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.     

Cytoskeletal dynamics in living plant cells

Microtubules and microfilaments have been imaged in living plant cells and their dynamic changes recorded during division, growth and development. Carboxyfluorescein labeled brain tubulin has been injected into cells that are maintained in an active state in a culture chamber on the microscope stage. Subsequent imaging with the confocal microscope reveals microtubules in the preprophase band, the mitotic apparatus, the phragmoplast, and the cortical array. The structural changes of these microtubules have been observed during transitional stages. In addition, their dynamic features are demonstrated by depolymerization in elevated calcium, low temperature, and in the drug oryzalin, and by repolymerization when returned to normal conditions. Examination of living Tradescantia stamen hair cells, which have been injected with fluorescent phalloidin to label the actin microfilaments, reveals hitherto undisclosed aspects of the preparation of the division site and dynamics of the phragmoplast cytoskeleton. During prophase microfilaments occur throughout the cell cortex, with those in the region of the preprophase band becoming transversely aligned. At nuclear envelope breakdown, these specifically disassemble, leaving a circumferential zone from which microfilaments remain absent throughout division. During cytokinesis microfilaments arise within the phragmoplast, oriented parallel to the microtubules, but excluded from the zone where the MTs overlap and where cell plate vesicles aggregate. The phragmoplast microfilaments, in a manner similar to microtubules, shorten in length, expand in girth, and eventually disassemble when the cell plate is complete.     

Imaging calcium dynamics in living plants using semi-synthetic recombinant aequorins

The genetic transformation of the higher plant Nicotiana plumbaginifolia to express the protein apoaequorin has recently been used as a method to measure cytosolic free calcium ([Ca2+]i) changes within intact living plants (Knight, M. R., A. K. Campbell, S. M. Smith, and A. J. Trewavas. 1991. Nature (Lond.). 352:524-526; Knight, M. R., S. M. Smith, and A. J. Trewavas. 1992. Proc. Natl. Acad. Sci. USA. 89:4967-4971). After treatment with the luminophore coelenterazine the calcium-activated photoprotein aequorin is formed within the cytosol of the cells of the transformed plants. Aequorin emits blue light in a dose-dependent manner upon binding free calcium (Ca2+). Thus the quantification of light emission from coelenterazine-treated transgenic plant cells provides a direct measurement of [Ca2+]i. In this paper, by using a highly sensitive photon-counting camera connected to a light microscope, we have for the first time imaged changes in [Ca2+]i in response to cold-shock, touch and wounding in different tissues of transgenic Nicotiana plants. Using this approach we have been able to observe tissue-specific [Ca2+]i responses. We also demonstrate how this method can be tailored by the use of different coelenterazine analogues which endow the resultant aequorin (termed semi-synthetic recombinant aeqorin) with different properties. By using h-coelenterazine, which renders the recombinant aequorin reporter more sensitive to Ca2+, we have been able to image relatively small changes in [Ca2+]i in response to touch and wounding: changes not detectable when standard coelenterazine is used. Reconstitution of recombinant aequorin with another coelenterazine analogue (e-coelenterazine) produces a semi-synthetic recombinant aequorin with a bimodal spectrum of luminescence emission. The ratio of luminescence at two wavelengths (421 and 477 nm) provides a simpler method for quantification of [Ca2+]i in vivo than was previously available. This approach has the benefit that no information is needed on the amount of expression, reconstitution or consumption of aequorin which is normally required for calibration with aequorin.     

Imaging living cells and tissues by two-photon excitation microscopy

Two-photon excitation microscopy provides attractive advantages over confocal microscopy for three-dimensionally resolved fluorescence imaging. Since two-photon excitation occurs only at the focal point of the microscope, it inherently provides three-dimensional resolution. This localization of excitation also minimizes photobleaching and photodamage, which are the ultimate limiting factors in imaging living cells. Furthermore, no pinhole is required to attain three-dimensional discrimination, so the efficiency of fluorescence collection is increased. These advantages allow experiments on thick living samples that would not be possible with other imaging techniques. The cost and complexity of the lasers required for two-photon excitation microscopy have limited its use, but appropriate turn-key lasers have now been introduced, and their cost should decrease. Finally, the recent introduction of commercial two-photon excitation laser-scanning microscope systems allows a much larger group of researchers access to this state-of-the-art methodology.     

Measuring calcium dynamics in living cells with genetically encodable calcium indicators

Genetically encoded calcium indicators (GECIs) allow researchers to measure calcium dynamics in specific targeted locations within living cells. Such indicators enable dissection of the spatial and temporal control of calcium signaling processes. Here we review recent progress in the development of GECIs, highlighting which indicators are most appropriate for measuring calcium in specific organelles and localized domains in mammalian tissue culture cells. An overview of recent approaches that have been undertaken to ensure that the GECIs are minimally perturbed by the cellular environment is provided. Additionally, the procedures for introducing GECIs into mammalian cells, conducting calcium imaging experiments, and analyzing data are discussed. Because organelle-targeted indicators often pose an additional challenge, we underscore strategies for calibrating GECIs in these locations.     

