Determination of supramolecular structure and spatial distribution of protein complexes in living cells

Raicu, Valerică; Stoneman, Michael R.; Fung, Russell; Melnichuk, Mike; Jansma, David B.; Pisterzi, Luca F.; Rath, Sasmita; Fox, Michael H.; Wells, James W.; Saldin, D. K.

doi:10.1038/nphoton.2008.291

Cited by 106 publications

(251 citation statements)

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“…Second, we carried out FRET spectrometry experiments to quantify the SERK1-EMS1 interaction via a two-photon microspectroscope (Raicu et al, 2009;Biener et al, 2013). FRET efficiencies were calculated using images generated by unmixing pixel-level spectra of donors upon excitation at the 860-nm wavelength (EMS1-cyan fluorescent protein [CFP]; Fig.…”

Section: Serk1 Interacts With Ems1mentioning

confidence: 99%

“…Acquisition of spectrally resolved fluorescence images, spectral unmixing of donor, acceptor, and autofluorescence signals, as well as determination of FRET efficiency were performed as described previously (Raicu and Singh, 2013;King et al, 2016). Briefly, cells coexpressing EMS1-CFP and SERK1-YFP (controls: cells expressing only EMS1-CFP or SERK1-YFP) were imaged using an optical microspectroscope (OptiMiS) with the line-scan excitation powered by a femtosecond laser emitting near-infrared light pulses with tunable emission wavelength between 780 and 1,040 nm (Raicu et al, 2009;Biener et al, 2013). The spectrally resolved images were then unmixed to generate two-dimensional fluorescence maps of donor and acceptor (Raicu and Singh, 2013).…”

Section: Fret Assaymentioning

confidence: 99%

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Two SERK Receptor-Like Kinases Interact with EMS1 to Control Anther Cell Fate Determination

Wang

Huang

et al. 2016

Plant Physiol.

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Cell signaling pathways mediated by leucine-rich repeat receptor-like kinases (LRR-RLKs) are essential for plant growth, development, and defense. The EMS1 (EXCESS MICROSPOROCYTES1) LRR-RLK and its small protein ligand TPD1 (TAPETUM DETERMINANT1) play a fundamental role in somatic and reproductive cell differentiation during early anther development in Arabidopsis (Arabidopsis thaliana). However, it is unclear whether other cell surface molecules serve as coregulators of EMS1. Here, we show that SERK1 (SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE1) and SERK2 LRR-RLKs act redundantly as coregulatory and physical partners of EMS1. The SERK1/2 genes function in the same genetic pathway as EMS1 in anther development. Bimolecular fluorescence complementation, Förster resonance energy transfer, and coimmunoprecipitation approaches revealed that SERK1 interacted biochemically with EMS1. Transphosphorylation of EMS1 by SERK1 enhances EMS1 kinase activity. Among 12 in vitro autophosphorylation and transphosphorylation sites identified by tandem mass spectrometry, seven of them were found to be critical for EMS1 autophosphorylation activity. Furthermore, complementation test results suggest that phosphorylation of EMS1 is required for its function in anther development. Collectively, these data provide genetic and biochemical evidence of the interaction and phosphorylation between SERK1/2 and EMS1 in anther development.

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Section: Serk1 Interacts With Ems1mentioning

confidence: 99%

Section: Fret Assaymentioning

confidence: 99%

Two SERK Receptor-Like Kinases Interact with EMS1 to Control Anther Cell Fate Determination

Wang

Huang

et al. 2016

Plant Physiol.

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“…The retention sequence that prevents surface expression of the GABA B1 receptor has been exploited to demonstrate that tetramers of the GABA B1 and GABA B2 receptors can be formed as a dimer of heterodimers (10). FRET efficiencies determined by means of spectral deconvolution at the level of single pixels have suggested that the Ste2p receptor forms tetramers in yeast (56). In the case of rhodopsin, atomic force microscopy has identified what appear to be extended rows of dimers in some preparations of native tissue (57).…”

mentioning

confidence: 99%

Oligomeric Size of the M2 Muscarinic Receptor in Live Cells as Determined by Quantitative Fluorescence Resonance Energy Transfer

Pisterzi

Jansma

Georgiou

et al. 2010

Journal of Biological Chemistry

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Fluorescence resonance energy transfer (FRET), measured by fluorescence intensity-based microscopy and fluorescence lifetime imaging, has been used to estimate the size of oligomers formed by the M 2 muscarinic cholinergic receptor. The approach is based on the relationship between the apparent FRET efficiency within an oligomer of specified size (n) and the pairwise FRET efficiency between a single donor and a single acceptor (E). The M 2 receptor was fused at the N terminus to enhanced green or yellow fluorescent protein and expressed in Chinese hamster ovary cells. Emission spectra were analyzed by spectral deconvolution, and apparent efficiencies were estimated by donor-dequenching and acceptor-sensitized emission at different ratios of enhanced yellow fluorescent protein-M 2 receptor to enhanced green fluorescent protein-M 2 receptor. The data were interpreted in terms of a model that considers all combinations of donor and acceptor within a specified oligomer to obtain fitted values of E as follows: n ‫؍‬ 2, 0.495 ؎ 0.019; n ‫؍‬ 4, 0.202 ؎ 0.010; n ‫؍‬ 6, 0.128 ؎ 0.006; n ‫؍‬ 8, 0.093 ؎ 0.005. The pairwise FRET efficiency determined independently by fluorescence lifetime imaging was 0.20 -0.24, identifying the M 2 receptor as a tetramer. The strategy described here yields an explicit estimate of oligomeric size on the basis of fluorescence properties alone. Its broader application could resolve the general question of whether G protein-coupled receptors exist as dimers or larger oligomers. The size of an oligomer has functional implications, and such information can be expected to contribute to an understanding of the signaling process.Much evidence now indicates that G protein-coupled receptors can exist as oligomers (1, 2), a development that has implications for all aspects of GPCR 4 -mediated signaling. Among the many questions prompted by the emergence of such structures is that of oligomeric size. Although commonly referred to as dimers, oligomers of GPCRs have been detected most often by means of coimmunoprecipitation or resonance energy transfer (3). As typically applied, neither technique can distinguish dimers from larger oligomers. The latter have been identified on the basis of their electrophoretic mobility (reviewed in Ref. 4), but the composition of the bands may be unclear, and the size under the conditions of electrophoresis may have little in common with that in the membrane. Larger oligomers also have been identified by approaches in which detection requires the colocalization of three or four proteins, each bearing a different tag (5-11), but such procedures place only a lower limit on the possible size of the array.There have been comparatively few attempts to examine the oligomeric status of a GPCR in a more quantitative and explicit manner. Measurements of BRET at different ratios of acceptor to donor have pointed to dimers of the melatonin receptor (12), the ␤ 1 -and ␤ 2 -adrenergic receptors (13), the M 1 , M 2 , and M 3 muscarinic receptors (14), and the neurotensin receptor (15)....

