Imaging-Based Screening Platform Assists Protein Engineering

Fabritius, Arne; Ng, David Y. W.; Kist, Andreas M.; Erdoğan, Mutlu; Portugues, Rubén; Griesbeck, Oliver

doi:10.1016/j.chembiol.2018.08.008

Cited by 26 publications

(24 citation statements)

References 39 publications

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“…Protein over-expression and purification was performed according to the protocol presented in (Fabritius et al, 2018). In brief, His-tagged proteins were over-expressed in E.coli BL21 (Invitrogen) in 50 mL cultures of auto inductive LB.…”

Section: Recombinant Expression and Protein Purificationmentioning

confidence: 99%

Phosphodiesterase 1 Bridges Glutamate Inputs with NO- and Dopamine-Induced Cyclic Nucleotide Signals in the Striatum

Betolngar¹,

Mota²,

Fabritius

et al. 2019

Cerebral Cortex

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The calcium-regulated phosphodiesterase 1 (PDE1) family is highly expressed in the brain, but its functional role in neurones is poorly understood. Using the selective PDE1 inhibitor Lu AF64196 and biosensors for cyclic nucleotides including a novel biosensor for cGMP, we analyzed the effect of PDE1 on cAMP and cGMP in individual neurones in brain slices from male newborn mice. Release of caged NMDA triggered a transient increase of intracellular calcium, which was associated with a decrease in cAMP and cGMP in medium spiny neurones in the striatum. Lu AF64196 alone did not increase neuronal cyclic nucleotide levels, but blocked the NMDA-induced reduction in cyclic nucleotides indicating that this was mediated by calcium-activated PDE1. Similar effects were observed in the prefrontal cortex and the hippocampus. Upon corelease of dopamine and NMDA, PDE1 was shown to down-regulate the D1-receptor mediated increase in cAMP. PDE1 inhibition increased long-term potentiation in rat ventral striatum, showing that PDE1 is implicated in the regulation of synaptic plasticity. Overall, our results show that PDE1 reduces cyclic nucleotide signaling in the context of glutamate and dopamine coincidence. This effect could have a therapeutic value for treating brain disorders related to dysfunctions in dopamine neuromodulation.

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Section: Recombinant Expression and Protein Purificationmentioning

confidence: 99%

Phosphodiesterase 1 Bridges Glutamate Inputs with NO- and Dopamine-Induced Cyclic Nucleotide Signals in the Striatum

Betolngar¹,

Mota²,

Fabritius

et al. 2019

Cerebral Cortex

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“…The current study focused on screening of Ca2+-sensitive proteins in E. coli colonies, which were previously shown as a robust platform for directed evolution purposes (Fabritius et al., 2018). Nevertheless, additional applications can be envisioned, such as screening proteins for their dopamine responses (Patriarchi et al., 2018, Sun et al., 2018), using a different cell substrate, optimizing OA signal of chromoproteins, or study the effects of nanobioconjugates influencing Ca2+ uptake using OA (Lyu et al., 2016).…”

Section: Discussionmentioning

confidence: 99%

“…In our previously published work we have shown the ability to automatically pick bacterial colonies from the dish based on their fluorescent properties (Fabritius et al., 2018). This capacity has not yet been implemented in the newly developed OA screening platform.…”

Section: Discussionmentioning

confidence: 99%

High-Throughput Platform for Optoacoustic Probing of Genetically Encoded Calcium Ion Indicators

et al. 2019

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SummaryFunctional optoacoustic (OA) imaging assisted with genetically encoded calcium ion indicators (GECIs) holds promise for imaging large-scale neuronal activity at depths and spatiotemporal resolutions not attainable with existing optical microscopic techniques. However, currently available GECIs optimized for fluorescence (FL) imaging lack sufficient contrast for OA imaging and respond at wavelengths having limited penetration into the mammalian brain. Here we present an imaging platform capable of rapid assessment and cross-validation between OA and FL responses of sensor proteins expressed in Escherichia coli colonies. The screening system features optimized pulsed light excitation combined with ultrasensitive ultrasound detection to mitigate photobleaching while further allowing the dynamic characterization of calcium ion responses with millisecond precision. Targeted probing of up to six individual colonies per second in both calcium-loaded and calcium-unloaded states was possible with the system. The new platform greatly facilitates optimization of absorption-based labels, thus setting the stage for directed evolution of OA GECIs.

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“…Other interesting reports include the development of mCarmine, a far-red FP with a 4-5-fold improved brightness over its ancestor mNeptune684 [16 ], and the improvement of FusionRed resulting in FusionRed-M, a variant with a twofold increased brightness [17 ]. Both optimization attempts introduced elements to automate the screening and selection procedure.…”

Section: Choosing and Creating The Best Fpsmentioning

confidence: 99%

Optimizing the fluorescent protein toolbox and its use

Duwé

Dedecker

2019

Current Opinion in Biotechnology

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Fluorescent proteins (FPs) and fluorescent protein-derived biosensors are indispensable tools in life sciences. They make it possible to probe the location, activity, or interaction of molecules of interest from a subcellular to a multicellular scale. The desire for high-resolution and multidimensional information imposes continuously increasing demands on the performance of the employed probes leading to a continued need for optimization and integration of additional features, such as phototransformable behavior. This review highlights the latest advances in FP engineering, which increase throughput and tailor the improvements directly to the envisioned experiments. Additionally, we discuss recent alternative approaches to introduce or alter phototransformable behavior and describe selected applications of phototransformable behavior in biosensors.

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Imaging-Based Screening Platform Assists Protein Engineering

Cited by 26 publications

References 39 publications

Phosphodiesterase 1 Bridges Glutamate Inputs with NO- and Dopamine-Induced Cyclic Nucleotide Signals in the Striatum

Phosphodiesterase 1 Bridges Glutamate Inputs with NO- and Dopamine-Induced Cyclic Nucleotide Signals in the Striatum

High-Throughput Platform for Optoacoustic Probing of Genetically Encoded Calcium Ion Indicators

Optimizing the fluorescent protein toolbox and its use

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