Changes In Living Cells Research Articles

Activatable Rotor for Quantifying Lysosomal Viscosity in Living Cells

We have developed Lyso-V, the first fluorescent probe of lysosomal viscosity. Because of its lysosome-actived fluorescence characteristics, Lyso-V has proved to be an ideal lysosomal tracer with high spatial and temporal resolution under laser confocal microscopy. More importantly, Lyso-V shows its practical applicability in real-time quantification of lysosomal viscosity changes in live cells through fluorescence lifetime imaging microscopy.

Predicting rates of cell state change caused by stochastic fluctuations using a data-driven landscape model

We develop a potential landscape approach to quantitatively describe experimental data from a fibroblast cell line that exhibits a wide range of GFP expression levels under the control of the promoter for tenascin-C. Time-lapse live-cell microscopy provides data about short-term fluctuations in promoter activity, and flow cytometry measurements provide data about the long-term kinetics, because isolated subpopulations of cells relax from a relatively narrow distribution of GFP expression back to the original broad distribution of responses. The landscape is obtained from the steady state distribution of GFP expression and connected to a potential-like function using a stochastic differential equation description (Langevin/Fokker-Planck). The range of cell states is constrained by a force that is proportional to the gradient of the potential, and biochemical noise causes movement of cells within the landscape. Analyzing the mean square displacement of GFP intensity changes in live cells indicates that these fluctuations are described by a single diffusion constant in log GFP space. This finding allows application of the Kramers' model to calculate rates of switching between two attractor states and enables an accurate simulation of the dynamics of relaxation back to the steady state with no adjustable parameters. With this approach, it is possible to use the steady state distribution of phenotypes and a quantitative description of the short-term fluctuations in individual cells to accurately predict the rates at which different phenotypes will arise from an isolated subpopulation of cells.

Using FLIM-FRET to Measure Conformational Changes of Transglutaminase Type 2 in Live Cells

Transglutaminase type 2 (TG2) is a ubiquitously expressed member of the transglutaminase family, capable of mediating a transamidation reaction between a variety of protein substrates. TG2 also has a unique role as a G-protein with GTPase activity. In response to GDP/GTP binding and increases in intracellular calcium levels, TG2 can undergo a large conformational change that reciprocally modulates the enzymatic activities of TG2. We have generated a TG2 biosensor that allows for quantitative assessment of TG2 conformational changes in live cells using Förster resonance energy transfer (FRET), as measured by fluorescence lifetime imaging microscopy (FLIM). Quantifying FRET efficiency with this biosensor provides a robust assay to quickly measure the effects of cell stress, changes in calcium levels, point mutations and chemical inhibitors on the conformation and localization of TG2 in living cells. The TG2 FRET biosensor was validated using established TG2 conformational point mutants, as well as cell stress events known to elevate intracellular calcium levels. We demonstrate in live cells that inhibitors of TG2 transamidation activity can differentially influence the conformation of the enzyme. The irreversible inhibitor of TG2, NC9, forces the enzyme into an open conformation, whereas the reversible inhibitor CP4d traps TG2 in the closed conformation. Thus, this biosensor provides new mechanistic insights into the action of two TG2 inhibitors and defines two new classes based on ability to alter TG2 conformation in addition to inhibiting transamidation activity. Future applications of this biosensor could be to discover small molecules that specifically alter TG2 conformation to affect GDP/GTP or calcium binding.

