Multifocal two-photon laser scanning microscopy combined with photo-activatable GFP for in vivo monitoring of intracellular protein dynamics in real time
TL;DR: The intracellular dynamics of the Arabidopsis MYB transcription factor LHY/CCA1-like 1 (LCL1) that contains both a nuclear import and a nuclear export signal was quantitatively investigated and an export-negative mutant of LCL1 remained trapped inside the nucleus.
About: This article is published in Journal of Structural Biology.The article was published on 2007-06-01. It has received 24 citations till now. The article focuses on the topics: Nuclear export signal & Nuclear transport.
Citations
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TL;DR: The structure, evolution, and function of GFP-like proteins and their numerous applications for in vivo imaging are focused on, with particular attention to recent techniques.
Abstract: Green fluorescent protein (GFP) from the jellyfish Aequorea victoria and its homologs from diverse marine animals are widely used as universal genetically encoded fluorescent labels. Many laboratories have focused their efforts on identification and development of fluorescent proteins with novel characteristics and enhanced properties, resulting in a powerful toolkit for visualization of structural organization and dynamic processes in living cells and organisms. The diversity of currently available fluorescent proteins covers nearly the entire visible spectrum, providing numerous alternative possibilities for multicolor labeling and studies of protein interactions. Photoactivatable fluorescent proteins enable tracking of photolabeled molecules and cells in space and time and can also be used for super-resolution imaging. Genetically encoded sensors make it possible to monitor the activity of enzymes and the concentrations of various analytes. Fast-maturing fluorescent proteins, cell clocks, and timers further expand the options for real time studies in living tissues. Here we focus on the structure, evolution, and function of GFP-like proteins and their numerous applications for in vivo imaging, with particular attention to recent techniques.
1,214 citations
TL;DR: The basic architecture of a multiphoton microscope capable of real-time analysis of cellular structure and function is discussed and the state-of-the-art technologies for the quantitative imaging of biological phenomena are summarized.
Abstract: The ability to dynamically image features deep within living organisms, permitting real-time analysis of cellular structure and function, is important for biological science. This Review article discusses multiphoton microscopy capable of such analysis, along with technologies that are pushing the limits of phenomena that can be quantitatively imaged.
441 citations
TL;DR: There is still room for further improvement of these important markers for live cell imaging, and special FP variants with low switching fatigue have been introduced in recent years.
Abstract: Fluorescent proteins (FPs) from the GFP family have become indispensable as marker tools for imaging live cells, tissues and entire organisms. A wide variety of these proteins have been isolated from natural sources and engineered to optimize their properties as genetically encoded markers. Here we review recent developments in this field. A special focus is placed on photoactivatable FPs, for which the fluorescence emission can be controlled by light irradiation at specific wavelengths. They enable regional optical marking in pulse-chase experiments on live cells and tissues, and they are essential marker tools for live-cell optical imaging with super-resolution. Photoconvertible FPs, which can be activated irreversibly via a photo-induced chemical reaction that either turns on their emission or changes their emission wavelength, are excellent markers for localization-based super-resolution microscopy (e.g., PALM). Patterned illumination microscopy (e.g., RESOLFT), however, requires markers that can be reversibly photoactivated many times. Photoswitchable FPs can be toggled repeatedly between a fluorescent and a non-fluorescent state by means of a light-induced chromophore isomerization coupled to a protonation reaction. We discuss the mechanistic origins of the effect and illustrate how photoswitchable FPs are employed in RESOLFT imaging. For this purpose, special FP variants with low switching fatigue have been introduced in recent years. Despite nearly two decades of FP engineering by many laboratories, there is still room for further improvement of these important markers for live cell imaging.
294 citations
TL;DR: Interestingly, several mutants in genes encoding components of the plant nuclear transport machinery were investigated, revealing differential sensitivity of plant-specific pathways to impaired nuclear transport, unravelling connections between nuclear transport and components of signalling and developmental pathways.
Abstract: Transport of macromolecules between the nucleus and the cytoplasm is an essential necessity in eukaryotic cells, since the nuclear envelope separates transcription from translation. In the past few years, an increasing number of components of the plant nuclear transport machinery have been characterised. This progress, although far from being completed, confirmed that the general characteristics of nuclear transport are conserved between plants and other organisms. However, plant-specific components were also identified. Interestingly, several mutants in genes encoding components of the plant nuclear transport machinery were investigated, revealing differential sensitivity of plant-specific pathways to impaired nuclear transport. These findings attracted attention towards plant-specific cargoes that are transported over the nuclear envelope, unravelling connections between nuclear transport and components of signalling and developmental pathways. The current state of research in plants is summarised in comparison to yeast and vertebrate systems, and special emphasis is given to plant nuclear transport mutants.
83 citations
Cites background or methods from "Multifocal two-photon laser scannin..."
...Nucleo-cytoplasmic shuttling of proteins can be visualised in vivo in real time using specific fluorescence microscopy techniques (Köster et al. 2005) or photoswitchable or photoactivatable fluorescent proteins (Martini et al. 2007)....
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...2005) or photoswitchable or photoactivatable fluorescent proteins (Martini et al. 2007)....
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TL;DR: Recent studies are uncovering the sophistication and complexity of the processes that use the canonical transport machinery in the service of a diversity of signaling pathways.
58 citations
References
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TL;DR: The fluorescence emission increased quadratically with the excitation intensity so that fluorescence and photo-bleaching were confined to the vicinity of the focal plane as expected for cooperative two-photon excitation.
