Molecular beacons: fluorogenic probes for living cell study

Molecular beacons are a new class of fluorescent probes that can report the presence of specific nucleic acids with high sensitivity and excellent specificity. In addition to their current wide applications in monitoring the progress of polymerase chain reactions, their unique properties make them promising probes for the detection and visualization of target biomolecules in living cells. This article is focused on our recent research in exploring the potential of using molecular beacon for living-cell studies in three important areas: the monitoring of mRNA in living cells, the development of ultrasmall DNA/RNA biosensors, and the novel approach of combining molecular beacon's signal transduction mechanism with aptamer's specificity for real-time protein detection. These applications demonstrate molecular beacon's unique properties in bioanalysis and bioassay development.     

Imaging signal transduction in living cells with GFP-based probes

Since the cloning and the eterologous expression of the Green Fluorescence Protein (GFP), a number of applications have been reported where protein location within the cell or gene expression is revealed by fluorescent imaging of living cells. Modified GFPs, however, can now be exploited not only as a fluorescent reporter but also as a dynamic marker of intracellular signalling events, such as fluctuations in the levels of the second messengers Ca2+ and cAMP, or as a probe for detecting changes in pH in various cell compartments. These genetically manipulated GFPs allow monitoring of the biochemistry of the cell in real time and thus offer the possibility to gain a more precise view of the functioning of live cells.     

Imaging the live plant cell

Observing a biological event as it unfolds in the living cell provides unique insight into the nature of the phenomenon under study. Capturing live cell data differs from imaging fixed preparations because living plants respond to the intense light used in the imaging process. In addition, live plant cells are inherently thick specimens containing colored and fluorescent molecules often removed when the plant is fixed and sectioned. For fixed cells, the straightforward goal is to maximize contrast and resolution. For live cell imaging, maximizing contrast and resolution will probably damage the specimen or rapidly bleach the probe. Therefore, the goals are different. Live cell imaging seeks a balance between image quality and the information content that comes with increasing contrast and resolution. That "lousy" live cell image may contain all the information needed to answer the question being posed--provided the investigator properly framed the question and imaged the cells appropriately. Successful data collection from live cells requires developing a specimen-mounting protocol, careful selection and alignment of microscope components, and a clear understanding of how the microscope system generates contrast and resolution. This paper discusses general aspects of modern live cell imaging and the special considerations for imaging live plant specimens.     

Imaging calcium dynamics in living plant cells and tissues

The central role of Ca²⁺ signalling in plants is now well established. Much of our recent research has been based on the premise that the direct demonstration of signal-response coupling via Ca²⁺ requires the imaging or measurement of cytosolic free Ca²⁺ in living cells. Methods (confocal microscopy, fluorescence ratio imaging and photon counting imaging) which we use for imaging Ca²⁺ with fluorescent dyes or recombinant aequorin, are described. Approaches for using dyes are now routine for many plant cells. However, the imaging Ca²⁺ in whole tissues of plants genetically transformed with the aequorin gene is a very new development. We predict that this method, first employed in our laboratory, will bring about a revolution in our understanding of Ca²⁺ signalling at the multicellular level.     