Benzofurazan Sulfides for Thiol Imaging and Quantification in Live Cells through Fluorescence Microscopy

Thiol groups play a significant role in various cellular functions. Cellular thiol concentrations can be affected by various physiological or pathological factors. A fluorescence imaging agent that can effectively and specifically image thiols in live cells through fluorescence microscopy is desirable for live cell thiol monitoring. Benzofurazan sulfides 1a-1e were synthesized and found to be thiol specific fluorogenic agents except 1d. They are not fluorescent but form strong fluorescent thiol adducts after reacting with thiols through a sulfide-thiol exchange reaction. On the other hand, they exhibit no reaction with other biologically relevant nucleophilic functional groups such as -NH(2), -OH, or -COOH revealing the specificity for the detection of thiols. Sulfide 1a was selected to confirm its ability to image cellular thiols through fluorescence microscopy. The compound was demonstrated to effectively image and quantify thiol changes in live cells through fluorescence microscopy using 430 and 520 nm as the excitation and emission wavelengths, respectively. The quantification results of total thiol in live cells obtained from fluorescence microscopy were validated by an high-pressure liquid chromatography/ultraviolet (HPLC/UV) total thiol assay method. The reagents and method will be of a great value to thiol redox-related research.

Poly(A)-targeting molecular beacons: Fluorescence resonance energy transfer-based in vitro quantitation and time-dependent imaging in live cells

Quantitation of poly(A)-RNA, time-dependent visualization of intracellular poly(A)+-RNA localization in living mammalian cells, and time-resolved intracellular binding dynamics of molecular beacons at the single-molecule level using a fluorescence resonance energy transfer (FRET)-based molecular beacon are described. FRET-based molecular beacons were designed as poly(A)-targeting probes to be oligonucleotides that contained Cy5 and Cy3 fluorescent dyes at the strand ends and a poly(A)-targeting sequence inside the strand. Our ratiometric analysis using poly(A)-targeting probes allowed for highly specific and wide-ranging detection (from 1.25nM to 0.5μM) of poly(A)-RNA, as well as for determination of Kd values, and revealed a distribution of the probe itself and localization of the target RNA sequence in cells. Furthermore, time-dependent FRET-mediated fluorescence changes at the single-molecule level caused by the folding-induced gradual conformation changes in live cells were observed.

Raman microspectroscopy of live cells under autophagy-inducing conditions

The role of autophagy in numerous physiological responses triggered by a variety of mechanisms both in states of health and disease has raised considerable interest in this cellular process. However, the current analytical tools to study autophagy are either invasive or require genetic manipulation. Raman microspectroscopy is a potentially quantitative analytical method that has been shown to be useful for the label-free, non-destructive analysis of living biological cells and tissues. We present in this study initial efforts to study autophagy using Raman spectroscopy. The response of adherent mouse and human cancer cells to starvation conditions (glutamine deprivation and amino acid deprivation) was probed by Raman spectroscopy and compared to fluorescence microscopy results using autophagy-specific markers. We also demonstrate the capability of Raman spectroscopy to monitor the recovery dynamics of starved cells and to probe the heterogeneity in the response to starvation that can arise in cell populations. Finally, this work suggests that the 718 cm(-1) Raman line associated with phospholipids may be a useful spectral marker indicative of an autophagic response to starvation stimuli. Overall, this study establishes the utility of Raman spectroscopy to non-invasively detect biologically relevant changes in live cells exposed to conditions known to trigger autophagy.

Dual colored mesoporous silica nanoparticles with pH activable rhodamine-lactam for ratiometric sensing of lysosomal acidity

Alteration of lysosome acidity has been implicated in many biological events ranging from apoptosis to cancer metastasis, etc. Mesoporous silica nanoparticles doped with acid activable rhodamine-lactam and fluorescein isothiocyanate (FITC) were developed for ratiometric sensing of lysosomal pH changes in live cells with flow cytometry.

