Dye Tetramethylrhodamine Methyl Research Articles

Optimized Automated Analysis of Live Neuronal Mitochondria Homeostasis Modulation by Isoform-Specific Retinoic Acid Receptors.

The complex mitochondrial network makes it very challenging to segment, follow, and analyze live cells. MATLAB tools allow the analysis of mitochondria in timelapse files, considerably simplifying and speeding up the process of image processing. Nonetheless, existing tools produce a large output volume, requiring individual manual attention, and basic experimental setups have an output of thousands of files, each requiring extensive and time-consuming handling. To address these issues, a routine optimization was developed, in both MATLAB code and live-script forms, allowing for swift file analysis and significantly reducing document reading and data processing. With a speed of 100 files/min, the optimization allows an overall rapid analysis. The optimization achieves the results output by averaging frame-specific data for individual mitochondria throughout time frames, analyzing data in a defined manner, consistent with those output from existing tools. Live confocal imaging was performed using the dye tetramethylrhodamine methyl ester, and the routine optimization was validated by treating neuronal cells with retinoic acid receptor (RAR) agonists, whose effects on neuronal mitochondria are established in the literature. The results were consistent with the literature and allowed further characterization of mitochondrial network behavior in response to isoform-specific RAR modulation. This new methodology allowed rapid and validated characterization of whole-neuron mitochondria network, but it also allows for differentiation between axon and cell body mitochondria, an essential feature to apply in the neuroscience field. Moreover, this protocol can be applied to experiments using fast-acting treatments, allowing the imaging of the same cells before and after treatments, transcending the field of neuroscience.

Differences in Mitochondrial Membrane Potential Identify Distinct Populations of Human Cardiac Mesenchymal Progenitor Cells.

Adult human cardiac mesenchymal progenitor cells (hCmPC) are multipotent resident populations involved in cardiac homeostasis and heart repair. Even if the mechanisms have not yet been fully elucidated, the stem cell differentiation is guided by the mitochondrial metabolism; however, mitochondrial approaches to identify hCmPC with enhanced stemness and/or differentiation capability for cellular therapy are not established. Here we demonstrated that hCmPCs sorted for low and high mitochondrial membrane potential (using a lipophilic cationic dye tetramethylrhodamine methyl ester, TMRM), presented differences in energy metabolism from preferential glycolysis to oxidative rates. TMRM-high cells are highly efficient in terms of oxygen consumption rate, basal and maximal respiration, and spare respiratory capacity compared to TMRM-low cells. TMRM-high cells showed characteristics of pre-committed cells and were associated with higher in vitro differentiation capacity through endothelial, cardiac-like, and, to a lesser extent, adipogenic and chondro/osteogenic cell lineage, when compared with TMRM-low cells. Conversely, TMRM-low showed higher self-renewal potential. To conclude, we identified two hCmPC populations with different metabolic profile, stemness maturity, and differentiation potential. Our findings suggest that metabolic sorting can isolate cells with higher regenerative capacity and/or long-term survival. This metabolism-based strategy to select cells may be broadly applicable to therapies.

Identification & characterization of leucine-rich repeat kinase 2 & parkin RBR E3 ubiquitin protein ligase variants in patients with Parkinson's disease.