Imaging phosphatidylinositol 4-phosphate dynamics in living plant cells

Polyphosphoinositides represent a minor group of phospholipids, accounting for less than 1% of the total. Despite their low abundance, these molecules have been implicated in various signalling and membrane trafficking events. Phosphatidylinositol 4-phosphate (PtdIns4 P ) is the most abundant polyphosphoinositide. ³²Pi-labelling studies have shown that the turnover of PtdIns4 P is rapid, but little is known about where in the cell or plant this occurs. Here, we describe the use of a lipid biosensor that monitors PtdIns4 P dynamics in living plant cells. The biosensor consists of a fusion between a fluorescent protein and a lipid-binding domain that specifically binds PtdIns4 P , i.e. the pleckstrin homology domain of the human protein phosphatidylinositol-4-phosphate adaptor protein-1 (FAPP1). YFP–PH_FAPP1 was expressed in four plant systems: transiently in cowpea protoplasts, and stably in tobacco BY-2 cells, Medicago truncatula roots and Arabidopsis thaliana seedlings. All systems allowed YFP–PH_FAPP1 expression without detrimental effects. Two distinct fluorescence patterns were observed: labelling of motile punctate structures and the plasma membrane. Co-expression studies with organelle markers revealed strong co-labelling with the Golgi marker STtmd–CFP, but not with the endocytic/pre-vacuolar marker GFP–AtRABF2b. Co-expression with the Ptdins3 P biosensor YFP–2 × FYVE revealed totally different localization patterns. During cell division, YFP–PH_FAPP1 showed strong labelling of the cell plate, but PtdIns3 P was completely absent from the newly formed cell membrane. In root hairs of M. truncatula and A. thaliana , a clear PtdIns4 P gradient was apparent in the plasma membrane, with the highest concentration in the tip. This only occurred in growing root hairs, indicating a role for PtdIns4 P in tip growth.     

Synthetic peptides as probes of plant cell signalling

Synthetic peptides have found increasing use in dissecting cell signalling pathways and have been employed as synthetic antigens, protein kinase and protease substrates. Recently, it has become evident that relatively short (10–30mer) peptides are able to mimic that part of the signalling protein to which their sequence corresponds. In particular, peptides corresponding to the C-terminus of Zea mays auxin binding protein, ZmABP1, were able to modulate ion channel function within Vicia guard cells. In this report, GTPS binding to NaCl-washed Zea microsomal membranes is shown to be stimulated by peptide A6.2, corresponding to the C-terminal 16 residues of ZmABP1, only when the membranes are reconstituted with soluble Zea protein fractions containing GP1 and G₀ homologues.     

Development and application of probes for labeling the actin cytoskeleton in living plant cells

The actin cytoskeleton is one of the most important components of eukaryotic cytoskeletons. It participates in numerous crucial procedures of cells and has been studied by using various methods. The development and application of appropriate probes for actin visualization is the first and foremost step for functional analysis of actin in vivo. Since the actin cytoskeleton is a highly dynamic and sensitive structure, methods previously used to visualize actin often harm cells and cannot reveal the native state of the actin cytoskeleton in living cells. The development of labeling technologies for living plant cells, especially the emergence and application of green fluorescent protein-tagged actin markers, has provided new insights into the structure and function of the actin cytoskeleton in vivo. There has been a number of probes for actin labeling in living plant cells though they each present different advantages and defects. In this review, we discuss and compare those widely used methods for actin visualization and analysis.     

Review: Fluorescent probes for living cells

The functional characteristics of fluorescent probes used for imaging and measuring dynamic processes in living cells are reviewed. Initial consideration is given to general design requirements for delivery, targeting, detectability and fluorescence readout, and current technologies for attaining them. Discussion then proceeds to the more application-specific properties of intracellurion indicators, membrane potential sensors, probes for proteins and lipids, and cell viability markers. 1998 © Chapman & Hall     

Nanoscale fluorescence correlation spectroscopy on intact living cell membranes with NSOM probes

Characterization of molecular dynamics on living cell membranes at the nanoscale is fundamental to unravel the mechanisms of membrane organization and compartmentalization. Here we demonstrate the feasibility of fluorescence correlation spectroscopy (FCS) based on the nanometric illumination of near-field scanning optical microscopy (NSOM) probes on intact living cells. NSOM-FCS applied to fluorescent lipid analogs allowed us to reveal details of the diffusion hidden by larger illumination areas. Moreover, the technique offers the unique advantages of evanescent axial illumination and straightforward implementation of multiple color excitation. As such, NSOM-FCS represents a powerful tool to study a variety of dynamic processes occurring at the nanometer scale on cell membranes.     

From pathogen genomes to host plant processes: the power of plant parasitic oomycetes

Recent pathogenomic research on plant parasitic oomycete effector function and plant host responses has resulted in major conceptual advances in plant pathology, which has been possible thanks to the availability of genome sequences.