Hepatic Ischemia Induced Immediate Oxidative Stress after Reperfusion and Determined the Severity of the Reperfusion-Induced Damage

Noninvasive evaluation of organ redox states provides invaluable information in many clinical settings. We evaluated a newly developed reduction/oxidation-sensitive green fluorescent protein (roGFP) probe that reports cellular redox potentials and their dynamic changes in live cells. On hypoxia/reoxygenation (H/R) of AML12 liver cells, roGFP indicated mild reduction during hypoxia, but immediate transient oxidation after reoxygenation. The roGFP probe confirmed the antioxidative effects of N-acetylcysteine, catalase, redox factor-1, and Mn-SOD/CuZn-SOD against H/R-induced cellular oxidative stress (OS). In a mouse liver ischemia/reperfusion (I/R) model, roGFP transduced by using an adenoviral vector revealed immediate reduction of the liver under ischemia, and two distinct peaks of OS: (a) early, observed within 60 min after reperfusion, similar to the in vitro study; and (b) later, at 24 h. The early peak levels paralleled the ischemic time up to 75 min and the postischemic liver injury (sGOT/GPT/LDH) in the later phase (6 and 24 h after I/R). The roGFP probe successfully indicated postischemic OS of the liver in living mice, accurately predicting postischemic liver injury. This probe may represent an effective OS marker indicating organ redox states and also predicting the damage/function.

Fluorescence resonance energy transfer sensors for quantitative monitoring of pentose and disaccharide accumulation in bacteria

BackgroundEngineering microorganisms to improve metabolite flux requires detailed knowledge of the concentrations and flux rates of metabolites and metabolic intermediates in vivo. Fluorescence resonance energy transfer sensors represent a promising technology for measuring metabolite levels and corresponding rate changes in live cells. These sensors have been applied successfully in mammalian and plant cells but potentially could also be used to monitor steady-state levels of metabolites in microorganisms using fluorimetric assays. Sensors for hexose and pentose carbohydrates could help in the development of fermentative microorganisms, for example, for biofuels applications. Arabinose is one of the carbohydrates to be monitored during biofuels production from lignocellulose, while maltose is an important degradation product of starch that is relevant for starch-derived biofuels production.ResultsAn Escherichia coli expression vector compatible with phage λ recombination technology was constructed to facilitate sensor construction and was used to generate a novel fluorescence resonance energy transfer sensor for arabinose. In parallel, a strategy for improving the sensor signal was applied to construct an improved maltose sensor. Both sensors were expressed in the cytosol of E. coli and sugar accumulation was monitored using a simple fluorimetric assay of E. coli cultures in microtiter plates. In the case of both nanosensors, the addition of the respective ligand led to concentration-dependent fluorescence resonance energy transfer responses allowing quantitative analysis of the intracellular sugar levels at given extracellular supply levels as well as accumulation rates.ConclusionThe nanosensor destination vector combined with the optimization strategy for sensor responses should help to accelerate the development of metabolite sensors. The new carbohydrate fluorescence resonance energy transfer sensors can be used for in vivo monitoring of sugar levels in prokaryotes, demonstrating the potential of such sensors as reporter tools in the development of metabolically engineered microbial strains or for real-time monitoring of intracellular metabolite during fermentation.

Imaging and manipulating phosphoinositides in living cells

Phosphoinositides are minor phospholipid constituents of virtually every biological membrane yet they play fundamental roles in controlling membrane-bound signalling events. Phosphoinositides are produced from phosphatidylinositol (PtdIns) by phosphorylation of one or more of three positions (3, 4 and 5) of the inositol headgroup located at the membrane cytoplasmic interface by distinct families of inositol lipid kinases. Intriguingly, many of the kinase reactions are catalysed by more than one form of the kinases even in simple organisms and these enzymes often assume non-redundant functions. A similar diversity is seen with inositide phosphatases, the enzymes that dephosphorylate phosphoinositides with a certain degree of specificity and the impairments of which are often linked to human diseases. This degree of multiplicity at the enzyme level together with the universal roles of these lipids in cell regulation assumes that inositol lipids are spatially and functionally restricted in specific membrane compartments. Studying the compartmentalized roles of these lipids at the cellular level represents a major methodological challenge. Over the last 10 years significant progress has been made in creating reagents that can monitor inositol lipid changes in live cells with fluorescence or confocal microscopy. New methods are also being developed to manipulate these lipids in specific membrane compartments in a regulated fashion. This article recalls some historical aspects of inositide research and describes the new methodological advances highlighting their great potential as well as the problems one can encounter with their use.

