绿色荧光蛋白——照亮生命科学的一盏明灯

绿色荧光蛋白的发现及应用具有划时代的重要意义,它不仅为当代生物学研究提供了极为实用的基本研究手段,并且在此基础上改造发展和发现了一系列荧光蛋白,拓展了应用范围。这使得对微观生物学的研究也可以进入一个时空结合,研究鲜活动态过程的新时代。本文主要回顾总结了绿色荧光蛋白的发现、优化改造及其应用。     

The dynamic microbe: green fluorescent protein brings bacteria to light

The demonstration that the green fluorescent protein (GFP) from the jellyfish Aequorea victoria required no jellyfish-specific cofactors and could be expressed as a fluorescent protein in heterologous hosts including both prokaryotes and eukaryotes sparked the development of GFP as one of the most common reporters in use today. Over the past several years, the utility of GFP as a reporter has been optimized through the isolation and engineering of variants with increased folding rates, different in vivo stabilities and colour variants with altered excitation and emission spectral properties. One of the great utilities of GFP is as a probe for characterizing spatial and temporal dynamics of gene expression, protein localization and protein-protein interactions in living cells. The innovative application of GFP as a reporter in bacteria has made a significant contribution to microbial cell biology. This review will highlight recent studies that demonstrate the potential of GFP for real-time analysis of gene expression, protein localization and the dynamics of signalling transduction pathways through protein-protein interactions.     

Green innovation for green light

<正>In darkness, plants undergo skotomorphogenesis, which is characterized by long hypocotyls and closed cotyledons. In contrast, light, such as far-red light, red light, and blue light,inhibits hypocotyl elongation. Plants sense different wavelengths of light through different photoreceptors: phytochromes perceive red/far-red light(600–750 nm);     

Cryptochromes integrate green light signals into the circadian system

Plants are acutely sensitive of their light environment, adapting their growth habit and prioritizing developmental decisions to maximize fecundity. In addition to providing an energy source and directional information, light quality also contributes to entrainment of the circadian system, an endogenous timing mechanism that integrates endogenous and environmental signalling cues to promote growth. Whereas plants' perception of red and blue portions of the spectrum are well defined, green light sensitivity remains enigmatic. In this study, we show that low fluence rates of green light are sufficient to entrain and maintain circadian rhythms in Arabidopsis and that cryptochromes contribute to this response. Importantly, green light responses are distinguishable from low blue light-induced phenotypes. These data suggest a distinct signalling mechanism enables entrainment of the circadian system in green light-enriched environments, such as those found in undergrowth and in densely planted monoculture.     

Using light to see and control membrane traffic

Cellular compartmentalization into discrete organelles is maintained by membrane trafficking including vesiculation and tubulation. Recent advances in superresolution imaging have begun to bring these small and dynamic events into focus. Most nanoscopes exploit, and are limited by, switching dyes ON and OFF. Using ground state depletion to switch dyes into long-lived dark states can exploit specific photophysical properties of dyes, such as redox potential or pK(a), and expand the repertoire of nanoscopy probes for multicolor imaging. Seeing is not enough, and new technologies based on homodimerization, heterodimerization and selective release can manipulate membrane trafficking in pulse-chase and light-controlled ways. Herein we highlight the utility and promise of these strategies and discuss their current limitations.     

Shedding light on disulfide bond formation: engineering a redox switch in green fluorescent protein.

To visualize the formation of disulfide bonds in living cells, a pair of redox-active cysteines was introduced into the yellow fluorescent variant of green fluorescent protein. Formation of a disulfide bond between the two cysteines was fully reversible and resulted in a >2-fold decrease in the intrinsic fluorescence. Inter conversion between the two redox states could thus be followed in vitro as well as in vivo by non-invasive fluorimetric measurements. The 1.5 A crystal structure of the oxidized protein revealed a disulfide bond-induced distortion of the beta-barrel, as well as a structural reorganization of residues in the immediate chromophore environment. By combining this information with spectroscopic data, we propose a detailed mechanism accounting for the observed redox state-dependent fluorescence. The redox potential of the cysteine couple was found to be within the physiological range for redox-active cysteines. In the cytoplasm of Escherichia coli, the protein was a sensitive probe for the redox changes that occur upon disruption of the thioredoxin reductive pathway.     