Confocal imaging of ionised calcium in living plant cells

Laser-scanning confocal microscopy has been used in conjunction with Fluo-3, a highly fluorescent visible wavelength probe for Ca2+, to visualize Ca2(+)-dynamics in the function of living plant cells. This combination has overcome many of the problems that have limited the use of fluorescence imaging techniques in the study of the role of cations (Ca2+ and H+) in plant cell physiology and enables these processes to be studied in single cells within intact plant tissue preparations. Maize coleoptiles respond to application of ionophores and plant growth hormones with elevations in cytosolic Ca2+ that can be resolved with a high degree of spatial resolution and can be interpreted quantitatively.     

Imaging dynamics of endogenous mitochondrial RNA in single living cells

We developed genetically encoded RNA probes for characterizing localization and dynamics of mitochondrial RNA (mtRNA) in single living cells. The probes consist of two RNA-binding domains of PUMILIO1, each connected with split fragments of a fluorescent protein capable of reconstituting upon binding to a target RNA. We designed the probes to specifically recognize a 16-base sequence of mtRNA encoding NADH dehydrogenase subunit 6 (ND6) and to be targeted into the mitochondrial matrix, which allowed real-time imaging of ND6 mtRNA localization in living cells. We showed that ND6 mtRNA is localized within mitochondria and concentrated particularly on mitochondrial DNA (mtDNA). Movement of the ND6 mtRNA is restricted but oxidative stress induces the mtRNA to disperse in the mitochondria and gradually decompose. These probes provide a means to study spatial and temporal mRNA dynamics in intracellular compartments in living mammalian cells.     

Visualization of PtdIns3P dynamics in living plant cells

To investigate PtdIns3P localization and function in plants, a fluorescent PtdIns3P-specific biosensor (YFP-2xFYVE) was created. On lipid dot blots it bound specifically and with high affinity to PtdIns3P. Transient expression in cowpea protoplasts labelled vacuolar membranes and highly motile structures undergoing fusion and fission. Stable expression in tobacco BY-2 cells labelled similar motile structures, but labelled vacuolar membranes hardly at all. YFP-2xFYVE fluorescence strongly co-localized with the pre-vacuolar marker AtRABF2b, partially co-localized with the endosomal tracer FM4-64, but showed no overlap with the Golgi marker STtmd-CFP. Treatment of cells with wortmannin, a PI3 kinase inhibitor, caused the YFP-2xFYVE fluorescence to redistribute into the cytosol and nucleus within 15 min. BY-2 cells expressing YFP-2xFYVE contained twice as much PtdIns3P as YFP-transformed cells, but this had no effect on cell-growth or stress-induced phospholipid signalling responses. Upon treatment with wortmannin, PtdIns3P levels were reduced by approximately 40% within 15 min in both cell lines. Stable expression of YFP-2xFYVE in Arabidopsis plants labelled different subcellular structures in root compared with shoot tissues. In addition labelling the motile structures common to all cells, YFP-2xFYVE strongly labelled the vacuolar membrane in leaf epidermal and guard cells, suggesting that cell differentiation alters the distribution of PtdIns3P. In dividing BY-2 cells, YFP-2xFYVE-labelled vesicles surrounded the newly formed cell plate, suggesting a role for PtdIns3P in cytokinesis. Together, these data show that YFP-2xFYVE may be used as a biosensor to specifically visualize PtdIns3P in living plant cells.     

Cytosolic inositol 1,4,5-trisphosphate dynamics during intracellular calcium oscillations in living cells

We developed genetically encoded fluorescent inositol 1,4,5-trisphosphate (IP3) sensors that do not severely interfere with intracellular Ca2+ dynamics and used them to monitor the spatiotemporal dynamics of both cytosolic IP3 and Ca2+ in single HeLa cells after stimulation of exogenously expressed metabotropic glutamate receptor 5a or endogenous histamine receptors. IP3 started to increase at a relatively constant rate before the pacemaker Ca2+ rise, and the subsequent abrupt Ca2+ rise was not accompanied by any acceleration in the rate of increase in IP3. Cytosolic [IP3] did not return to its basal level during the intervals between Ca2+ spikes, and IP3 gradually accumulated in the cytosol with a little or no fluctuations during cytosolic Ca2+ oscillations. These results indicate that the Ca2+ -induced regenerative IP3 production is not a driving force of the upstroke of Ca2+ spikes and that the apparent IP3 sensitivity for Ca2+ spike generation progressively decreases during Ca2+ oscillations.     

