (top) and SHG (bottom) images for the three probes. (b) Signal/noise for the fluorescence and SHG for the initial frame and shortly after beginning acquisition. Error bars represent the mean ± SE (n = 10). Scale bar is 10 μm.To test whether Di-4 would report a rapid change in membrane organization, we applied a single nanosecond-duration pulsed electric field (nsPEF) to the labeled cell. These ultrabrief, high-intensity (MV/m) pulses differ from longer (μs-ms), lower-intensity (kV/m) pulses traditionally associated with electroporation in induced cellular response (3,11,12). Through selective uptake of small ions (Ca²⁺, Ti⁺) with limited uptake of propidium iodide, nsPEF have been previously postulated to cause nanopores (<2 nm diameter) in the plasma membrane. In contrast with a previous study observing poration resulting from traditional electroporation (13), the brevity of this apparently novel cellular insult allows for the decoupling of the mechanical effects of the pulse on the membrane from the electrical effects of the pulse itself. A single pulse, generated by a custom pulse generator, was delivered to the cells using a pair of 125-μm diameter tungsten electrodes, separated edge-to-edge by 150 μm, as previously described in Ibey et al. (14). For maximum visualization of changes in the SHG signal, a half-wave plate was placed before the scan head to align the polarization of the laser such that the brightest signals from the plasma membrane were at the poles facing the electrodes.The Di-4 SHG signal in response to a single 16.7 kV/cm, 600-ns nsPEF is shown in Fig. 2. Before the pulse, the intensity of the SHG signal is high at each of these poles. Immediately after the pulse, the SHG intensity drops by ∼50% on the side of the cell facing the anodic electrode, whereas little intensity is lost at the other pole. This response is plotted in Fig. 2 (pulse applied at 2 s), where it is apparent that the response is near instantaneous with little recovery in signal in the 5 s postexposure. The SHG response matches the previously observed effect of this stimulus, where ion uptake displayed a polar dependence and persisted for a number of minutes (11,12). Images taken 5 min after an nsPEF exposure are also shown in Fig. 2. These images confirm the eventual recovery of the cell and the corresponding return of SHG signal to preexposure levels.Open in a separate windowFigure 2(a) SHG images showing drop in signal on the anodic (or A-pole) of the cell. (b) Time trace of SHG response with the electrical pulse applied at 2 s that shows a near-instantaneous drop in the SHG signal at the anodic pole of the cell. (c) SHG image preexposure, immediately postexposure, and then 5-min postexposure showing recovery of the SHG signal.To decouple membrane disturbance from environmental changes around the membrane, we compared the SHG response to the simultaneously acquired fluorescence signal. Because fluorescence is not subject to the strict orientation requirement of SHG, the plasma membrane fluorescence signal provides an indication of the membrane fluidity and/or potential. Despite the dramatic shift in SHG intensity on the anodic pole upon the electrical pulse exposure (Fig. 3 a), the fluorescence channels display little response from the equivalent membrane sections with the exception of photobleaching and a slight increase in signal in both emission bands on the anodic side (Fig. 3, b and c). The shading in these graphs represents the mean ± SE for six cells. Although this slight increase may indicate that a small amount of dye is simply diffusing in or out of the membrane upon exposure, the fluorescence response is not as rapid or as lasting as the SHG response. Change in membrane fluidity or voltage can also be quantized using these fluorescence signals and a value known as the generalized polarization (GP) (4),GP=I515−570−I620−680I515−570+I620−680.(1)As with the raw intensity of the individual signals, the GP value for the membrane (Fig. 3 d) shows no significant shift, indicating that the membrane is likely not transitioning between a more raft- and fluidlike state. Thus, it seems likely that the dye was initially aligned in the tightly-packed ordered membrane so that the probes were able to generate a SHG photon. As shown in Fig. 3 e, we postulate that upon electrical pulse exposure, the membrane was disrupted by the formation with nanopores giving the probe molecules the flexibility to disorient within the membrane. The resulting alignment of the probes is more isotropic in nature, thereby limiting the probes probability of producing a SHG photon. The fluorescence signal remained, however, indicating that the probes remained active in the membrane.Open in a separate windowFigure 3(a) Average SHG signal showing the dramatic drop in signal on the anodic pole at the pulse application (2 s). (b and c) Simultaneous TPF signals showing nearly no instantaneous change at the pulse application. (d) GP showing no observable changes in the membrane potential or fluidity after the pulse. (Shaded areas) Fit to the mean ± SE for each trace (n = 6). (e) Conceptualization of the hypothesized membrane disruption underlying the observed change in SHG response.Thus, by taking advantage of the selection criteria of SHG, we were able to successfully use the SHG probe, Di-4, to monitor rapid disruption of the plasma membrane. Because SHG can only be generated when the probes are aligned in the plasma membrane, the SHG signal diminishes significantly upon disruption. The simultaneous collection of the multiphoton-excited fluorescence signal was advantageous in that it demonstrated that the probes did not simply diffuse out of the membrane, did not appear to be energetically disrupted by the electric pulse, and showed that the membrane changes were not simply a change in lipid order. We believe that this technique holds tremendous potential for use in the study of how external stimuli interact with and change the orientation of biological membranes. Such knowledge may allow for further understanding of how manipulation of cells and biological systems can be achieved using external stimuli.     

