Peptide-based targeting of fluorophores to organelles in living cells

Peptides carrying organelle-specific import or retention sequences can target the fluorophore BODIPY(581/591) to the nucleus, peroxisomes, endoplasmic reticulum (ER), or the trans-Golgi network (TGN). The peroxisomal peptide contains the PTS1 sequence AKL. For targeting to the ER or TGN, the peptides carry the retention sequences KDEL and SDYQRL, respectively. A peptide carrying the nuclear leader sequence of the simian virus SV40 large tumor antigen, KKKRK, was used to direct the fluorophore to the nucleus. The fluorescent peptides for peroxisomes, ER, and the TGN spontaneously incorporate into living fibroblasts at 37 degrees C and accumulate in their target organelles within minutes. The uptake is still significant at 4 degrees C, indicating that endocytosis is not required for internalization. The highly charged nuclear peptide (net charge +4) does not spontaneously internalize. However, by transient permeabilization of the plasma membrane, this fluorescent peptide was found to rapidly accumulate in the nucleus. These fluorescent peptides open new opportunities to follow various aspects of specific organelles such as their morphology, biogenesis, dynamics, degradation, and their internal parameters (pH, redox).     

Experimental approaches for addressing fundamental biological questions in living, functioning cells with single molecule precision

In recent years, single molecule experimentation has allowed researchers to observe biological processes at the sensitivity level of single molecules in actual functioning, living cells, thereby allowing us to observe the molecular basis of the key mechanistic processes in question in a very direct way, rather than inferring these from ensemble average data gained from traditional molecular and biochemical techniques. In this short review, we demonstrate the impact that the application of single molecule bioscience experimentation has had on our understanding of various cellular systems and processes, and the potential that this approach has for the future to really address very challenging and fundamental questions in the life sciences.     

Visualization of RNA-protein interactions in living cells: FMRP and IMP1 interact on mRNAs

Protein expression depends significantly on the stability, translation efficiency and localization of mRNA. These qualities are largely dictated by the RNA-binding proteins associated with an mRNA. Here, we report a method to visualize and localize RNA-protein interactions in living mammalian cells. Using this method, we found that the fragile X mental retardation protein (FMRP) isoform 18 and the human zipcode-binding protein 1 ortholog IMP1, an RNA transport factor, were present on common mRNAs. These interactions occurred predominantly in the cytoplasm, in granular structures. In addition, FMRP and IMP1 interacted independently of RNA. Tethering of FMRP to an mRNA caused IMP1 to be recruited to the same mRNA and resulted in granule formation. The intimate association of FMRP and IMP1 suggests a link between mRNA transport and translational repression in mammalian cells.     

光学显微镜技术在活细胞单分子检测中的应用

单分子检测是一门以高度的时间以及空间分辨率研究生物单分子的技术。近来,科学技术的探索发展使我们可以观察、检测甚至操纵单个分子并且研究它们的构象变化和动力学行为。这一发展使得以前被传统系综研究体系平均化所隐藏的新信息被揭示出来。单分子检测技术的发展已经揭开了生命科学研究的新篇章。在本文中,我们将介绍有关活细胞中单分子检测技术的发展以及活细胞内单分子检测的现状。     

A transient decrease of electrochemical gradient stabilizes DNA structural change in single mitochondria of living cells

The effect of controlled and reversible perturbation of the electrochemical gradient on the structural changes of mitochondrial DNA has been studied in living cells by fluorescence microscopy. Electrochemical gradient perturbations were induced by the protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone and quantified by measuring the mitochondrial membrane potential using tetramethyl rhodamine methyl ester. Under our experimental conditions, we have shown that ethidium fluorescence was mainly due to ethidium molecules intercalated in mtDNA. Ethidium fluorescence variations have been used to probe DNA structural changes. This showed that: i) electrochemical gradient perturbations induced mtDNA structural change; ii) this change was readily reversible following a total but short collapse of the electrochemical gradient; iii) in contrast, a short and weak perturbation of the electrochemical gradient stabilized the mtDNA structural change; and iv) the degree of weak depolarization varied from cell to cell, showing the necessity of studying the effect of energetic perturbations at the level of an individual cell.     