Protein dynamics in living cells

A protein's structure is most often used to explain its function, but function also depends on dynamics. To date, protein dynamics have been studied only in vitro under dilute solution conditions where solute concentrations are typically less than 10 g/L, yet proteins function in a crowded environment where the solute concentration can exceed 400 g/L. Does the intracellular environment affect protein dynamics? The answer will help in assessing the biological significance of the NMR-derived dynamics data collected to date. We investigated fast protein dynamics inside living Escherichia coli by using in-cell NMR. The backbone dynamics of apocytochrome b5 were quantified using {1H}-15N nuclear Overhauser effect (nOe) measurements, which characterize motions on the pico- to nanosecond time scale. The overall trend of backbone dynamics remains the same in cells. Some of the nOe values differ, but most of the differences track the increased intracellular viscosity rather than a change in dynamics. Therefore, it appears that dilute solution steady-state {1H}-15N nOe measurements provide biologically relevant information about pico- to nanosecond backbone motion in proteins.     

Imaging molecular interactions in living cells

Hormones integrate the activities of their target cells through receptor-modulated cascades of protein interactions that ultimately lead to changes in cellular function. Understanding how the cell assembles these signaling protein complexes is critically important to unraveling disease processes, and to the design of therapeutic strategies. Recent advances in live-cell imaging technologies, combined with the use of genetically encoded fluorescent proteins, now allow the assembly of these signaling protein complexes to be tracked within the organized microenvironment of the living cell. Here, we review some of the recent developments in the application of imaging techniques to measure the dynamic behavior, colocalization, and spatial relationships between proteins in living cells. Where possible, we discuss the application of these different approaches in the context of hormone regulation of nuclear receptor localization, mobility, and interactions in different subcellular compartments. We discuss measurements that define the spatial relationships and dynamics between proteins in living cells including fluorescence colocalization, fluorescence recovery after photobleaching, fluorescence correlation spectroscopy, fluorescence resonance energy transfer microscopy, and fluorescence lifetime imaging microscopy. These live-cell imaging tools provide an important complement to biochemical and structural biology studies, extending the analysis of protein-protein interactions, protein conformational changes, and the behavior of signaling molecules to their natural environment within the intact cell.     

Imaging protein-protein interactions in living cells

The complex organization of plant cells makes it likely that the molecular behaviour of proteins in the test tube and the cell is different. For this reason, it is essential though a challenge to study proteins in their natural environment. Several innovative microspectroscopic approaches provide such possibilities, combining the high spatial resolution of microscopy with spectroscopic techniques to obtain information about the dynamical behaviour of molecules. Methods to visualize interaction can be based on FRET (fluorescence detected resonance energy transfer), for example in fluorescence lifetime imaging microscopy (FLIM). Another method is based on fluorescence correlation spectroscopy (FCS) by which the diffusion rate of single molecules can be determined, giving insight into whether a protein is part of a larger complex or not. Here, both FRET- and FCS-based approaches to study protein-protein interactions in vivo are reviewed.     

Enrichment for microbes living in association with plant tissues

AIMS: To investigate a cultivation-independent method of enrichment for microbes living in association with plant tissues. METHODS AND RESULTS: A large quantity of leaves or seeds was enzymatically hydrolyzed, and the pellets were collected by differential centrifugation. Enzyme concentration, buffer and incubation time were optimized for release of plant-associated microbes. The relative abundance of plant nuclear DNA and bacterial DNA in the enriched sample was estimated by PCR amplification of genome-specific marker genes. The efficiency of microbe enrichment was estimated from the proportion of bacterium-derived clones and their restriction fragment length polymorphism (RFLP) types as detected by 16S rRNA gene-based techniques. With a higher ratio of bacterial to plant nuclear DNA, the enriched samples showed a considerably enhanced proportion of bacterium-derived clones and a wider sequence diversity of those clones. CONCLUSIONS: The method described here proved to be remarkably effective in enriching for bacteria living in association with plant tissues. SIGNIFICANCE AND IMPACT OF THE STUDY: The method can be applied to study plant-associated microbes in the field of environmental molecular ecology and environmental metagenomics.     

Studying protein dynamics in living cells

Since the advent of the green fluorescent protein, the subcellular localization, mobility, transport routes and binding interactions of proteins can be studied in living cells. Live cell imaging, in combination with photobleaching, energy transfer or fluorescence correlation spectroscopy are providing unprecedented insights into the movement of proteins and their interactions with cellular components. Remarkably, these powerful techniques are accessible to non-specialists using commercially available microscope systems.