The mechanism of extracellular stimulation of nerve cells on an electrolyte-oxide-semiconductor capacitor

Extracellular excitation of neurons is applied in studies of cultured networks and brain tissue, as well as in neuroprosthetics. We elucidate its mechanism in an electrophysiological approach by comparing voltage-clamp and current-clamp recordings of individual neurons on an insulated planar electrode. Noninvasive stimulation of neurons from pedal ganglia of Lymnaea stagnalis is achieved by defined voltage ramps applied to an electrolyte/HfO₂/silicon capacitor. Effects on the smaller attached cell membrane and the larger free membrane are distinguished in a two-domain-stimulation model. Under current-clamp, we study the polarization that is induced for closed ion channels. Under voltage-clamp, we determine the capacitive gating of ion channels in the attached membrane by falling voltage ramps and for comparison also the gating of all channels by conventional variation of the intracellular voltage. Neuronal excitation is elicited under current-clamp by two mechanisms: Rising voltage ramps depolarize the free membrane such that an action potential is triggered. Falling voltage ramps depolarize the attached membrane such that local ion currents are activated that depolarize the free membrane and trigger an action potential. The electrophysiological analysis of extracellular stimulation in the simple model system is a basis for its systematic optimization in neuronal networks and brain tissue.     

Two mechanisms of synaptic vesicle recycling in rat brain nerve terminals

KCl and 4-aminopyridine (4-AP) evoke glutamate release from rat brain cortical nerve terminals by voltage clamping or by Na(+) channel-generated repetitive action potentials, respectively. Stimulation by 4-AP but not KCl is largely mediated by protein kinase C (PKC). To determine whether KCl and 4-AP utilise the same mechanism to release glutamate, we correlated glutamate release with release of the hydrophobic synaptic vesicle (SV) marker FM2-10. A strong correlation was observed for increasing concentrations of KCl and after application of phorbol 12-myristate 13-acetate (PMA) or staurosporine. The parallel increase in exocytosis measured by two approaches suggested it occurred by a PKC-independent mechanism involving complete fusion of SVs with the plasma membrane. At low concentrations of 4-AP, alone or with staurosporine, glutamate and FM2-10 release also correlated. However, higher concentrations of 4-AP or of 4-AP plus PMA greatly increased glutamate release but did not further increase FM2-10 release. This divergence suggests that 4-AP recruits an additional mechanism of release during strong stimulation that is PKC dependent and is superimposed upon the first mechanism. This second mechanism is characteristic of kiss-and-run, which is not detectable by styryl dyes. Our data suggest that glutamate release in nerve terminals occurs via two mechanisms: (1) complete SV fusion, which is PKC independent; and (2) a kiss-and-run-like mechanism, which is PKC dependent. Recruitment of a second release mechanism may be a widespread means to facilitate neurotransmitter release in central neurons.     

Behavioural and morphological evidence for the involvement of glial cell activation in delta opioid receptor function: implications for the development of opioid tolerance

The molecular identity and pharmacological properties of mechanically gated ion channels in sensory neurons are poorly understood. We show that FM1-43, a styryl dye used to fluorescently label cell membranes, permeates mechanosensitive ion channels in cultured dorsal root ganglion neurons, resulting in blockade of three previously defined subtypes of mechanically activated currents. Blockade and dye uptake is voltage dependent and regulated by external Ca²⁺. The structurally related larger dye FM3-25 inhibited mechanically activated currents to a lesser degree and did not permeate the channels. In vivo, FMI-43 decreases pain sensitivity in the Randall-Selitto test and increases the withdrawal threshold from von Frey hairs, together suggesting that the channels expressed at the cell body in culture mediate mechanosensation in the intact animal. These data give further insight into the mechanosensitive ion channels expressed by somatosensory neurons and suggest FM dyes are an interesting tool for studying them.     