Background & objectives:Parkinson's disease (PD) is a motor disorder that affects movement. More than 24 loci and 28 associated genes have been identified to be associated with this disease. The present study accounts for the contribution of two candidates, leucine-rich repeat kinase 2 (LRRK2) and parkin RBR E3 ubiquitin protein ligase (PRKN) in the PD patients, and their characterization in silico and in vitro.Methods:A total of 145 sporadic PD cases and 120 ethnically matched healthy controls were enrolled with their informed consent. Mutation screening was performed by direct DNA sequencing of the targeted exons of LRRK2 and all exons flanking introns of PRKN. The effect of the pathogenic PRKN variants on a drug (MG-132) induced loss of mitochondrial membrane potential (△ΨM) was measured by a fluorescent dye tetramethylrhodamine methyl ester (TMRM).Results:Twelve and 20 genetic variants were identified in LRRK2 and PRKN, respectively. Interestingly, five out of seven exonic LRRK2 variants were synonymous. Further assessment in controls confirmed the rarity of two such p.Y1527 and p.V1615. Among the pathogenic missense variations (as predicted in silico) in PRKN, two were selected (p.R42H and p.A82E) for their functional study in vitro, which revealed the reduced fluorescence intensity of TMRM as compared to wild type, in case of p.R42H but not the other.Interpretation & conclusions:About 6.2 per cent of the cases (9/145) in the studied patient cohort were found to carry pathogenic (as predicted in silico) missense variations in PRKN in heterozygous condition but not in case of LRRK2 which was rare. The presence of two rare synonymous variants of LRRK2 (p.Y1527 and p.V1615) may support the phenomenon of codon bias. Functional characterization of selected PRKN variations revealed p.R42H to cause disruption of mitochondrial membrane potential (△ΨM) rendering cells more susceptible to cellular stress.

Abstract 202: Low Mitochondrial Membrane Potential Identified a Novel Population of Human Cardiac Progenitor Cells with the Capacity for Myocardial Repair

As resident cardiac stem/progenitor cells (CPCs) stay in the hypoxia niche, have less metabolic activity and lower mitochondrial membrane potential, the aim of this study was to characterize a new subset of CPCs with lower mitochondrial membrane potential and examine their potential for myocardial repair. Following the live cell staining with a lipophilic cationic dye tetramethylrhodamine methyl ester (TMRM) at a final concentration of 25 nm, a novel subset of hCPCs with the lowest ~3% TMRM fluorescence were sorted from right atrial appendage derived cells (RAACs) in patients with cardiomyopathy, and named as ΔΨ m low -hCPCs, which are self-renewable, and characterized as positive (>90%) for CD90, CD44, CD140b, CD172a, however, negative (<5%) for CD34, CD45, CD54, CD117, CD133. The increased expression of pluripotent genes (LIN28, NANOG, & OCT3/4) and cardiac lineage genes (GATA4, MEF2C, MYOCD, & NKX2.5), as well as the decreased expression of cell cycle inhibitor (CDKN2A & CDKN1A) were observed in ΔΨ m low -hCPCs comparing with the non-sorted RAACs. Interestingly, ΔΨ m low -hCPCs exclusively express heme oxygenase-1 (HO-1). And CPCs positive for HO-1 and NKX2.5/GATA4 were found in the intact human heart tissue sections. ΔΨ m low -hCPCs are able to differentiate into all three cardiac lineage cells in both in vitro and in vivo conditions, and display superior ability for survival, proliferation and release of cytoprotective cytokines compared to the RAACs. At 35 days, compared with the vehicle and control cell groups, transplantation of ΔΨ m low -hCPCs significantly improve the cardiac structure and function following myocardial infarction in the immunodeficient mouse model, as indicated by the increased left ventricular (LV) ejection fraction, increased positive and negative LV dP/dt, reduced LV remodeling, and reduced infarct size. The immunohistochemistry studies showed the significant increased cardiac proliferation, newly formed cardiomyocytes, angiogenesis, and decreased cell apoptosis in the ΔΨ m low -hCPCs treated group. In summary, we identified a new hCPC population that might represent a primitive progenitor cell population within human hearts. The existence of these cells opens new opportunities for myocardial repair.