Metabolic control and compartmentation in single living cells

Microspectrofluorometry of cell coenzymes (NAD(P)H, flavins) in conjunction with sequential microinjections into the same cell of metabolites and modifiers, reveals aspects of the regulatory mechanisms of transient redox changes of mitochondrial and extramitochondrial nicotinamide adenine dinucleotides. The injection of ADP in the course of an NAD(P)H transient produced by glycolytic (e.g. glucose 6-phosphate, G6P) or mitochondrial (e.g. malate) substrate leads to sharp reoxidation (state III, Chance and Williams, 1955), followed by a spontaneous state III to IV transition, and an ultimate return to original redox steady state. The response to ADP alone is biphasic, i.e. a small oxidation-reduction transient followed by a larger reverse transient. Similarities between responses to injected ATP and ADP suggest possible intracellular interconversions. Sequential injections of glycolytic and Krebs cycle substrates into the same cell, produce a two-step NAD(P) response, possibly revealing the intracellular compartmentation of this coenzyme. A two-step NAD(P)H response to sequentially injected fructose 1,6-diphosphate and G6P indicates the dynamic or even structural compartmentation of glycolytic phosphate esters in separate intracellular pools. The intracellular regulation and compartmentation of bioenergetic pathways and cell-to-cell metabolic inhomogeneities provide the basis on which the quantitative biochemistry of the intact living cell may be reconciled with these in situ findings.     

Visualizing single fluorophores in live cells

The methods have been described that can be used to visualize single fluorescent molecules in live cells: laser epifluorescent, confocal, near-field, two-photon, and total internal reflection microscopy. Each method has its own advantages and limitations. We showed that total internal reflection microscopy is a method of choice for single fluorophore visualisation near substrate-medium interface. It can be used to study receptors, ion channels, and many cytoskeleton or signalling molecules located at or in close proximity to basal cell membrane. It was shown that it is very important to use rigorous criteria for single fluorophore identification since these objects emit a limited number of photons before irreversible photo-bleaching, and their fluorescence is often obscured by cell auto-fluorescence and out-of-focus fluorescence. Methods used for lateral mobility studies of single molecules floating on cell membrane were also described.     

Visualization of mitochondria and nuclei in living plant cells by the use of a potential-sensitive fluorescent dye

Abstract Cationic potential-sensitive dyes have previously been used to selectively stain mitochondria in living animal cells (Johnson, Walsh & Chen, 1980; Johnson et al., 1981). The present work demonstrates that the cyanine dye 3,3′-dihexyloxacarbocyanine iodide (DiOC₆(3)) can also be used as a mitochondrial stain in living plant cells. The stained mitochondria were easily visualized by fluorescence microscopy. The accumulation of DiOC₆(3) in mitochondria seemed to be potential-dependent since it was prevented by protonophores, valinomycin and inhibitors of electron transport. It was often observed that DiOC₆(3) also stained the nuclear membrane of some cells. This fluorescence, limited to the perinuclear region, was possibly due to a potential across one or both nuclear membranes, although it was not completely dissipated by any of the ionophores or inhibitors tested. Our observations demonstrate the usefulness of using DiOC₆(3) for studying relative membrane potentials of plant mitochondria and, perhaps, other organelles and membrane systems in living plant cells.     

Switchable fluorophores for protein labeling in living cells

Numerous synthetic fluorophores have been developed that can switch their spectroscopic properties upon interaction with other molecules or by irradiation with light. In recent years, protein-labeling techniques have been introduced that permit the specific attachment of such molecules to proteins of interest in living cells. We review here how the attachment of switchable fluorophores to selected proteins of interest via self-labeling protein tags enables new applications in different areas of biology and discuss how these molecules could be further improved.     