Endocytosis and membrane turnover in the germ tube of uromyces fabae

We have used the fluorescent dye FM4-64 as a tracer to demonstrate bulk membrane internalization (endocytosis) and redistribution of the dye within the cytoplasm of the germ tube of the rust fungus Uromyces fabae. Staining of the hyphal membrane was detected 4 s after application of FM4-64 and reached a maximum after 1 min. The highest fluorescence intensity occurred in the apex. Subsequently, staining of the plasma membrane decreased and a subapical region of the fungal protoplast (5-20 &mgr;m from the tip) displayed increasing fluorescence with a maximum after 5 min. Fluorescence in the subapical region was redistributed to an area in the hyphal tip, which corresponds to the accumulation of apical vesicles, after 10-15 min and subsequently to a cytoplasmic region in front of the two nuclei (35-45 &mgr;m from the tip). We conclude from our measurements of membrane fluorescence that the turnover time from endocytosis to secretion of the dye amounts to 15 min. The uptake of the dye into the cytoplasm, but not membrane loading, could be inhibited completely with 5 mM NaN3 or by a temperature shift to 4 degreesC. This is the first evidence for endocytosis in a fungal germ tube. Copyright 1998 Academic Press.     

Visualization of membrane domains in Escherichia coli

Bacterial membrane and nucleoids were stained concurrently by the lipophilic styryl dye FM 4-64 [N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino)phenyl) hexatrienyl)pyridinium dibromide] and 4',6-diamidino-2-phenylindole (DAPI), respectively, and studied using fluorescence microscopy imaging. Observation of plasmolysed cells indicated that FM 4-64 stained the inner membrane preferentially. In live Escherichia coli pbpB cells and filaments, prepared on wet agar slabs, an FM 4-64 staining pattern developed in the form of dark bands. In dividing cells, the bands occurred mainly at the constriction sites and, in filaments, between partitioning nucleoids. The FM 4-64 pattern of dark bands in filaments was abolished after inhibiting protein synthesis with chloramphenicol. It is proposed that the staining patterns reflect putative membrane domains formed by DNA-membrane interactions and have functional implications in cell division.     

EAAT2 density at the astrocyte plasma membrane and Ca(2 + )-regulated exocytosis

We studied whether regulated exocytosis affects the glutamate transporter density in cultured astrocytes, in which the expression of a fluorescently labeled excitatory amino acid transporter 2 (EAAT2-EGFP) predominantly labeled the plasma membrane. The addition of ionomycin that elevates cytosolic Ca(2+) strongly increased the fluorescence of FM 4-64 membrane area dye, confirming the presence of regulated exocytosis in transfected astrocytes. However, concomitant with Ca(2+)-dependent FM 4-64 fluorescence increase, ionomycin induced a significant steady-state decrease in EAAT2-EGFP fluorescence. This is likely due to a secondary inner filter effect since,(i) in the absence of FM 4-64, ionomycin stimulation was ineffective in changing the EAAT2-EGFP fluorescence, and (ii) fluorescence changes in FM 4-64 and EAAT2-EGFP were inversely correlated. To test whether subcellular EAAT2-EGFP structures are translocated from the cytoplasm to the plasma membrane during ionomycin stimulation, EAAT2-EGFP fluorescence was monitored locally at the plasma membrane and a few microns away in the adjacent cytoplasm. Measurements revealed sites with an increase in EAAT2-EGFP plasma membrane fluorescence correlated with a fluorescence decrease beneath the plasma membrane, and sites with plasma membrane fluorescence decrease correlated with fluorescence increase within the adjacent cytoplasm. The sites of rapid translocation/retrieval of EAAT2-EGFP structures to/from the plasma membrane appeared to be distributed in a punctuate pattern around the cell perimeter. The density of EAAT2-EGFP was regulated in a Ca(2+)-dependent manner, since in the absence of extracellular Ca(2+) local translocation/retrieval events were absent, revealing rapid surface density regulation of EAAT2 in astrocytes by regulated exo/endocytosis.     