Conformational Regulation of α4β1-Integrin Affinity by Reducing Agents: “INSIDE-OUT” SIGNALING IS INDEPENDENT OF AND ADDITIVE TO REDUCTION-REGULATED INTEGRIN ACTIVATION

The alpha(4)beta(1)-integrin (very late antigen-4 (VLA-4), CD49d/CD29) is an adhesion receptor involved in the interaction of lymphocytes, dendritic cells, and stem cells with the extracellular matrix and endothelial cells. This and other integrins have the ability to regulate their affinity for ligands through a process termed "inside-out" signaling that affects cell adhesion avidity. Several mechanisms are known to regulate integrin affinity and conformation: conformational changes induced by separation of the C-terminal tails, divalent ions, and reducing agents. Recently, we described a fluorescent LDV-containing small molecule that was used to monitor VLA-4 affinity changes in live cells (Chigaev, A., Blenc, A. M., Braaten, J. V., Kumaraswamy, N., Kepley, C. L., Andrews, R. P., Oliver, J. M., Edwards, B. S., Prossnitz, E. R., Larson, R. S., and Sklar, L. A. (2001) J. Biol. Chem. 276, 48670-48678). Using the same molecule, we also developed a fluorescence resonance energy transfer-based assay to probe the "switchblade-like" opening of VLA-4 upon activation. Here, we investigated the effect of reducing agents on the affinity and conformational state of the VLA-4 integrin simultaneously with cell activation initiated by inside-out signaling through G protein-coupled receptors or Mn(2+) in live cells in real time. We found that reducing agents (dithiothreitol and 2,3-dimercapto-1-propanesulfonic acid) induced multiple states of high affinity of VLA-4, where the affinity change was accompanied by an extension of the integrin molecule. Bacitracin, an inhibitor of the reductive function of the plasma membrane, diminished the effect of dithiothreitol, but had no effect on inside-out signaling. Based on this result and differences in the kinetics of integrin activation, we conclude that conformational activation of VLA-4 by inside-out signaling is independent of and additive to reduction-regulated integrin activation.

Vibrational imaging of lipid droplets in live fibroblast cells with coherent anti-Stokes Raman scattering microscopy

A new vibrational imaging method based on coherent anti-Stokes Raman scattering (CARS) has been used for high-speed, selective imaging of neutral lipid droplets (LDs) in unstained live fibroblast cells. LDs have a high density of C-H bonds and show a high contrast in laser-scanning CARS images taken at 2,845 cm-1, the frequency for aliphatic C-H vibrations. The contrast from LDs was confirmed by comparing CARS and Oil Red O (ORO)-stained fluorescence images. The fluorescent labeling processes were examined with CARS microscopy. It was found that ORO staining of fixed cells caused aggregation of LDs, whereas fixing with formaldehyde or staining with Nile Red did not affect LDs. CARS microscopy was also used to monitor the 3T3-L1 cell differentiation process, revealing that there was an obvious clearance of LDs at the early stage of differentiation. After that, the cells started to differentiate and reaccumulate LDs in the cytoplasm in a largely unsynchronized manner. Differentiated cells formed small colonies surrounded by undifferentiated cells that were devoid of LDs. These observations demonstrate that CARS microscopy can follow dynamic changes in live cells with chemical selectivity and noninvasiveness. CARS microscopy, in tandem with other techniques, provides exciting possibilities for studying LD dynamics under physiological conditions without perturbation of cell functions.

