GFP technology for live cell imaging
Introduction
Fluorescent proteins (FPs) have become key tools for the investigation of plant cell function and development. As genomics projects generate the ‘parts list’ of plant genes and proteins, there is an increasing need to understand where gene products reside within the cell, what other molecules they interact with, how the molecular assemblies are constructed, and how individual molecules and assemblies behave in response to developmental and environmental cues. Fluorescent probes are instrumental in addressing these questions because they permit the specific labeling of biological molecules and cellular structures, and can be sensitively detected by a variety of optical techniques. The relatively non-invasive nature of fluorescent detection permits the visualization of these labels in living tissues, thus allowing transient and dynamic events to be visualized and measured.
Historically, fluorescent probes with molecular specificity were difficult to create and apply. Plant cells in particular provided special challenges for these tools. To create a fluorescently labeled protein it was necessary to purify the protein in an active form, chemically conjugate the fluorophore, and then re-introduce the protein to a single cell by micro-injection. The thick cell wall and high turgor of plant cells have made micro-injection notoriously challenging, time consuming and unreliable, especially the microinjection of large molecules and proteins. Even fluorescent probes that do not require injection are sometimes problematic in plant tissue: they might stain the cell wall in addition to the target compartment, fail to load into the cytosol, or be sequestered into the wrong subcellular compartment.
The discovery of the green fluorescent protein (GFP) from the jellyfish Aequorea, and its many natural and mutated spectral variants, has dramatically changed this picture. GFP is a relatively bright and particularly stable fluorophore [1], with the outstanding property that it is genetically encoded. The GFP fluorophore can therefore be manipulated with the commonly accessible tools of molecular biology to create genetic fusions to any variety of protein sequences. These fusions can be expressed by one of the available arsenal of regulatory sequences. This flexibility allows fluorescently tagged proteins to be expressed in cell types that are difficult or impossible to micro-inject, and on a tissue scale that is not conceivable with microinjection. Further, the genetic nature of GFP and its variants permits novel strategies for generating cellular probes and the use of fluorescent markers in living tissue.
The goal of this review is to highlight the variety of questions that are being addressed in living cells using FPs, emphasizing examples from the recent plant cell biology literature and recent technological advances.
Section snippets
Dissecting cell pathways with FP markers and genetics
There has been a proliferation of studies using fluorescent proteins to dissect membrane trafficking pathways 2., 3.••, 4., 5.••, 6.••. Fluorescent proteins are used both to mark protein cargos and to identify trafficking compartments. In combination with molecular genetic manipulation, FP markers have proven particularly productive in analyzing the function of small GTP-binding proteins in trafficking events. Markers are typically expressed transiently in tissue culture cells along with an
Measuring subcellular dynamics
Fluorescent protein markers have made it possible to ask new questions about how proteins and organelles behave, and to study these behaviors in a quantitative fashion. Although the dynamics of the plant cortical cytoskeleton as a whole had been explored before the discovery of FP markers [7], it had not been possible to measure the contribution of individual microtubule behaviors. Two recent studies used FP markers to analyze the dynamics of individual plant cortical microtubules. Shaw et al.
Discovery of new subcellular structures
Imaging of FP fusions has allowed new subcellular structures to be discovered. FP fusions to phytochrome photoreceptors have revealed novel discreet locations within the cell nucleus at which photoreceptor proteins accumulate, and these structures have been termed speckles 12., 13., 14., 15.••. Fusions to multiple photoreceptor proteins translocate from the cytosol to the nucleus in response to stimulation by light, and accumulate in the speckles [15••]. The fact that these structures are
Measuring protein interactions and conformational change
One of the most powerful applications of FPs is in the measurement of changes in protein activity in vivo. These changes include protein translocation (as described for phytochromes above), the interaction of proteins with other proteins and molecules, and protein conformational changes. The measurement of many of these changes depends on a phenomenon known as fluorescence energy transfer (FRET) [17]. Resonance transfer occurs when part of the energy of an excited donor is transferred to an
Biosensors
Conformation-dependent FRET is not only useful for observing a protein of interest but has been exploited to create a new generation of sensors for small molecules (Figure 2). It has long been a goal of cell biologists to visualize changes in metabolites and signal molecules at the cellular level, but the spectrum of available optical sensors has been limited by the ability of chemists to design reporter dyes. FP technology is opening the door to the utilization of biologically evolved sensors
New fluorescent-protein variants
As interest in the applications of FPs grows, FPs with new properties are being developed or discovered as naturally occurring variants. Among the most interesting of the new FPs are variants that have useful photoactivation properties 45.••, 46.••, 47.••. Wildtype GFP is normally a mixed population of protein in two fluorophore states, neutral and anionic. The neutral form is responsible for a major absorption peak at 397 nm and the anionic form for a minor peak at 475 nm. Intense illumination
Conclusions and perspectives
FP technology promises to play a growing role in plant cell biology. Several large-scale screens are underway to generate to fluorescent cell probes and to gain clues as to gene function [48••]. Oparka and colleagues [48••] recently reported an extension of the type of screen first reported by Cutler et al. [49] in which random cDNA libraries are fused to GFP and screened for localization patterns in plant cells. Escobar et al. [48••] report a large number of intriguing localization patterns,
References and recommended reading
Papers of particular interest, published within the annual period of review, have been highlighted as:
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of special interest
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of outstanding interest
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Dual-colour imaging with GFP variants
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Fluorescence resonance energy transfer (FRET) microscopy imaging of live cell protein localizations
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A defined range of guard cell calcium oscillation parameters encodes stomatal movements
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Molecular dynamics in living cells observed by fluorescence correlation spectroscopy with one- and two-photon excitation
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Three-dimensional structural dynamics of myosin V by single-molecule fluorescence polarization
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A rab1 GTPase is required for transport between the endoplasmic reticulum and Golgi apparatus and for normal Golgi movement in plants
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ADP-ribosylation factor 1 of Arabidopsis plays a critical role in intracellular trafficking and maintenance of endoplasmic reticulum morphology in Arabidopsis
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Redistribution of membrane proteins between the Golgi apparatus and endoplasmic reticulum in plants is reversible and not dependent on cytoskeletal networks
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Rha1, an Arabidopsis Rab5 homolog, plays a critical role in the vacuolar trafficking of soluble cargo proteins
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Arf1 GTPase plays roles in the protein traffic between the endoplasmic reticulum and the Golgi apparatus in tobacco and Arabidopsis cultured cells
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Quantification of microtubule dynamics in living plant cells using fluorescence redistribution after photobleaching
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Sustained microtubule treadmilling in Arabidopsis cortical arrays
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Alteration of microtubule dynamic instability during preprophase band formation revealed by yellow fluorescent protein-CLIP170 microtubule plus-end labeling
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Chromatin of endoreduplicated pavement cells has greater range of movement than that of diploid guard cells in Arabidopsis thaliana
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Interphase chromosomes undergo constrained diffusional motion in living cells
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Missense mutation in the PAS2 domain of phytochrome A impairs subnuclear localization and a subset of responses
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Light-dependent translocation of a phytochrome B–GFP fusion protein to the nucleus in transgenic Arabidopsis
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Functional interaction of phytochrome B and cryptochrome 2
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Nucleocytoplasmic partitioning of the plant photoreceptors phytochrome A, B, C, D, and E is regulated differentially by light and exhibits a diurnal rhythm
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A complex and mobile structure forms a distinct subregion within the continuous vacuolar membrane in young cotyledons of Arabidopsis
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The photomorphogenesis regulator DET1 binds the amino-terminal tail of histone H2B in a nucleosome context
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