Inactivation of acetylcholinesterase by various fluorophores

The inhibition of recombinant mouse acetylcholinesterase (rMAChE) and electric eel acetylcholinesterase (EEAChE) by seven, structurally different chromophore-based (dansyl, pyrene, dabsyl, diethylamino- and methoxycoumarin, Lissamine rhodamine B, and Texas Red) propargyl carboxamides or sulfonamides was studied. Diethylaminocoumarin, Lissamine, and Texas Red amides inhibited rMAChE with IC₅₀ values of 1.00 μM, 0.05 μM, and 0.70 μM, respectively. Lissamine and Texas Red amides inhibited EEAChE with IC₅₀ values of 3.57 and 10.4 μM, respectively. The other chromophore amides did not inhibit either AChE. The surprising inhibitory potency of Lissamine was examined in further detail against EEAChE and revealed a mixed-type inhibition with K_i?=?11.7 μM (competitive) and K_i′?=?24.9 μM (noncompetitive), suggesting that Lissamine binds to free enzyme and enzyme–substrate complex.     

Fluorescence microscopy of the endomembrane system of living plant cells

Abstract The fluorochrome Auramine O has been evaluated as a fluorescent probe for components of the endomembrane system of living plant cells. At 0.001% w/v the compound did not inhibit seedling growth or cytoplasmic streaming but stained the nuclear envelope, endoplasmic reticulum and Golgi apparatus. The three-dimensional, structural interrelationships of these organelles in living tissues could be resolved after minimal tissue preparation. The method is also a valuable control treatment for use in conjunction with electron microscope fixation procedures. It provides a rapid means of examining dynamic changes in the endomembrane system associated with cell development and differentiation and could have application in monitoring the effects of applied physiological or chemical stress.     

Automatic detection of single fluorophores in live cells

Recent developments in light microscopy enable individual fluorophores to be observed in aqueous conditions. Biological molecules, labeled with a single fluorophore, can be localized as isolated spots of light when viewed by optical microscopy. Total internal reflection fluorescence microscopy greatly reduces background fluorescence and allows single fluorophores to be observed inside living cells. This advance in live-cell imaging means that the spatial and temporal dynamics of individual molecules can be measured directly. Because of the stochastic nature of single molecule behavior a statistically meaningful number of individual molecules must be detected and their separate trajectories in space and time stored and analyzed. Here, we describe digital image processing methods that we have devised for automatic detection and tracking of hundreds of molecules, observed simultaneously, in vitro and within living cells. Using this technique we have measured the diffusive behavior of pleckstrin homology domains bound to phosphoinositide phospholipids at the plasma membrane of live cultured mammalian cells. We found that mobility of these membrane-bound protein domains is dominated by mobility of the lipid molecule to which they are attached and is highly temperature dependent. Movement of PH domains isolated from the tail region of myosin-10 is consistent with a simple random walk, whereas, diffusion of intact PLC-delta1 shows behavior inconsistent with a simple random walk. Movement is rapid over short timescales but much slower at longer timescales. This anomalous behavior can be explained by movement being restricted to membrane regions of 0.7 microm diameter.     