Use of a fusion protein between GFP and an actin-binding domain to visualize transient filamentous-actin structures

Many important processes in eukaryotic cells involve changes in the quantity, location and the organization of actin filaments [1–3]. We have been able to visualize these changes in live cells using a fusion protein (GFP–ABD) comprising the green fluorescent protein (GFP) of Aequorea victoria and the 25 kDa highly conserved actin-binding domain (ABD) from the amino terminus of the actin cross-linking protein ABP-120 [4]. In live cells of the soil amoeba Dictyostelium that were expressing GFP–ABD, the three-dimensional architecture of the actin cortex was clearly visualized. The pattern of GFP–ABD fluorescence in these cells coincided with that of rhodamine–phalloidin, indicating that GFP–ABD specifically binds filamentous (F) actin. On the ventral surface of non-polarized vegetative cells, a broad ring of F actin periodically assembled and contracted, whereas in polarized cells there were transient punctate F-actin structures; cells cycled between the polarized and non-polarized morphologies. During the formation of pseudopods, an increase in fluorescence intensity coincided with the initial outward deformation of the membrane. This is consistent with the models of pseudopod extension that predict an increase in the local density of actin filaments. In conclusion, GFP–ABD specifically binds F actin and allows the visualization of F-actin dynamics and cellular behavior simultaneously.

A genetically engineered, protein-based optical biosensor of myosin II regulatory light chain phosphorylation.

Myosin II is an important motor in the contraction of smooth and striated muscle as well as in a variety of non-muscle cell motile events including cytokinesis, cortical contractions during migration of fibroblasts, and capping of receptors. Phosphorylation of the 20-kDa light chain by myosin light chain kinase is part of the regulation of smooth muscle and mammalian nonmuscle myosin II. We designed, characterized, and tested the use of a protein-based optical biosensor to monitor this phosphorylation "switch." A regulatory light chain was genetically engineered to contain a single cysteine at amino acid position 18. The mutant light chain (Cys18.LC20), reacted with the fluorophore acrylodan, responded to phosphorylation of serine 19 with a fluorescence emission quenching of 60% and a 28-nm red-shift. When the acrylodan-labeled mutant light chain (AC-Cys18.LC20) was exchanged into turkey gizzard myosin II, it exhibited a 25% fluorescence emission quenching and a 10-nm red-shift upon phosphorylation of serine 19. The myosin II optical biosensor exhibited nearly control levels of the rate of phosphorylation, K+ATPase activity, and in vitro motility. The acrylodan-labeled light chain was exchanged into the A-bands of chicken pectoralis myofibrils in situ to demonstrate the localization and activity of the biosensor in a highly ordered contractile system. Fluorometry and quantitative fluorescence microscopic imaging experiments demonstrated that AC-Cys18.LC20 exchanged myofibrils expressed a phosphorylation-dependent fluorescence change. Labeled light chains were also incorporated into stress fibers of living fibroblasts and smooth muscle cells. This general approach of combining molecular biology and fluorescence spectroscopy to create novel protein-based optical biosensors should provide valuable tools for investigations with model systems and solution studies and ultimately yield important information about temporal-spatial chemical and molecular changes in live cells.

Measurements of DNA distribution changes in live cells

It has long been recognized by pathologists that many diseases can be diagnosed by examination of stained cell preparations. Particularly important information is found in the cell nucleus. For example, in the diagnosis of cancerous and pre-cancerous states in cervical cells, nuclear size, shape, and distribution of DNA have been used to classify the cells. Many other examples can be found in the examination and diagnosis of malignant diseases and other abnormalities.In experimental cell biology, one also finds numerous examples where critical information regarding cell status is found in the nuclear DNA. One of the most commonly used assays is that of determining DNA amount and/or degree of condensation in order to define cell cycle position. Effects of exposure to damaging agents such as ionizing radiation or toxic chemicals are also frequently assessed through examination of changes in the cell nucleus. Endpoints such as chromosome aberrations (breaks, translocations, etc.) and formation of micronuclei are commonly used in such assays.

Intracellular pH.

Intracellular pH.