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 The advent of optogenetics provided researchers Alerter l'administrateur Recommander à un ami Lien de l'article 

The advent of optogenetics provided researchers with powerful tools that manipulate living systems with exquisite spatiotemporal precision using non-invasive visible light. The plethora of optical reporters to probe changes in cellular structure, second messenger levels, post-translational modifications and even conformation of individual proteins was until recently matched by few optical actuators, mostly chemically caged compounds released by ultraviolet light. Synthetic biology has now provided numerous designer channels and proteins that can be activated in different cells and distinct subcellular regions for precisely defined periods or using temporally patterned inputs. Nevertheless, the design of an optogenetic regulator for a new target is often a strenuous multidisciplinary task guided by combinations of crystallography, directed evolution, library screening, molecular modelling and/or mutational interweaving strategies6,7,8,9,10. It therefore remains challenging for a typical research lab to implement new optogenetic designs for new proteins and pathways.

Here we develop and evaluate a simple protocol for developing optogenetic regulators of new targets, specifically MAP kinases JNK and p38, for which the currently available tools are limited in spatiotemporal precision. We demonstrate that a LOV2-based regulator of JNK, OptoJNKi, can be applied as an optopharmacological tool without the need for gene transfer, and provide detailed protocols to successfully produce and transfer recombinant OptoJNKi into neurons. To target MAP kinases, we made use of known D-domain interactions55,56. We found AsLOV2Jα sterically constrained JNK inhibitor peptides, showing considerable dark/lit-state JNK-binding ratios for the optimal peptide (~30-fold, Fig. 1f,g). This was surprising considering reported difficulties achieving steric hindrance-based light regulation of either proteins or short peptides6,8,9. Molecular dynamic simulation suggested that phenylalanine at or near the C terminus could be ‘caged’ by a novel non-polar LOV2 domain pocket formed between residues of the Iβ-sheet and the Aβ/Bβ loop (Fig. 9). This distal pocket might cooperate with the dark-state α-helical form of the Jα sequence to constrain and thereby generate steric hindrance of fused inhibitor peptide that can be released by light, leading to LED Tube China regulation of JNK that we observed. In support of this, a point mutation in this pocket releases the JIP sequence (without disrupting protein stability as judged by soluble expression level) so that dark-state binding to JNK became indistinguishable from lit-state binding (Fig. 9d).

These structural considerations suggested a simple protocol

  Aucun commentaire | Ecrire un nouveau commentaire Posté le 06-06-2017 à 08h28

 GAL4-cJun luciferase reporter assays Alerter l'administrateur Recommander à un ami Lien de l'article 

GAL4-cJun luciferase reporter assays in cerebellar granule neurons
Cerebellar granule neurons were transfected with a firefly luciferase reporter plasmid driven by five GAL4 elements in tandem, pGL3-G5E4Δ38 (12.5% of total DNA), pcDNA3-GAL4cJun plasmid encoding the transactivation domain of c-Jun (residues 5–105) fused to the DNA-binding domain of GAL4 (residues 1–147; 12.5% of total DNA), and pRL-CMV (12.5% of total DNA) expressing sea pansy luciferase as an internal standard against which signals were normalized24. CMV was selected for normalization because pcDNA3-GAL4cJun is also driven by CMV promoter. To activate the JNK pathway, either pEBG-ΔMEKK1 (10% of total DNA), a plasmid encoding MAPK-kinase kinase 1 (MEKK1) kinase domain (amino acids 1,174–1,493) was co-transfected, or cells were subjected to 4?h of WTS. Differentially targeted mCherry-OptoJNKi constructs or empty vector (mCherry-C1) were co-transfected as indicated. For WTS experiments, all OptoJNKi constructs were transfected at 40% of total DNA. For the specific case of ΔMEKK1-co-expression experiments, mCherry-3xNLS-OptoJNKi was transfected at 40% of total DNA but pilot titration evaluations indicated light-independent artefacts at high levels of the NES-targeted form; by transfecting at only 2% of total DNA, we avoided effects in dark conditions, as can be seen in the figures. We did not find a suitable percentage level for use of H2B-OptoJNKi in ΔMEKK1-transfected cells, suggesting a possible unexpected interaction between these two constructs, so we were unable to use H2B-OptoJNKi in this specific JNK-activation model, even though there was no evidence of dark effects of H2B-OptoJNKi in the WTS model. This emphasizes the need for careful titration of constructs and inclusions of appropriate controls in every model. But this is not unique to OptoJNKi as the risks of overexpression are well known, potentially affecting use of ΔMEKK1 and most other constructs. It should be noted that, in cerebellar neuron cultures as used here, the 10% level of ΔMEKK1 plasmid used had little impact on overall expression as judged by Renilla luciferase internal control signal, but it substantially increased the GAL4-cJun-driven firefly luciferase response. For the ΔMEKK1 plates, 2?h after transfection, wells were individually illuminated as indicated. Illumination was carried out using blue LEDs (peak 465–467?nm) with 500?Hz pulsed-width modulation to achieve 0.6?mW?cm?2 incident power on the well bottom (duty cycle modulation was set according to measured LED Tube China at 100% duty cycle using an Ex-Cite XR2100 power meter from Lumen Dynamics/LDGI, Mississauga, Ontario, Canada). The illumination was delivered in pulses of 1.5?s length or as indicated. Pulses were repeated with periodicity and for time windows as indicated in the figures. For ΔMEKK1 experiments, dual luciferase reporter assay was carried out on samples lysed 18–20?h after transfection. For the WTS plates, conditioned cell culture medium was replaced by minimal essential medium (cat# 11700) 20?h after transfection and wells were maintained in a cell culture incubator (Cytomat 2C, Thermo) for a further 4?h before lysis, under the illumination conditions described. Dual luciferase reagent (Promega) was used to assay firefly (reporter) and Renilla (internal standard) luciferase activities in samples lysed in passive-lysis buffer (Promega) according to the manufacturer’s instructions24.

  Aucun commentaire | Ecrire un nouveau commentaire Posté le 06-06-2017 à 08h37

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