Visualization of myosin in living cells

Myosin light chains labeled with rhodamine are incorporated into myosin-containing structures when microinjected into live muscle and nonmuscle cells. A mixture of myosin light chains was prepared from chicken skeletal muscle, labeled with the fluorescent dye iodoacetamido rhodamine, and separated into individual labeled light chains, LC-1, LC-2, and LC-3. In isolated rabbit and insect myofibrils, the fluorescent light chains bound in a doublet pattern in the A bands with no binding in the cross-bridge-free region in the center of the A bands. When injected into living embryonic chick myotubes and cardiac myocytes, the fluorescent light chains were also incorporated along the complete length of the A band with the exception of the pseudo-H zone. In young myotubes (3-4 d old), myosin was localized in aperiodic as well as periodic fibers. The doublet A band pattern first appeared in 5-d-old myotubes, which also exhibited the first signs of contractility. In 6-d and older myotubes, A bands became increasingly more aligned, their edges sharper, and the separation between them (I bands) wider. In nonmuscle cells, the microinjected fluorescent light chains were incorporated in a striated pattern in stress fibers and were absent from foci and attachment plaques. When the stress fibers of live injected cells were disrupted with DMSO, fluorescently labeled myosin light chains were present in the cytoplasm but did not enter the nucleus. Removal of the DMSO led to the reformation of banded, fluorescent stress fibers within 45 min. In dividing cells, myosin light chains were concentrated in the cleavage furrow and became reincorporated in stress fibers after cytokinesis. Thus, injected nonmuscle cells can disassemble and reassemble contractile fibers using hybrid myosin molecules that contain muscle light chains and nonmuscle heavy chains. Our experiments demonstrate that fluorescently labeled myosin light chains from muscle can be readily incorporated into muscle and nonmuscle myosins and then used to follow the dynamics of myosin distribution in living cells.     

Multicolor fluorescence nanoscopy in fixed and living cells by exciting conventional fluorophores with a single wavelength

Current far-field fluorescence nanoscopes provide subdiffraction resolution by exploiting a mechanism of fluorescence inhibition. This mechanism is implemented such that features closer than the diffraction limit emit separately when simultaneously exposed to excitation light. A basic mechanism for such transient fluorescence inhibition is the depletion of the fluorophore ground state by transferring it (via a triplet) in a dark state, a mechanism which is workable in most standard dyes. Here we show that microscopy based on ground state depletion followed by individual molecule return (GSDIM) can effectively provide multicolor diffraction-unlimited resolution imaging of immunolabeled fixed and SNAP-tag labeled living cells. Implemented with standard labeling techniques, GSDIM is demonstrated to separate up to four different conventional fluorophores using just two detection channels and a single laser line. The method can be expanded to even more colors by choosing optimized dichroic mirrors and selecting marker molecules with negligible inhomogeneous emission broadening.     

Visualization of mRNA translation in living cells

The role of mRNA localization is presumably to effect cell asymmetry by synthesizing proteins in specific cellular compartments. However, protein synthesis has never been directly demonstrated at the sites of mRNA localization. To address this, we developed a live cell method for imaging translation of beta-actin mRNA. Constructs coding for beta-actin, containing tetracysteine motifs, were transfected into C2C12 cells, and sites of nascent polypeptide chains were detected using the biarsenial dyes FlAsH and ReAsH, a technique we call translation site imaging. These sites colocalized with beta-actin mRNA at the leading edge of motile myoblasts, confirming that they were translating. beta-Actin mRNA lacking the sequence (zipcode) that localizes the mRNA to the cell periphery, eliminated the translation there. A pulse-chase experiment on living cells showed that the recently synthesized protein correlated spatially with the sites of its translation. Additionally, localization of beta-actin mRNA and translation activity was enhanced at cell contacts and facilitated the formation of intercellular junctions.     

Long-range tertiary interactions in single hammerhead ribozymes bias motional sampling toward catalytically active conformations

Enzymes generally are thought to derive their functional activity from conformational motions. The limited chemical variation in RNA suggests that such structural dynamics may play a particularly important role in RNA function. Minimal hammerhead ribozymes are known to cleave efficiently only in ～ 10-fold higher than physiologic concentrations of Mg(2+) ions. Extended versions containing native loop-loop interactions, however, show greatly enhanced catalytic activity at physiologically relevant Mg(2+) concentrations, for reasons that are still ill-understood. Here, we use Mg(2+) titrations, activity assays, ensemble, and single molecule fluorescence resonance energy transfer (FRET) approaches, combined with molecular dynamics (MD) simulations, to ask what influence the spatially distant tertiary loop-loop interactions of an extended hammerhead ribozyme have on its structural dynamics. By comparing hammerhead variants with wild-type, partially disrupted, and fully disrupted loop-loop interaction sequences we find that the tertiary interactions lead to a dynamic motional sampling that increasingly populates catalytically active conformations. At the global level the wild-type tertiary interactions lead to more frequent, if transient, encounters of the loop-carrying stems, whereas at the local level they lead to an enrichment in favorable in-line attack angles at the cleavage site. These results invoke a linkage between RNA structural dynamics and function and suggest that loop-loop interactions in extended hammerhead ribozymes-and Mg(2+) ions that bind to minimal ribozymes-may generally allow more frequent access to a catalytically relevant conformation(s), rather than simply locking the ribozyme into a single active state.     

