Photoconversion in orange and red fluorescent proteins

Kremers, Gert‐Jan; Hazelwood, Kristin L.; Murphy, Christopher S.; Davidson, Michael W.; Piston, David W.

doi:10.1038/nmeth.1319

Cited by 134 publications

(144 citation statements)

References 20 publications

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“…When these fluorescent proteins are illuminated, i.e. with many more photons than are generated in the bioluminescent reaction, they turn out to be photolabile and undergo photoconversion, which has been documented for GFP (Patterson and LippincottSchwartz 2002), YFP (Valentin et al 2005;Kirber et al 2007) and recently also for orange and red fluorescent proteins (Kremers et al 2009). Photoconversion can also take place during the FRET process.…”

Section: Discussionmentioning

confidence: 93%

Time-resolved FRET fluorescence spectroscopy of visible fluorescent protein pairs

et al. 2009

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Section: Discussionmentioning

confidence: 93%

Time-resolved FRET fluorescence spectroscopy of visible fluorescent protein pairs

et al. 2009

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“…37 These cells maintain properties of post-mitotic neurons, including membrane excitability, and have been used previously to study neuronal responses to hypoxic stress. MKP-1-induced toxicity was performed by seeding 4 Â 10 4 cells to 12-mm glass coverslips and transfecting with either control plasmid (pWay21 or pSG5), the nuclear chromatin reporter mOrange-H2B 38 or constructs expressing forms of MKP or variants of the bZIP factor C/EBPb. Total amounts of DNA and lipid were kept at a ratio of 400 ng to 1 ml of Lipofectamine 2000.…”

Section: Methodsmentioning

confidence: 99%

MKP-1 antagonizes C/EBP β activity and lowers the apoptotic threshold after ischemic injury

et al. 2012

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The dual specificity phosphatase MAPK phosphatase-1 (MKP-1) feeds back on MAP kinase signaling to regulate metabolic, inflammatory and survival responses. MKP-1 is widely expressed in the central nervous system (CNS) and induced after ischemic stress, although its function in these contexts remains unclear. Here we report that MKP-1 activated several cell death factors, including BCL2 and adenovirus E1B 19 kDa interacting protein 3, and caspases 3 and 12 culminating in apoptotic cell death in vitro. MKP-1 also exerted inhibitory effects on the bZIP transcription factor CCAAT/enhancer-binding protein (C/EBPb), previously shown to have neuroprotective properties. These effects included reduced expression of the full-length C/EBPb variant and hypo-phosphorylation at the MEK-ERK1/2-sensitive Thr 188 site. Notably, enforced expression C/EBPb rescued cells from MKP-1-induced toxicity. Studies performed in knock-out mice indicate that the MKP-1 activity is required to exclude C/EBPb from the nucleus basally, and that MKP-1 antagonizes C/EBPb expression after global forebrain ischemia, particularly within the vulnerable CA1 sector of the hippocampus. Overall, MKP-1 appears to lower the cellular apoptotic threshold by inhibiting C/EBPb and enhancing both BH3 protein expression and cellular caspase activity. Thus, although manipulation of the MKP-1-C/EBPb axis could have therapeutic value in ischemic disorders, our observations using MKP-1 catalytic mutants suggest that approaches geared towards inhibiting MKP-1's phosphatase activity alone may be ineffective.

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“…iRFP (Filonov et al 2011) in particular is extremely bright and truly far red (emitting at a wavelength close to 700 nm) . Additionally, Orange2 (Shaner et al 2008;Kremers et al 2009) is a red fluorophore which can be photo-converted to far red with a 488-nm laser pulse.…”

Section: Imaging: Basicsmentioning

confidence: 99%

Imaging stress

Brielle

Gura

Kaganovich

2015

Cell Stress and Chaperones

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Recent innovations in cell biology and imaging approaches are changing the way we study cellular stress, protein misfolding, and aggregation. Studies have begun to show that stress responses are even more variegated and dynamic than previously thought, encompassing nano-scale reorganization of cytosolic machinery that occurs almost instantaneously, much faster than transcriptional responses. Moreover, protein and mRNA quality control is often organized into highly dynamic macromolecular assemblies, or dynamic droplets, which could easily be mistaken for dysfunctional Baggregates,^but which are, in fact, regulated functional compartments. The nano-scale architecture of stressresponse ranges from diffraction-limited structures like stress granules, P-bodies, and stress foci to slightly larger quality control inclusions like juxta nuclear quality control compartment (JUNQ) and insoluble protein deposit compartment (IPOD), as well as others. Examining the biochemical and physical properties of these dynamic structures necessitates live cell imaging at high spatial and temporal resolution, and techniques to make quantitative measurements with respect to movement, localization, and mobility. Hence, it is important to note some of the most recent observations, while casting an eye towards new imaging approaches that offer the possibility of collecting entirely new kinds of data from living cells. StressProtecting the cell from protein-associated damage is a matter of having the correct proteins at the right place at the right time: the cellular environment changes rapidly in different folding and stress conditions in order to avoid catastrophic consequences of too many misfolded polypeptides or not enough functional proteins. When a cell is exposed to stresses such as heat-shock, cold-shock, osmotic stress, starvation, or amino-acid analogues which cause rampant mutations, the cellular response propagates across the entire network of protein biogenesis.Although much research has focused on how these stresses affect protein synthesis, we are only now beginning to look closely at how stress effects protein localization and distribution. Whereas expression takes time, and is more difficult to accomplish under stress, protein localization is substantially more dynamic and therefore changes in protein distribution can be affected quickly, in time to deal with the stress. Hence, regulation of protein distribution deserves careful attention in the study of stress response. Indeed, recent work has shown that in the single cell eukaryote Saccharomyces cerevisiae more than half of the proteome radically changes its localization under different stress condition (Breker et al. 2013). By altering the spatial positioning of proteins, cells can change Electronic supplementary material The online version of this article

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Photoconversion in orange and red fluorescent proteins

Cited by 134 publications

References 20 publications

Time-resolved FRET fluorescence spectroscopy of visible fluorescent protein pairs

Time-resolved FRET fluorescence spectroscopy of visible fluorescent protein pairs

MKP-1 antagonizes C/EBP β activity and lowers the apoptotic threshold after ischemic injury

Imaging stress

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