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“…Such a prGO based 'FRET on fiber' configuration, bridging the FRET and the fiber-optic sensing technology, may serve as a platform for the realization of series of integrated 'FRET on Fiber' sensors for on-line environmental, chemical, and biomedical detection, with excellent compactness, high sensitivity, good selectivity and fast response Biochemical detection with both high sensitivity and selectivity is of great importance in medical, chemical, environmental and security areas. During the past decades, various optical biochemical detection methods based on diverse technologies have been reported and still developing fast [1][2][3][4][5][6][7] . In particular, the fluorescence resonance energy transfer (FRET) is a highly popular method due to its biochemical universality and naturally exceptional selectivity [8][9][10] .…”

mentioning

confidence: 99%

“…During the past decades, various optical biochemical detection methods based on diverse technologies have been reported and still developing fast [1][2][3][4][5][6][7] . In particular, the fluorescence resonance energy transfer (FRET) is a highly popular method due to its biochemical universality and naturally exceptional selectivity [8][9][10] .…”

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confidence: 99%

Partially reduced graphene oxide based FRET on fiber optic interferometer for biochemical detection

Yao

et al. 2017

SPIE Proceedings

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Fluorescent resonance energy transfer (FRET) with naturally exceptional selectivity is a powerfultechnique and widely used in chemical and biomedical analysis. However, it is still challenging for conventional FRET to perform as a high sensitivity compact sensor. Here we propose a novel 'FRET on Fiber' concept, in which a partially reduced graphene oxide (prGO) film is deposited on a fiberoptic modal interferometer, acting as both the fluorescent quencher for the FRET and the sensitive cladding for optical phase measurement due to refractive index changes in biochemical detection. The target analytes induced fluorescence recovery with good selectivity and optical phase shift with high sensitivity are measured simultaneously. The functionalized prGO film coated on the fiber-optic interferometer shows high sensitivities for the detections of metal ion, dopamine and single-stranded DNA (ssDNA), with detection limits of 1.2 nM, 1.3 μM and 1 pM, respectively. Such a prGO based 'FRET on fiber' configuration, bridging the FRET and the fiber-optic sensing technology, may serve as a platform for the realization of series of integrated 'FRET on Fiber' sensors for on-line environmental, chemical, and biomedical detection, with excellent compactness, high sensitivity, good selectivity and fast response Biochemical detection with both high sensitivity and selectivity is of great importance in medical, chemical, environmental and security areas. During the past decades, various optical biochemical detection methods based on diverse technologies have been reported and still developing fast [1][2][3][4][5][6][7] . In particular, the fluorescence resonance energy transfer (FRET) is a highly popular method due to its biochemical universality and naturally exceptional selectivity [8][9][10] . However, as a key biochemical detection technique, the optical signal of the conventional FRET method based on fluorescent intensity measurement is just collected from dispersions or solutions in free-space. Hence, it is quite challenging to replace the conventional FRET instrument with a compact FRET probe for on-line biochemical detection, limiting the applications of the FRET technology considerably. Optical fiber sensors have great potential to solve such a problem, as they are compact and suitable for on-line applications [11][12][13][14][15] . Furthermore, high sensitivity could be achieved with a fiber-optic interferometer based on optical phase measurement due to refractive index changes 16,17 . Hence, by combining the FRET and fiber-optic sensing technologies, we could pave a new way for the realization of all-fiber based FRET sensors, which can be expected to have high impact on the area of the biochemical detection due to their excellent compactness, high sensitivity, good selectivity, and fast response.The emergence of graphene materials provides the possibility to bridge such a combination 18,19 , where graphene or graphene oxide acting as a naturally fluorescent quencher can be used to improve the fluorescence transfer efficienc...

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Determination of supramolecular structure and spatial distribution of protein complexes in living cells

Cited by 106 publications

References 44 publications

Two SERK Receptor-Like Kinases Interact with EMS1 to Control Anther Cell Fate Determination

Two SERK Receptor-Like Kinases Interact with EMS1 to Control Anther Cell Fate Determination

Oligomeric Size of the M2 Muscarinic Receptor in Live Cells as Determined by Quantitative Fluorescence Resonance Energy Transfer

Partially reduced graphene oxide based FRET on fiber optic interferometer for biochemical detection

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