Post-Golgi protein traffic in the plant secretory pathway

The Golgi apparatus in plants is organized as a multitude of individual stacks that are motile in the cytoplasm and in close association with the endoplasmic reticulum (ER) (Boevink et al. in Plant J 15:441–447, 1998). These stacks operate as a sorting centre for cargo molecules, providing modification and redirection to other organelles as appropriate. In the post-Golgi direction, these include vacuole and plasma membrane, and specialized transport routes to each are required to prevent mislocalization. Recent evidence in plant cells points to the existence of post-Golgi organelles that function as intermediate stations for efficient protein traffic, as well as to the influence of small GTPases such as Rabs and ARFs on post-Golgi trafficking. This review focuses on the latest findings on post-Golgi trafficking routes and on the involvement of GTPases and their effectors on the trafficking of proteins in the plant secretory pathway. Sally L. Hanton and Loren A. Matheson have contributed equally to this work.     

Imaging the environment of green fluorescent protein

An emerging theme in cell biology is that cell surface receptors need to be considered as part of supramolecular complexes of proteins and lipids facilitating specific receptor conformations and distinct distributions, e.g., at the immunological synapse. Thus, a new goal is to develop bioimaging that not only locates proteins in live cells but can also probe their environment. Such a technique is demonstrated here using fluorescence lifetime imaging of green fluorescent protein (GFP). We first show, by time-correlated single-photon counting, that the fluorescence decay of GFP depends on the local refractive index. This is in agreement with the Strickler Berg formula, relating the Einstein A and B coefficients for absorption and spontaneous emission in molecules. We then quantitatively image, by wide-field time-gated fluorescence lifetime imaging, the refractive index of the environment of GFP. This novel approach paves the way for imaging the biophysical environment of specific GFP-tagged proteins in live cells.     

Calcium-dependent protein kinase in the green algaChara

Cytoplasmic streaming in the characean algae is inhibited by micromolar rises in the level of cytosolic free Ca²⁺, but both the mechanism of action and the molecular components involved in this process are unknown. We have used monoclonal antibodies against soybean Ca²⁺-dependent protein kinase (CDPK), a kinase that is activated by micromolar Ca²⁺ and co-localizes with actin filaments in higher-plant cells (Putnam-Evans et al., 1989, Cell Motil. Cytoskel.12, 12–22) to identify and localize its characean homologue. Immunoblot analysis revealed that CDPK inChara corralina Klein ex. Wild shares the same relative molecular mass (51–55 kDa) as the kinase purified from soybean, and after electrophoresis in denaturing gels is capable of phosphorylating histone III-S in a Ca²⁺-dependent manner. Immunofluorescence microscopy localized CDPK inChara to the subcortical actin bundles and the surface of small organelles and other membrane components of the streaming endoplasm. The endoplasmic sites carrying CDPK were extracted from internodal cells by vacuolar perfusion with 1 mM ATP or 10^–4 M Ca²⁺. Both the localization of CDPK and its extraction from internodal cells by perfusion with ATP or high Ca²⁺ are properties similar to that reported for the heavy chain of myosin inChara (Grolig et al., 1988, Eur. J. Cell Biol.47, 22–31). Based on its endoplasmic location and inferred enzymatic properties, we suggest that CDPK may be a putative element of the signal-transduction pathway that mediates the rapid Ca²⁺-induced inhibition of streaming that occurs in the characean algae.Abbreviations CDPK calcium-dependent protein kinase - kDa kilodalton - mAb monoclonal antibody - M_r relative molecular mass - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis We thank Dr. Richard Williamson (Plant Cell Biology Group, Research School of Biological Sciences, The Australian National University) for valuable discussions during the course of this research. This work was supported by funds from a Queen Elizabeth II Fellowship awarded to D.W.McC. and U.S. Department of Agriculture (88-37261-4199) and National Research Inititive Competitive Grants Program (91-37304-6654) grants to A.C.H.     

Phototransformation of green fluorescent protein with UV and visible light leads to decarboxylation of glutamate 222.

Wild type green fluorescent protein (GFP) from Aequorea victoria absorbs predominantly at 398 nm. Illumination with UV (254 nm) or visible (390 nm) light transforms this state (GFP(398)) into one absorbing at 483 nm (GFP(483)). Here we show that this photoconversion of GFP is a one-photon process that is paralleled by decarboxylation of Glu 222. We propose a mechanism in which decarboxylation is due to electron transfer between the gamma-carboxylate of Glu 222 and the p-hydroxybenzylidene-imidazolidinone chromophore of GFP, followed by reverse transfer of an electron and a proton to the remaining carbon side chain atom of Glu 222. Oxidative decarboxylation of a gamma-carboxylate represents a new type of posttranslational modification that may also occur in enzymes with high-potential reaction intermediates.     

