Green to red photoconversion of GFP for protein tracking in vivo

Sattarzadeh, Amirali; Saberianfar, Reza; Zipfel, Warren R.; Menassa, Rima; Hanson, Maureen R.

doi:10.1038/srep11771

Cited by 29 publications

(18 citation statements)

References 29 publications

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“…A recent study revealed that many GCaMPs are inherently photoconvertible to red emission by 400-nm light, with the red species retaining up to 41% of the responsiveness of the original green form 110 . These findings add to earlier observations of green-to-red photoconversion in GFP derivatives 111 , although the photoconversion may be less efficient than in fluorescent proteins evolved for photoconvertibility. Finally, a unique GECI, CAMPARI, undergoes green-to-red photoconversion by 400-nm light only when bound to calcium 112 .…”

Section: Indicatorssupporting

confidence: 81%

Genetically encoded indicators of neuronal activity

2016

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Experimental efforts to understand how the brain represents, stores and processes information require high-fidelity recordings of multiple different forms of neural activity within functional circuits. Thus, creating improved technologies for large-scale recordings of neural activity in the live brain is a crucial goal in neuroscience. Over the past two decades, the combination of optical microscopy and genetically encoded fluorescent indicators has become a widespread means of recording neural activity in nonmammalian and mammalian nervous systems, transforming brain research in the process. In this review, we describe and assess different classes of fluorescent protein indicators of neural activity. We first discuss general considerations in optical imaging and then present salient characteristics of representative indicators. Our focus is on how indicator characteristics relate to their use in living animals and on likely areas of future progress.

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Section: Indicatorssupporting

confidence: 81%

Genetically encoded indicators of neuronal activity

2016

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“…The photoconvertible Dendra2 protein has proven to be a valuable tool that can be used to measure protein turnover while having minimal influence on the turnover rate kinetics of the fused protein (Hamer et al, 2010;Sattarzadeh et al, 2015). The photoconvertible Dendra2 protein has proven to be a valuable tool that can be used to measure protein turnover while having minimal influence on the turnover rate kinetics of the fused protein (Hamer et al, 2010;Sattarzadeh et al, 2015).…”

Section: Resultsmentioning

confidence: 99%

“…In patients with the C9orf72 NRE, there is an accumulation of specific DPRs in tissue, but little is known whether this could be in part due to intrinsic DPR turnover rates. The photoconvertible Dendra2 protein has proven to be a valuable tool that can be used to measure protein turnover while having minimal influence on the turnover rate kinetics of the fused protein (Hamer et al, 2010;Sattarzadeh et al, 2015). Therefore, we cotransfected C9 DPR reporters with NES-mIFP in HEK293T cells, photoconverted the Dendra2 from fluorescent green to red ( Fig EV3), and then monitored the decay of red fluorescent intensity longitudinally for sense and antisense C9 DPRs (Fig EV3).…”

Section: Resultsmentioning

confidence: 99%

Repeat‐associated non‐ AUG translation in C9orf72‐ ALS / FTD is driven by neuronal excitation and stress

et al. 2019

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Nucleotide repeat expansions (NREs) are prevalent mutations in a multitude of neurodegenerative diseases. Repeat‐associated non‐AUG (RAN) translation of these repeat regions produces mono or dipeptides that contribute to the pathogenesis of these diseases. However, the mechanisms and drivers of RAN translation are not well understood. Here we analyzed whether different cellular stressors promote RAN translation of dipeptide repeats (DPRs) associated with the G4C2 hexanucleotide expansions in C9orf72, the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). We found that activating glutamate receptors or optogenetically increasing neuronal activity by repetitive trains of depolarization induced DPR formation in primary cortical neurons and patient derived spinal motor neurons. Increases in the integrated stress response (ISR) were concomitant with increased RAN translation of DPRs, both in neurons and different cell lines. Targeting phosphorylated‐PERK and the phosphorylated‐eif2α complex reduces DPR levels revealing a potential therapeutic strategy to attenuate DPR‐dependent disease pathogenesis in NRE‐linked diseases.

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“…UAS:Kaede) larvae were anesthetized, mounted in agarose and exposed to UV light for approximately 3 min to irreversibly convert Kaede protein from green to red, which can be stable for several days (Sattarzadeh, Saberianfar, Zipfel, Menassa, & Hanson, 2015). To The mpz:mEGFP reporter co-localized with known oligodendrocyte markers.…”

Section: Resultsmentioning

confidence: 99%

A novel myelin protein zero transgenic zebrafish designed for rapid readout of in vivo myelination

Preston

Finseth

Bourne

et al. 2019

Glia

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Demyelination occurs following many neurological insults, most notably in multiple sclerosis (MS). Therapeutics that promote remyelination could slow the neurological decline associated with chronic demyelination; however, in vivo testing of candidate small molecule drugs and signaling cascades known to impact myelination is expensive and labor intensive. Here, we describe the development of a novel zebrafish line which uses the putative promoter of Myelin Protein Zero (mpz), a major structural protein in myelin, to drive expression of Enhanced Green Fluorescent Protein (mEGFP) specifically in the processes and nascent internodes of myelinating glia. We observe that changes in fluorescence intensity in Tg(mpz:mEGFP) larvae are a reliable surrogate for changes in myelin membrane production per se in live larvae following bath application of drugs. These changes in fluorescence are strongly predictive of changes in myelin‐specific mRNAs [mpz, 36K and myelin basic protein (mbp)] and protein production (Mbp). Finally, we observe that certain drugs alter nascent internode number and length, impacting the overall amount of myelin membrane synthesized and a number of axons myelinated without significantly changing the number of myelinating oligodendrocytes. These studies demonstrate that the Tg(mpz:mEGFP) reporter line responds effectively to positive and negative small molecule regulators of myelination, and could be useful for identifying candidate drugs that specifically target myelin membrane production in vivo. Combined with high throughput cell‐based screening of large chemical libraries and automated imaging systems, this transgenic line is useful for rapid large scale whole animal screening to identify novel myelinating small molecule compounds in vivo.

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Green to red photoconversion of GFP for protein tracking in vivo

Cited by 29 publications

References 29 publications

Genetically encoded indicators of neuronal activity

Genetically encoded indicators of neuronal activity

Repeat‐associated non‐ AUG translation in C9orf72‐ ALS / FTD is driven by neuronal excitation and stress

A novel myelin protein zero transgenic zebrafish designed for rapid readout of in vivo myelination

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