Probing structural stability of chromatin assembly sorted from living cells

Post-translational modifications of the histone tails and other chromatin binding proteins affect the stability of chromatin structure. In this study, we have purified chromatin from live cell nuclei using a fluorescence activated cell sorter (FACS) and studied the structural stability of this self-assembled structure. Using total internal reflection fluorescence (TIRF) microscopy, we map the effect of covalent modifications on the interaction of histone-DNA complex, by measuring the dissociation rates of histones from the chromatin fiber in the presence of different salt concentrations. Dynamic force spectroscopy (DFS) experiments were carried out to measure the structural disintegration of large chromatin globules under force. The characteristic rupture of multiple linkages in the large chromatin globules show differences in the stiffness of the higher order structure of chromatin with altered epigenetic states. Our studies reveal a direct correlation between histone modifications and the structural stability of higher order chromatin assembly.     

Visualization of gap junction mobility in living cells

In order to study the dynamics of gap junctions in living cells, a cDNA was expressed in hepatocellular carcinoma-derived PLC cells coding for chimerical polypeptide Cx.EGFP-1, which consists of rat connexin32 and enhanced green fluorescent protein (EGFP). Cx.EGFP-1 was integrated into gap junctions, and the emitted epifluorescence reliably reported the distribution of the chimera. Therefore, stably transfected PLC clone PCx-9 was used to examine the dynamic behavior of gap junctions by time-lapse fluorescence microscopy. The pleomorphic fluorescent junctional plaques were highly motile within the plasma membrane. They often fused with each other or segregated into smaller patches, and fluctuation of fluorescence was detected within individual gap junctions. Furthermore, the uptake of junctional fragments into the cytoplasm of live cells was documented as originating from dynamic invaginations that form long tubulovesicular structures that pinch off. Endocytosis and subsequent lysosomal degradation, however, appeared to contribute only a little to the rapid gap junction turnover (determined half-life of 3.3 h for Cx.EGFP-1), since most cytoplasmic Cx.EGFP-1 fluorescence did not colocalize with the endocytosed fluid phase marker horseradish peroxidase or the receptor-specific endocytotic ligand transferrin and since it was distinct from lysosomes. Disassembly of gap junctions was monitored in the presence of the translation-inhibitor cycloheximide and showed increased endocytosis and continuous reduction of junctional plaques. Highly motile cytoplasmic microvesicles, which were detectable as multiple, weakly fluorescent puncta in all movies, are proposed to contribute significantly to gap junction morphogenesis by the transport of small subunits between biosynthetic, degradative, and recycling compartments.     

Visualization of microtubules in living cells of transgenicArabidopsis thaliana

Summary Microtubules (MTs) were visualized in living cells of several tissues in transgenicArabidopsis thaliana. The transformed Arabidopsis plant was obtained by infecting it withAgrobacterium tumefaciens carrying the GFP-TUA6 plasmid. The fluorescence of the MTs was due to the fluorescence of GFP-TUA6 that was polymerized into the MTs. The distribution patterns of the visualized MTs in the living epidermal cells of leaves was similar to that in fixed epidermal cells. The actual destruction of MTs by oryzalin was observed in a living cell. Cytochalasin B exerts no effect on the distribution pattern of MTs. The fluorescence intensity of MTs was different among cells in different tissues.