EAAT2 density at the astrocyte plasma membrane and Ca2 + -regulated exocytosis

We studied whether regulated exocytosis affects the glutamate transporter density in cultured astrocytes, in which the expression of a fluorescently labeled excitatory amino acid transporter 2 (EAAT2-EGFP) predominantly labeled the plasma membrane. The addition of ionomycin that elevates cytosolic Ca²⁺ strongly increased the fluorescence of FM 4-64 membrane area dye, confirming the presence of regulated exocytosis in transfected astrocytes. However, concomitant with Ca²⁺-dependent FM 4-64 fluorescence increase, ionomycin induced a significant steady-state decrease in EAAT2-EGFP fluorescence. This is likely due to a secondary inner filter effect since,(i) in the absence of FM 4-64, ionomycin stimulation was ineffective in changing the EAAT2-EGFP fluorescence, and (ii) fluorescence changes in FM 4-64 and EAAT2-EGFP were inversely correlated. To test whether subcellular EAAT2-EGFP structures are translocated from the cytoplasm to the plasma membrane during ionomycin stimulation, EAAT2-EGFP fluorescence was monitored locally at the plasma membrane and a few microns away in the adjacent cytoplasm. Measurements revealed sites with an increase in EAAT2-EGFP plasma membrane fluorescence correlated with a fluorescence decrease beneath the plasma membrane, and sites with plasma membrane fluorescence decrease correlated with fluorescence increase within the adjacent cytoplasm. The sites of rapid translocation/retrieval of EAAT2-EGFP structures to/from the plasma membrane appeared to be distributed in a punctuate pattern around the cell perimeter. The density of EAAT2-EGFP was regulated in a Ca²⁺-dependent manner, since in the absence of extracellular Ca²⁺ local translocation/retrieval events were absent, revealing rapid surface density regulation of EAAT2 in astrocytes by regulated exo/endocytosis.     

Vacuolar H+-ATPase activity is required for endocytic and secretory trafficking in Arabidopsis

In eukaryotic cells, compartments of the highly dynamic endomembrane system are acidified to varying degrees by the activity of vacuolar H(+)-ATPases (V-ATPases). In the Arabidopsis thaliana genome, most V-ATPase subunits are encoded by small gene families, thus offering potential for a multitude of enzyme complexes with different kinetic properties and localizations. We have determined the subcellular localization of the three Arabidopsis isoforms of the membrane-integral V-ATPase subunit VHA-a. Colocalization experiments as well as immunogold labeling showed that VHA-a1 is preferentially found in the trans-Golgi network (TGN), the main sorting compartment of the secretory pathway. Uptake experiments with the endocytic tracer FM4-64 revealed rapid colocalization with VHA-a1, indicating that the TGN may act as an early endosomal compartment. Concanamycin A, a specific V-ATPase inhibitor, blocks the endocytic transport of FM4-64 to the tonoplast, causes the accumulation of FM4-64 together with newly synthesized plasma membrane proteins, and interferes with the formation of brefeldin A compartments. Furthermore, nascent cell plates are rapidly stained by FM4-64, indicating that endocytosed material is redirected into the secretory flow after reaching the TGN. Together, our results suggest the convergence of the early endocytic and secretory trafficking pathways in the TGN.     

Ectopic expression of an activated RAC in Arabidopsis disrupts membrane cycling

Rho GTPases regulate the actin cytoskeleton, exocytosis, endocytosis, and other signaling cascades. Rhos are subdivided into four subfamilies designated Rho, Racs, Cdc42, and a plant-specific group designated RACs/Rops. This research demonstrates that ectopic expression of a constitutive active Arabidopsis RAC, AtRAC10, disrupts actin cytoskeleton organization and membrane cycling. We created transgenic plants expressing either wild-type or constitutive active AtRAC10 fused to the green fluorescent protein. The activated AtRAC10 induced deformation of root hairs and leaf epidermal cells and was primarily localized in Triton X-100-insoluble fractions of the plasma membrane. Actin cytoskeleton reorganization was revealed by creating double transgenic plants expressing activated AtRAC10 and the actin marker YFP-Talin. Plants were further analyzed by membrane staining with N-[3-triethylammoniumpropyl]-4-[p-diethylaminophenylhexatrienyl] pyridinium dibromide (FM4-64) under different treatments, including the protein trafficking inhibitor brefeldin A or the actin-depolymeryzing agents latrunculin-B (Lat-B) and cytochalasin-D (CD). After drug treatments, activated AtRAC10 did not accumulate in brefeldin A compartments, but rather reduced their number and colocalized with FM4-64-labeled membranes in large intracellular vesicles. Furthermore, endocytosis was compromised in root hairs of activated AtRAC10 transgenic plants. FM4-64 was endocytosed in nontransgenic root hairs treated with the actin-stabilizing drug jasplakinolide. These findings suggest complex regulation of membrane cycling by plant RACs.     