Use of the fluorescent dye tetramethylrhodamine methyl ester perchlorate for mitochondrial membrane potential assessment in human spermatozoa

Mitochondrial membrane potential (ΔΨm) is an indicator of sperm quality and its evaluation complements the standard semen analysis. The fluorescent dye JC-1 has been widely used to assess sperm ΔΨm; however, some problems have been detected under certain experimental conditions. Another fluorescent compound, tetramethylrhodamine methyl ester perchlorate (TMRM), has been used in somatic cells and bovine spermatozoa but not in human spermatozoa. TMRM accumulates in hyperpolarised mitochondria and the fluorescence intensity of this compound correlates with ΔΨm. Thus, the aim of this study was to evaluate and validate the usefulness of the fluorescent dye TMRM for measuring sperm ΔΨm. The results showed that TMRM accurately detects sperm populations displaying either high or low ΔΨm. Moreover, TMRM was able to measure sperm ΔΨm under the experimental conditions in which JC-1 had previously presented difficulties. Differences in ΔΨm according to sperm and semen quality were properly detected and a positive correlation between ΔΨm and conventional semen parameters was observed. Finally, a positive correlation was found between the ΔΨm measurement by TMRM and by the widely used JC-1. In conclusion, TMRM is a simple, time-effective method, easy to set in laboratories equipped with flow cytometry technology, and can accurately detect changes in ΔΨm with efficiency comparable to JC-1 without its limitations.

Mitochondrial Membrane Potential Identifies Cells with Enhanced Stemness for TCR and CAR based Adoptive Immunotherapy of Cancer

Abstract Long-term survival and anti-tumor immunity critically depends on their metabolic fitness but approaches to select metabolically robust T cells for T cell based immunotherapy remains unknown. We utilized a lipophilic cationic dye (tetramethylrhodamine methyl ester) to identify and isolate metabolically robust T cells based on mitochondrial membrane potential (ΔΨm). Cells with lower membrane-potential (low ΔΨm) had a molecular profile of memory precursors and displayed an enhanced ability to enter the memory pool as compared to cells displaying higher mitochondrial potential (high ΔΨm) characteristic of short lived effectors. Interestingly, we also found that multiple distinct negative inhibitory receptors such as PD1, CTLA4, BTLA, LAG3 and TIM3 were enriched in the high ΔΨm subset compared to the low-ΔΨm subset. Comprehensive metabolomics profiling demonstrated global features of metabolic fitness (increased lipid oxidation) in low ΔΨm sorted CD8+ T cells. Transfer of low ΔΨm T cells was associated with superior long-term in vivo persistence as evidenced by 100 fold increase in the frequency of T cells 300 days after adoptive transfer, augmented autoimmunity and an enhanced capacity to eradicate established cancer compared with high ΔΨm cells. Mechanistically, we found that the IL2 driven mTOR activation caused increased mitochondrial oxygen consumption and accumulation of higher level of ROS. Surprisingly, use of Ψm to enrich for cells with superior metabolic features was observed within central memory, effector (Tc17, Th1, and Th17) T cells and long term hematopoietic stem cells (LT HSC). This novel metabolism based approach may be applicable to therapies involving transfer of lymphocytes for treatment of advanced cancer.

Mitochondrial Membrane Potential Identifies Cells with Enhanced Stemness for Cellular Therapy

Long-term survival and antitumor immunity of adoptively transferred CD8(+) T cells is dependent on their metabolic fitness, but approaches to isolate therapeutic T cells based on metabolic features are not well established. Here we utilized a lipophilic cationic dye tetramethylrhodamine methyl ester (TMRM) to identify and isolate metabolically robust T cells based on their mitochondrial membrane potential (ΔΨm). Comprehensive metabolomic and gene expression profiling demonstrated global features of improved metabolic fitness in low-ΔΨm-sorted CD8(+) T cells. Transfer of these low-ΔΨm T cells was associated with superior long-term in vivo persistence and an enhanced capacity to eradicate established tumors compared with high-ΔΨm cells. Use of ΔΨm-based sorting to enrich for cells with superior metabolic features was observed in CD8(+), CD4(+) T cell subsets, and long-term hematopoietic stem cells. This metabolism-based approach to cell selection may be broadly applicable to therapies involving the transfer of HSC or lymphocytes for the treatment of viral-associated illnesses and cancer.