Photosynthetic activity of far-red light in green plants

We have found that long-wavelength quanta up to 780 nm support oxygen evolution from the leaves of sunflower and bean. The far-red light excitations are supporting the photochemical activity of photosystem II, as is indicated by the increased chlorophyll fluorescence in response to the reduction of the photosystem II primary electron acceptor, Q_A. The results also demonstrate that the far-red photosystem II excitations are susceptible to non-photochemical quenching, although less than the red excitations. Uphill activation energies of 9.8 ± 0.5 kJ mol⁻¹ and 12.5 ± 0.7 kJ mol⁻¹ have been revealed in sunflower leaves for the 716 and 740 nm illumination, respectively, from the temperature dependencies of quantum yields, comparable to the corresponding energy gaps of 8.8 and 14.3 kJ mol⁻¹ between the 716 and 680 nm, and the 740 and 680 nm light quanta. Similarly, the non-photochemical quenching of far-red excitations is facilitated by temperature confirming thermal activation of the far-red quanta to the photosystem II core. The observations are discussed in terms of as yet undisclosed far-red forms of chlorophyll in the photosystem II antenna, reversed (uphill) spill-over of excitation from photosystem I antenna to the photosystem II antenna, as well as absorption from thermally populated vibrational sub-levels of photosystem II chlorophylls in the ground electronic state. From these three interpretations, our analysis favours the first one, i.e., the presence in intact plant leaves of a small number of far-red chlorophylls of photosystem II. Based on analogy with the well-known far-red spectral forms in photosystem I, it is likely that some kind of strongly coupled chlorophyll dimers/aggregates are involved. The similarity of the result for sunflower and bean proves that both the extreme long-wavelength oxygen evolution and the local quantum yield maximum are general properties of the plants.     

Photosynthetic activity of far-red light in green plants

We have found that long-wavelength quanta up to 780 nm support oxygen evolution from the leaves of sunflower and bean. The far-red light excitations are supporting the photochemical activity of photosystem II, as is indicated by the increased chlorophyll fluorescence in response to the reduction of the photosystem II primary electron acceptor, Q(A). The results also demonstrate that the far-red photosystem II excitations are susceptible to non-photochemical quenching, although less than the red excitations. Uphill activation energies of 9.8+/-0.5 kJ mol(-1) and 12.5+/-0.7 kJ mol(-1) have been revealed in sunflower leaves for the 716 and 740 nm illumination, respectively, from the temperature dependencies of quantum yields, comparable to the corresponding energy gaps of 8.8 and 14.3 kJ mol(-1) between the 716 and 680 nm, and the 740 and 680 nm light quanta. Similarly, the non-photochemical quenching of far-red excitations is facilitated by temperature confirming thermal activation of the far-red quanta to the photosystem II core. The observations are discussed in terms of as yet undisclosed far-red forms of chlorophyll in the photosystem II antenna, reversed (uphill) spill-over of excitation from photosystem I antenna to the photosystem II antenna, as well as absorption from thermally populated vibrational sub-levels of photosystem II chlorophylls in the ground electronic state. From these three interpretations, our analysis favours the first one, i.e., the presence in intact plant leaves of a small number of far-red chlorophylls of photosystem II. Based on analogy with the well-known far-red spectral forms in photosystem I, it is likely that some kind of strongly coupled chlorophyll dimers/aggregates are involved. The similarity of the result for sunflower and bean proves that both the extreme long-wavelength oxygen evolution and the local quantum yield maximum are general properties of the plants.     

Interactions of green or red light with blue light on the dark closure of Albizzia pinnules

The interactions of green or red light with blue light on the dark closing of Albizzia julibrissin Durazz. pinnules have been investigated. Irradiations at 430, 450 and 470 nm progressively delay dark closing with increasing photon fluence rates. Red or green light alone has no effect. However, when the blue fluence rate is low, both red and green light interact with it and increase the delaying effect of the blue light. When the blue fluence rate is high, green light interacts with it to negate some of the effectiveness of the blue light, while red light has no effect. This is similar to results obtained previously with far-red light. It is suggested that the same unidentified photoreceptor is operating in both the far-red and blue regions. The results also indicate the presence of a blue-only absorbing photoreceptor whose action is increased by phytochrome.