Spectral selectivity of FM-FM neurons in the auditory cortex of the echolocating bat,Myotis lucifugus

1. Spectral sensitivity was examined in delay-sensitive neurons in the auditory cortex of the awake FM bat, Myotis lucifugus. FM stimuli sweeping 60 kHz downward in 4 ms were used as simulated pulse-echo pairs to measure delay-dependent responses. At each neuron's best delay, the pulse and/or echo were divided into 4 FM quarters (Ist, IInd, IIIrd, and IVth), each sweeping 15 kHz in 1 ms, and quarters essential for delay sensitivity were determined for both pulse and echo. 2. For the pulse, the IVth quarter was essential for delay sensitivity in the majority of neurons. For the echo, the essential quarter for most neurons was the IInd, IIIrd, or IVth. 3. Different quarters of the pulse and echo were essential for delay sensitivity in 68% of the neurons examined. 4. This study provides neurophysiological evidence linking both spectral and temporal processing in delay-sensitive neurons of Myotis. Since spectral cues can provide target-shape information, sensitivity to both spectral and temporal parameters in single neurons may endow these neurons in FM bats with the potential for target analysis other than echo-ranging.     

FM dyes label sterol-rich plasma membrane domains and are internalized independently of the cytoskeleton in characean internodal cells

We applied the endocytic markers FM1-43, FM4-64 and filipin to internodal cells of the green alga Chara corallina. Both FM dyes stained stable, long-living plasma membrane patches with a diameter of up to 1 microm. After 5 min, FM dyes labeled cortical, trembling structures up to 500 nm in size. After 15 min, FM dyes localized to endoplasmic organelles up to 1 microm in diameter, which migrated actively along actin bundles or participated in cytoplasmic mass streaming. After 30-60 min, FM fluorescence appeared in the membrane of small, endoplasmic vacuoles but not in that of the central vacuole. Some of the FM-labeled organelles were also stained by neutral red and lysotracker yellow, indicative of acidic compartments. Filipin, a sterol-specific marker, likewise labeled plasma membrane domains which co-localized with the FM patches. However, internalization of filipin could not be observed. KCN, cytochalasin D, latrunculin B and oryzalin had no effect on size, shape and distribution of FM- and filipin-labeled plasma membrane domains. Internalization of FM dyes was inhibited by KCN but not by drugs which interfere with the actin or microtubule cytoskeleton. Our data indicate that the plasma membrane of characean internodal cells contains discrete domains which are enriched in sterols and probably correspond to clusters of lipid rafts. The inhibitor experiments suggest that FM uptake is active but independent of actin filaments, actin polymerization and microtubules. The possible function of the sterol-rich, FM labeled plasma membrane areas and the significance of actin-independent FM internalization (via endocytosis or energy-dependent flippases) are discussed.     

Characterization of cholesterol crystals in atherosclerotic plaques using stimulated Raman scattering and second-harmonic generation microscopy

Cholesterol crystals (ChCs) have been identified as a major factor of plaque vulnerability and as a potential biomarker for atherosclerosis. Yet, due to the technical challenge of selectively detecting cholesterol in its native tissue environment, the physiochemical role of ChCs in atherosclerotic progression remains largely unknown. In this work, we demonstrate the utility of hyperspectral stimulated Raman scattering (SRS) microscopy combined with second-harmonic generation (SHG) microscopy to selectively detect ChC. We show that despite the polarization sensitivity of the ChC Raman spectrum, cholesterol monohydrate crystals can be reliably discriminated from aliphatic lipids, from structural proteins of the tissue matrix and from other condensed structures, including cholesteryl esters. We also show that ChCs exhibit a nonvanishing SHG signal, corroborating the noncentrosymmetry of the crystal lattice composed of chiral cholesterol molecules. However, combined hyperspectral SRS and SHG imaging reveals that not all SHG-active structures with solidlike morphologies can be assigned to ChCs. This study exemplifies the merit of combining SRS and SHG microscopy for an enhanced label-free chemical analysis of crystallized structures in diseased tissue.     