PKA Phosphorylation of NCLX Reverses Mitochondrial Calcium Overload and Depolarization, Promoting Survival of PINK1-Deficient Dopaminergic Neurons

SUMMARYMitochondrial Ca2+ overload is a critical, preceding event in neuronal damage encountered during neurodegenerative and ischemic insults. We found that loss of PTEN-induced putative kinase 1 (PINK1) function, implicated in Parkinson disease, inhibits the mitochondrial Na+/Ca2+ exchanger (NCLX), leading to impaired mitochondrial Ca2+ extrusion. NCLX activity was, however, fully rescued by activation of the protein kinase A (PKA) pathway. We further show that PKA rescues NCLX activity by phosphorylating serine 258, a putative regulatory NCLX site. Remarkably, a constitutively active phosphomimetic mutant of NCLX (NCLXS258D) prevents mitochondrial Ca2+ overload and mitochondrial depolarization in PINK1 knockout neurons, thereby enhancing neuronal survival. Our results identify an mitochondrial Ca2+ transport regulatory pathway that protects against mitochondrial Ca2+ overload. Because mitochondrial Ca2+ dyshomeostasis is a prominent feature of multiple disorders, the link between NCLX and PKA may offer a therapeutic target.

Mitochondrial depolarization and asystole in the globally ischemic rabbit heart: coordinated response to interventions affecting energy balance.

Mitochondrial membrane potential (ΔΨm) depolarization has been implicated in the loss of excitability (asystole) during global ischemia, which is relevant for the success of defibrillation and resuscitation after cardiac arrest. However, the relationship between ΔΨm depolarization and asystole during no-flow ischemia remains unknown. We applied spatial Fourier analysis to confocally recorded fluorescence emitted by ΔΨm-sensitive dye tetramethylrhodamine methyl ester. The time of ischemic ΔΨm depolarization (tmito_depol) was defined as the time of 50% decrease in the magnitude of spectral peaks reflecting ΔΨm. The time of asystole (tasys) was determined as the time when spontaneous and induced ventricular activity ceased to exist. Interventions included tachypacing (150 ms), myosin II ATPase inhibitor blebbistatin (heart immobilizer), and the combination of blebbistatin and the inhibitor of glycolysis iodoacetate. In the absence of blebbistatin, confocal images were obtained during brief perfusion with hyperkalemic solution and after the contraction failed between 7 and 15 min of ischemia. In control, tmito_depol and tasys were 24.4 ± 6.0 and 26.0 ± 5.0 min, respectively. Tachypacing did not significantly affect either parameter. Blebbistatin dramatically delayed tmito_depol and tasys (51.4 ± 8.6 and 45.7 ± 5.3 min, respectively; both P < 0.0001 vs. control). Iodoacetate combined with blebbistatin accelerated both events (tmito_depol, 12.7 ± 1.8 min; and tasys, 6.5 ± 1.1 min; both P < 0.03 vs. control). In all groups pooled together, tasys was strongly correlated with tmito_depol (R(2) = 0.845; P < 0.0001). These data may indicate a causal relationship between ΔΨm depolarization and asystole or a similar dependence of the two events on energy depletion during ischemia. Our results urge caution against the use of blebbistatin in studies addressing pathophysiology of myocardial ischemia.

High-Content Imaging Assays for Identifying Compounds that Generate Superoxide and Impair Mitochondrial Membrane Potential in Adherent Eukaryotic Cells.

Reactive oxygen species (ROS) are constantly produced in cells as a result of aerobic metabolism. When there is an excessive production of ROS and the cell's antioxidant defenses are overwhelmed, oxidative stress occurs. The superoxide anion is a type of ROS that is produced primarily in mitochondria but is also generated in other regions of the cell including peroxisomes, endoplasmic reticulum, plasma membrane, and cytosol. Here, a high-content imaging assay using the dye dihydroethidium is described for identifying compounds that generate superoxide in eukaryotic cells. A high-content imaging assay using the fluorescent dye tetramethylrhodamine methyl ester is also described to identify compounds that impair mitochondrial membrane potential in eukaryotic cells. The purpose of performing both assays is to identify compounds that (1) generate superoxide at lower concentrations than they impair mitochondrial membrane potential, (2) impair mitochondrial membrane potential at lower concentrations than they generate superoxide, (3) generate superoxide and impair mitochondrial function at similar concentrations, and (4) do not generate superoxide or impair mitochondrial membrane potential during the duration of the assays.