Abstract: Molecular excitation by the simultaneous absorption of two photons provides intrinsic three-dimensional resolution in laser scanning fluorescence microscopy. The excitation of fluorophores having single-photon absorption in the ultraviolet with a stream of strongly focused subpicosecond pulses of red laser light has made possible fluorescence images of living cells and other microscopic objects. The fluorescence emission increased quadratically with the excitation intensity so that fluorescence and photo-bleaching were confined to the vicinity of the focal plane as expected for cooperative two-photon excitation. This technique also provides unprecedented capabilities for three-dimensional, spatially resolved photochemistry, particularly photolytic release of caged effector molecules.
8,905 citations
Additional excerpts
...The intrinsic limitation of excitation to the microscope’s focal plane, provided by the axial sectioning capability of 2PLSM (Denk et al., 1990; Diaspro, 2002), is also responsible for the spatial conWnement and selectivity of the 2P-activation of pa-GFP....
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TL;DR: Multiphoton microscopy has found a niche in the world of biological imaging as the best noninvasive means of fluorescence microscopy in tissue explants and living animals and its use is now increasing exponentially.
Abstract: Multiphoton microscopy (MPM) has found a niche in the world of biological imaging as the best noninvasive means of fluorescence microscopy in tissue explants and living animals. Coupled with transgenic mouse models of disease and 'smart' genetically encoded fluorescent indicators, its use is now increasing exponentially. Properly applied, it is capable of measuring calcium transients 500 microm deep in a mouse brain, or quantifying blood flow by imaging shadows of blood cells as they race through capillaries. With the multitude of possibilities afforded by variations of nonlinear optics and localized photochemistry, it is possible to image collagen fibrils directly within tissue through nonlinear scattering, or release caged compounds in sub-femtoliter volumes.
3,738 citations
"Multifocal two-photon laser scannin..." refers background in this paper
...…for activation and detection is reasoned by the speciWc cross sections of pa-GFP, but it also provides the advantage of a higher penetration depth, negligible one-photon cross sections, and less potential for cellular damage and Xuorescent background (Zipfel et al., 2003a; Zipfel et al., 2003b)....
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TL;DR: It is concluded that CRM1 is an export receptor for leucine-rich nuclear export signals and a model for the role of RanGTP inCRM1 function and in nuclear export in general is discussed.
2,068 citations
"Multifocal two-photon laser scannin..." refers background in this paper
...LMB covalently modiWes the nuclear export receptor XPO1 (in humans designated CRM1; Fornerod et al., 1997; Fukuda et al., 1997) thus speciWcally inhibiting nuclear export of cargo macromolecules that bind to this nuclear transport receptor (Kudo et al., 1999)....
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TL;DR: Applications involving a range of intrinsic molecules and molecular assemblies that enable direct visualization of tissue morphology, cell metabolism, and disease states such as Alzheimer's disease and cancer are compiled and demonstrated.
Abstract: Multicolor nonlinear microscopy of living tissue using two- and three-photon-excited intrinsic fluorescence combined with second harmonic generation by supermolecular structures produces images with the resolution and detail of standard histology without the use of exogenous stains. Imaging of intrinsic indicators within tissue, such as nicotinamide adenine dinucleotide, retinol, indoleamines, and collagen provides crucial information for physiology and pathology. The efficient application of multiphoton microscopy to intrinsic imaging requires knowledge of the nonlinear optical properties of specific cell and tissue components. Here we compile and demonstrate applications involving a range of intrinsic molecules and molecular assemblies that enable direct visualization of tissue morphology, cell metabolism, and disease states such as Alzheimer's disease and cancer.
1,730 citations
"Multifocal two-photon laser scannin..." refers background in this paper
...…for activation and detection is reasoned by the speciWc cross sections of pa-GFP, but it also provides the advantage of a higher penetration depth, negligible one-photon cross sections, and less potential for cellular damage and Xuorescent background (Zipfel et al., 2003a; Zipfel et al., 2003b)....
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TL;DR: A photoactivatable variant of the Aequorea victoria green fluorescent protein is reported that, after intense irradiation with 413-nanometer light, increases fluorescence 100 times when excited by 488-nanometers light and remains stable for days under aerobic conditions.
Abstract: We report a photoactivatable variant of the Aequorea victoria green fluorescent protein (GFP) that, after intense irradiation with 413-nanometer light, increases fluorescence 100 times when excited by 488-nanometer light and remains stable for days under aerobic conditions. These characteristics offer a new tool for exploring intracellular protein dynamics by tracking photoactivated molecules that are the only visible GFPs in the cell. Here, we use the photoactivatable GFP both as a free protein to measure protein diffusion across the nuclear envelope and as a chimera with a lysosomal membrane protein to demonstrate rapid interlysosomal membrane exchange.
1,605 citations
"Multifocal two-photon laser scannin..." refers background or methods in this paper
...…detection of pa-GFP implies spectral activation at » 408 nm, which induces a photoconversion resulting in a shift of the absorption maximum of the Xuorescent protein to »504 nm with a maximum of emission at » 517 nm (Patterson and Lippincott-Schwartz, 2002; Patterson and Lippincott-Schwartz, 2004)....
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...Selective detection of pa-GFP implies spectral activation at » 408 nm, which induces a photoconversion resulting in a shift of the absorption maximum of the Xuorescent protein to »504 nm with a maximum of emission at » 517 nm (Patterson and Lippincott-Schwartz, 2002; Patterson and Lippincott-Schwartz, 2004)....
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...The development of a photo-activatable variant of GFP (Patterson and Lippincott-Schwartz, 2002) created a powerful tool for real-time measurement of protein dynamics in living cells....
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