Wavelength- and Time-Dependence of Potentiometric Non-linear Optical Signals from Styryl Dyes

Second harmonic generation (SHG) imaging microscopy is an important emerging technique for biological research, complementing existing one- and two-photon fluorescence (2PF) methods. A non-linear phenomenon employing light from mode-locked Ti:sapphire or fiber-based lasers, SHG results in intrinsic optical sectioning without the need for a confocal aperture. Furthermore, as a second-order process SHG is confined to loci lacking a center of symmetry, a constraint that is readily satisfied by lipid membranes with only one leaflet stained by a dye. Of particular interest is “resonance-enhanced” SHG from styryl dyes in cellular membranes and the possibility that SHG is sensitive to transmembrane potential. We have previously confirmed this, using simultaneous voltage-clamping and non-linear imaging of cells to find that SHG is up to four times more sensitive to potential than fluorescence. In this work, we have extended these results in two directions. First, with a range of wavelengths available from a mode-locked Ti:sapphire laser and a fiber-based laser, we have more fully investigated SHG and 2PF voltage-sensitivity from ANEP and ASTAP chromophores, obtaining SHG sensitivity spectra that are consistent with resonance enhancements. Second, we have modified our system to coordinate the application of voltage-clamp steps with non-linear image acquisition to more precisely characterize the time dependence of SHG and 2PF voltage sensitivity, finding that, at least for some dyes, SHG responds more slowly than fluorescence to changes in transmembrane potential.     

Calcium and voltage dependence of single Ca2+-activated K+ channels from cultured hippocampal neurons of rat

Calcium and voltage dependence of the Ca2+-activated K+ channel, K(Ca), was studied at the single-channel level in cultured hippocampal neurons from rat. The K(Ca) channel has approx. 220 pS conductance in symmetrical 150 mM K+, and is gated both by voltage and by Ca2+ ions. For a fixed Ca2+ concentration at the inner membrane surface, [Ca]i, channel open probability, Po, increases e-fold for 14 mV positive change in membrane potential. At a fixed membrane potential (0 mV), channel activity is first observed at [Ca]i = 10(-6) M, and increases with Ca2+ concentration approximating an absorption isotherm with power 1.4. The [Ca]i required to half activate (Po = 0.5) the channel is 4.10(-6) M. When compared to other preparations, the K(Ca) channel from hippocampal neurons reported here shows the lowest Ca2+ sensitivity and the highest voltage sensitivity. These findings are interpreted in evolutionary terms.     

Calcium-induced calcium release contributes to somatic secretion of serotonin in leech Retzius neurons

We analyzed the contribution of calcium (Ca2+)-induced Ca2+ release to somatic secretion in serotonergic Retzius neurons of the leech. Somatic secretion was studied by the incorporation of fluorescent dye FM1-43 upon electrical stimulation with trains of 10 impulses and by electron microscopy. Quantification of secretion with FM1-43 was made in cultured neurons to improve optical resolution. Stimulation in the presence of FM1-43 produced a frequency-dependent number of fluorescent spots. While a 1-Hz train produced 19.5+/-5.0 spots/soma, a 10-Hz train produced 146.7+/-20.2 spots/soma. Incubation with caffeine (10 mM) to induce Ca2+ release from intracellular stores without electrical stimulation and external Ca2+, produced 168+/-21.7 spots/soma. This staining was reduced by 49% if neurons were preincubated with the Ca2+- ATPase inhibitor thapsigargin (200 nM). Moreover, in neurons stimulated at 10 Hz in the presence of ryanodine (100 microM) to block Ca2+-induced Ca2+ release, FM1-43 staining was reduced by 42%. In electron micrographs of neurons at rest or stimulated at 1 Hz in the ganglion, endoplasmic reticulum lay between clusters of dense core vesicles and the plasma membrane. In contrast, in neurons stimulated at 20 Hz, the vesicle clusters were apposed to the plasma membrane and flanked by the endoplasmic reticulum. These results suggest that Ca2+-induced Ca2+ release produces vesicle mobilization and fusion in the soma of Retzius neurons, and supports the idea that neuronal somatic secretion shares common mechanisms with secretion by excitable endocrine cells.