Thyroid hormones can increase astrocyte fatty acid oxidation and survival; a possible mechanism for decreased tissue necrosis after cerebral stroke

Enhancement of astrocyte metabolism in mice significantly decreases lesion size after stroke. This was achieved after purinergic stimulation, but we know that thyroid hormones (TH) can also increase mitochondrial metabolism. Acute treatment with triiodothyronine (T3) increases long‐chain fatty acid oxidation (FAO) in several tissues to improve metabolism. Because astrocytes also use fatty acids for energy, we hypothesized that THs increase astrocyte FAO and improve survival after oxidative stress or stroke.ResultsNanomolar concentrations of either diiodothyronine (T2) or T3 increase astrocyte FAO of [3H]palmitate into 3H2O by over 30% (p&lt;0.01). The astrocyte mitochondrial membrane potential was measured using the potential sensitive dye tetramethyl rhodamine methyl ester. Acute T2 treatment (15 minutes) tripled the dye intensity (p&lt;0.05), indicating improved mitochondrial membrane potential. These data are consistent with increased energy production upon TH treatment. Astrocytes were stressed with hydrogen peroxide, and viability was measured using the vital dye calcien‐AM. T2 and T3 each increased astrocyte viability 25% (p&lt;0.01). To test if TH is protective in vivo, we induced focal ischemia in mice. Administration of T2 decreased the necrotic volume in the brain by half (p=0.02) 24 hours post‐stroke. Future testing will determine if TH‐mediated survival of astrocytes is dependent on FAO.

P2Y1R-Initiated, IP3R-Dependent Stimulation of Astrocyte Mitochondrial Metabolism Reduces and Partially Reverses Ischemic Neuronal Damage in Mouse

Glia-based neuroprotection strategies are emerging as promising new avenues to treat brain damage. We previously reported that activation of the glial-specific purinergic receptor, P2Y(1)R, reduces both astrocyte swelling and brain infarcts in a photothrombotic mouse model of stroke. These restorative effects were dependent on astrocyte mitochondrial metabolism. Here, we extend these findings and report that P2Y(1)R stimulation with the purinergic ligand 2-methylthioladenosine 5' diphosphate (2MeSADP) reduces and partially reverses neuronal damage induced by photothrombosis. In vivo neuronal morphology was confocally imaged in transgenic mice expressing yellow fluorescent protein under the control of the Thy1 promoter. Astrocyte mitochondrial membrane potentials, monitored with the potential sensitive dye tetra-methyl rhodamine methyl ester, were depolarized after photothrombosis and subsequently repolarized when P2Y(1)Rs were stimulated. Mice deficient in the astrocyte-specific type 2 inositol 1,4,5 trisphosphate (IP(3)) receptor exhibited aggravated ischemic dendritic damage after photothrombosis. Treatment of these mice with 2MeSADP did not invoke an intracellular Ca(2+) response, did not repolarize astrocyte mitochondria, and did not reduce or partially reverse neuronal lesions induced by photothrombotic stroke. These results demonstrate that IP(3)-Ca(2+) signaling in astrocytes is not only critical for P2Y(1)R-enhanced protection, but suggest that IP(3)-Ca(2+) signaling is also a key component of endogenous neuroprotection.

Mitochondrial activation at the onset of contractions in isolated myofibres during successive contractile periods

At the onset of skeletal muscle repetitive contractions, there is a significant delay in the time to achieve oxidative phosphorylation steady state. The purpose of the present study was to examine the factors that limit oxidative phosphorylation at the onset of contractions. NAD(P)H was measured in real time during two contractile periods (2 min each) separated by 5 min of rest in intact single muscle fibres (n = 7) isolated from Xenopus laevis. The fibres were then loaded with the dye tetramethylrhodamine methyl ester perchlorate (TMRM) to evaluate the kinetics of the mitochondrial membrane potential (Δψ (m)) during two further successive contractile periods. At the onset of contractions in the first period, NAD(P)H exhibited a time delay (14.1 ± 1.3 s) before decreasing toward a steady state. In contrast, Δψ(m) decreased immediately after the first contraction and started to be reestablished after 10.7 ± 0.9 s, with restoration to the pre-stimulation values after approximately 32 s. In the second contractile period (5 min after the first), NAD(P)H decreased immediately (i.e. no time delay) after the first contraction and had a significantly shorter time constant compared to the first contractile bout (3.3 ± 0.3 vs. 5.0 ± 0.2 s, P < 0.05). During the second bout, Δψ(m) remained unchanged from pre-stimulation values. These results suggest: (1) that at the onset of contractions, oxidative phosphorylation is primarily limited by the activity of the electron transport chain complexes rather than by a limited level of substrates; and (2) when the muscle is 'primed' by previous contractile activity, the faster enhancement of the cellular respiratory rate is due to intrinsic factors within the myofibre.

116 Matrix metalloproteinase inhibition attenuates reperfusion injury, independently of and additive to mitochondrial permeability transition pore inhibition

While matrix-metalloproteinase (MMP) inhibitors appear to protect against myocardial ischaemia/reperfusion injury, the mechanisms are poorly understood. We hypothesised that cardioprotection resultant from MMP inhibition is independent of mitochondrial permeability transition...

Pulsing of Membrane Potential in Individual Mitochondria: A Stress-Induced Mechanism to Regulate Respiratory Bioenergetics in Arabidopsis

Mitochondrial ATP synthesis is driven by a membrane potential across the inner mitochondrial membrane; this potential is generated by the proton-pumping electron transport chain. A balance between proton pumping and dissipation of the proton gradient by ATP-synthase is critical to avoid formation of excessive reactive oxygen species due to overreduction of the electron transport chain. Here, we report a mechanism that regulates bioenergetic balance in individual mitochondria: a transient partial depolarization of the inner membrane. Single mitochondria in living Arabidopsis thaliana root cells undergo sporadic rapid cycles of partial dissipation and restoration of membrane potential, as observed by real-time monitoring of the fluorescence of the lipophilic cationic dye tetramethyl rhodamine methyl ester. Pulsing is induced in tissues challenged by high temperature, H(2)O(2), or cadmium. Pulses were coincident with a pronounced transient alkalinization of the matrix and are therefore not caused by uncoupling protein or by the opening of a nonspecific channel, which would lead to matrix acidification. Instead, a pulse is the result of Ca(2+) influx, which was observed coincident with pulsing; moreover, inhibitors of calcium transport reduced pulsing. We propose a role for pulsing as a transient uncoupling mechanism to counteract mitochondrial dysfunction and reactive oxygen species production.

Methods to Assess Beta Cell Death Mediated by Cytotoxic T Lymphocytes

Type 1 diabetes (T1D) is a T cell mediated autoimmune disease. During the pathogenesis, patients become progressively more insulinopenic as insulin production is lost, presumably this results from the destruction of pancreatic beta cells by T cells. Understanding the mechanisms of beta cell death during the development of T1D will provide insights to generate an effective cure for this disease. Cell-mediated lymphocytotoxicity (CML) assays have historically used the radionuclide Chromium 51 ((51)Cr) to label target cells. These targets are then exposed to effector cells and the release of (51)Cr from target cells is read as an indication of lymphocyte-mediated cell death. Inhibitors of cell death result in decreased release of (51)Cr. As effector cells, we used an activated autoreactive clonal population of CD8(+) Cytotoxic T lymphocytes (CTL) isolated from a mouse stock transgenic for both the alpha and beta chains of the AI4 T cell receptor (TCR). Activated AI4 T cells were co-cultured with (51)Cr labeled target NIT cells for 16 hours, release of (51)Cr was recorded to calculate specific lysis Mitochondria participate in many important physiological events, such as energy production, regulation of signaling transduction, and apoptosis. The study of beta cell mitochondrial functional changes during the development of T1D is a novel area of research. Using the mitochondrial membrane potential dye Tetramethyl Rhodamine Methyl Ester (TMRM) and confocal microscopic live cell imaging, we monitored mitochondrial membrane potential over time in the beta cell line NIT-1. For imaging studies, effector AI4 T cells were labeled with the fluorescent nuclear staining dye Picogreen. NIT-1 cells and T cells were co-cultured in chambered coverglass and mounted on the microscope stage equipped with a live cell chamber, controlled at 37°C, with 5% CO(2;), and humidified. During these experiments images were taken of each cluster every 3 minutes for 400 minutes. Over a course of 400 minutes, we observed the dissipation of mitochondrial membrane potential in NIT-1 cell clusters where AI4 T cells were attached. In the simultaneous control experiment where NIT-1 cells were co-cultured with MHC mis-matched human lymphocyte Jurkat cells, mitochondrial membrane potential remained intact. This technique can be used to observe real-time changes in mitochondrial membrane potential in cells under attack of cytotoxic lymphocytes, cytokines, or other cytotoxic reagents.

Spontaneous Oscillations in Mitochondrial Membrane Potential of Cultured Neurons Did Not Correlate With Cytosolic Calcium Concentration

We used two-photon imaging of the fluorescent dye tetramethyl-rhodamine methyl ester (TMRM) to visualize spontaneous oscillations of mitochondrial membrane potential in cultured rat hippocampal neurons. TMRM-loaded cells displayed two distinct forms of spontaneous oscillation when imaged in artificial cerebral spinal fluid (ACSF) at room temperature: fast oscillation of individual mitochondria (flickering) corresponding to periods of depolarization lasting several seconds, and slow oscillation of groups of mitochondria (wave) corresponding to depolarizations lasting 1-3 minutes. Similar types of spontaneous oscillation have been reported previously using isolated mitochondria and cultured cells, although the underlying cause(s) of the oscillations is unclear. In isolated mitochondria, flickering can be triggered by mitochondrial Ca uptake (Biophys. J. 87: 3585, 2004) and waves can be induced by local Ca elevation (Cell 89: 1145, 1997). We tested whether Ca could exert similar effects in cultured hippocampal neurons by testing cells loaded with both TMRM and a Ca-sensitive fluorophore (fura-2 or fluo-3). Cellular fluorescence was imaged repeatedly every second for up to 60 minutes. Under our conditions (in ACSF at room temperature), neurons displayed spontaneous oscillations in both TMRM and Ca-sensitive dyes, but there was no correlation between these signals. Furthermore, some neurons displayed fast and slow TMRM oscillations without alteration in Ca levels, while others exhibited fast Ca oscillations (lasting 3-8 sec) but no TMRM oscillations. In addition, Ca oscillations were often synchronized in adjacent neurons, whereas TMRM oscillations were not. These results indicate that oscillations in cytosolic calcium levels do not appear to be directly coupled to oscillations in mitochondrial membrane potential in cultured hippocampal neurons. This does not rule out the possibility that Ca might influence mitochondrial oscillations through an indirect action.

Nonequivalence of Membrane Voltage and Ion-Gradient as Driving Forces for the Bacterial Flagellar Motor at Low Load

Many bacterial species swim using flagella. The flagellar motor couples ion flow across the cytoplasmic membrane to rotation. Ion flow is driven by both a membrane potential ( V m) and a transmembrane concentration gradient. To investigate their relation to bacterial flagellar motor function we developed a fluorescence technique to measure V m in single cells, using the dye tetramethyl rhodamine methyl ester. We used a convolution model to determine the relationship between fluorescence intensity in images of cells and intracellular dye concentration, and calculated V m using the ratio of intracellular/extracellular dye concentration. We found V m = −140 ± 14 mV in Escherichia coli at external pH 7.0 ( pH ex), decreasing to −85 ± 10 mV at pH ex 5.0. We also estimated the sodium-motive force (SMF) by combining single-cell measurements of V m and intracellular sodium concentration. We were able to vary the SMF between −187 ± 15 mV and −53 ± 15 mV by varying pH ex in the range 7.0–5.0 and extracellular sodium concentration in the range 1–85 mM. Rotation rates for 0.35- μm- and 1- μm-diameter beads attached to Na +-driven chimeric flagellar motors varied linearly with V m. For the larger beads, the two components of the SMF were equivalent, whereas for smaller beads at a given SMF, the speed increased with sodium gradient and external sodium concentration.

Interfacing SH-SY5Y human neuroblastoma cells with SU-8 microstructures

Microwell structures were fabricated using SU-8 photoresist for engineering a quasi-three-dimensional (quasi-3D) microenvironment for cultured neuronal cells. SH-SY5Y human neuroblastoma cells were successfully integrated into microwells of a nominal diameter of 100 μm, with or without 10-μm wide microchannels connecting neighboring microwells, in an aspect ratio (ratio of structure depth over width) of approximately 1. With the help of polyethylene glycol stamping and laminin coating, a neuronal-like network was achieved by integrating populations of SH-SY5Y cells with a microwell network pattern. Resting membrane potential establishment was evaluated with confocal microscopy and the potentiometric fluorescent dye tetramethylrhodamine methyl ester. It was found that the intra/extracellular fluorescent intensity ratio ( R) was 2.4 ± 1.4 [ n (number of cells measured) = 112] for SH-SY5Y cells on flat SU-8 substrates on day 5 into differentiation, which was not significantly different from the ratio on day 13 into differentiation, 2.0 ± 1.8 ( n = 104) ( P > 0.05). For cells in the microwell network structures, R was 4.8 ± 4.7 ( n = 51) and 3.9 ± 3.2 ( n = 62) on days 5 and 13 into differentiation, respectively ( P > 0.5). Cells within the network structures had higher R ratios than on flat substrates, for either day 5 or 13 into differentiation ( P < 0.01). These results demonstrated that the well network structures, or topographically patterned substrates, were more suitable formats for promoting SH-SY5Y cell resting membrane potential establishment than flat substrates, suggesting the potential to control cellular function through substrate topography engineering.

Formation of actin-ADF/cofilin rods transiently retards decline of mitochondrial potential and ATP in stressed neurons

When neurons in culture are transiently stressed by inhibition of ATP synthesis, they rapidly form within their neurites rodlike actin inclusions that disappear when the insult is removed. Oxidative stress, excitotoxic insults, and amyloid beta-peptide oligomers also induce rods. Immunostaining of neurites indicates that these rods also contain the majority of the actin filament dynamizing proteins, actin-depolymerizing factor (ADF) and cofilin (AC). If the rods reappear within 24 h after the stress is removed, the neurite degenerates distal to the rod but with no increase in neuronal death. Here, rods were generated in cultured rat E18 hippocampal cells by overexpression of a green fluorescent protein chimera of AC. Surprisingly, we have found that, for a short period (approximately 60 min) immediately after initial rod formation, the loss of mitochondrial membrane potential (Delta Psi(m)) and ATP in neurites with rods is slower than in neurites without them. The Delta Psi(m) was monitored with the fluorescent dye tetramethylrhodamine methyl ester, and ATP was monitored with the fluorescent ion indicator mag-fura 2. Actin in rods is less dynamic than is filamentous actin in other cytoskeletal structures. Because Delta Psi(m) depends on cellular ATP and because ATP hydrolysis associated with actin filament turnover is responsible for a large fraction of neuronal energy consumption (approximately 50%), the formation of rods transiently protects neurites by slowing filament turnover and its associated ATP hydrolysis.