NF-2dB Transpert in Neurons RHD p65 p65 wt EEKRKRTY -IC74 NLSmut EETR TTY Dynami
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NF-2dB Transpert in Neurons RHD p65 p65 wt EEKRKRTY -IC74 NLSmut EETR TTY Dynami Figure 2. The functional NLS) of p65 is essen al for it nteraction with dy /dynactin in vitro. (A Nur nca lization gna of wild-typs NF-xB p65 and NLS mutant NILSmut he NLS to NF- p65 consists of a stretch of a basic amno acids O alization gnal es ard lyri res. The three pnine re introduced irto the NIS (depicted in yellc wh. The carstnict 12as tanged with polyhistidine mutatirn ays. (B) Polyhisticire-tagged wild-type p65 ard p65 with mutant NLS NLSmut) purified enable purifi ation from bacteri al extracts and pulHGawn a from barterial extracts were immabilized separately cn nirkel-caaten matrix and incubate with the rat brain extact.The bound was elinten from the matrices, factionated by SDS-PAGE ard examined by Western blat with anti-C71 and anti oymactin p50 artibodies. As expected, wilc-typ with dyrei /dynactin in vitro. In contrast, p65 with m utart NLS failed to form this complex p65 at Jciate doi: 10.137 nurral.pone.0000589.g002. ubcellular redistribution of NF-NB p65 as response to a gure 3C), Fireny Luciferase has a half-life of only 3 utamate stimulus, Unstimulated neurons were shown to have igh levels of mammalian cells, makin muitable to monitor pressio activated nuclear NF-CP to basal marries over long period of ime vith out limitation, synap to hich at least partially can be suppres by Pharmacological the accumulation of residual luciferase. Beca use of this, we could nhibitors of ction potential geameration, cetect rapid increasc cironal gene expression utanmate antagonists d type G anne blockers Fot th reason we treated even weeks after reporter gene transfection. Lucierase a 33ayg Were neurons with activity antagonists APV, CNQX and nimodipine performed using Dual Luciferase Assay System (P romsga. 24 h prior to glutamate stimulation. Interestingly, cultures with Glutamate application increased NF-xB-dependent activity mearly nhibitor pre naubation had potenti NE-KB artivation after 4-fold compared to untreated contro Co transfection of the gh tamate stim e interpreted his effort ncT cased plastmid significanty decreased the activit ation expre synaptic strength in compensation for lost synaptic activit ty. In with inhibitors s, preincubatio above was sed to bloc basal NF- P activi ty and to providc stable Microtubule-perturbing drugs inhibit neuronal baseline and maximal response. Additionally, Leptomycin B, an transport of NF-KB tor of nuclear expor was used dr the stinnulation to trap Mierotubul are components of the cytoskeleton an are serving t reached the n aclaus, thus mitting small amounts to be tuctural element for the directed movement of the datinc more readily detected. To inhi ibit newly synthesized IKP to Structurally, microtubule ytoplaam, neurons were exposed t motor protein transport NE-NB back to th polymers of co- and A-huhulin dim Wr nsed microtabile. 30 LLM sornycin for lh before and during experimen really disrupting drugs vincristine and colchicine in order to suppress the solated and dissociated hippocampal nauron. dynein-dependent transport to study redistri butio of NF-KB pl 65 ith dynamitin or mo vsator and after plating left to mature foe from distal nuronal nucl ghtamate 8 dat processes to ys in culture hid culturing tine waa be sufficie stimulation. Fig 4A hovia a microtubule network visualized by develop mature processes high nd level of synaptic hibulim immunostain Treatment of hippocampal nerron connectivity. At 8 days culture, mature neurons were subiected th 20 nM colchicine or 10 nM vinrigtine Trsulted in efficient o a 5 min pulse vith .300 glutamate. Cell were then washed depolymerisation, as shown by the diffuse tubulin staining and the and placed back in an incubator. To determine the optimal assay loss of the well-organi ized microhibule pattern cen in untreated o achieve maximal NF-POB nuclear accumulat run Iron cells. To detemine whether dy nein is involved neuronal NF-KB bated up to 3 The maximu response was transport, we stimulated neurons with utamate (as described achieved at 20 e observations are in general agreeme above) and then exami the sibcchular of NF-KB with previous studies After thi ncubation time neurons were immunostained for p55 iFigure 3A. As expected, in u nstimulated p55 by immunostaining (Figure 4B) 90 min after glutamate stimulation, 80% of the cells displayed significant redistibution nen manal chtmmes p65 was mainly local vithin th cytoplasm of p65 from nerona processes and cell body to the micex. In ncluding prouimal and distal neuronal procesges. In contrast glutamate treatment result in redistribution of NF-KB of culture microtubule disrupting contra Pre treatme drugs resulted in the suppression of the p65 moveme We next distal processes to the nucleis. Gon th the hypothesis of istent antific the average nudear/dendri ratio of p65 inm dy ein-dependent NF transport, overe ion of dynar XPI nearly completely blocked th relocatio Figure 3B shows tha intensity Figu In orde. to confirm data there was a signuicant reduction of nuclear/dendrit is ratio of NE. obtained by microscopy, the subcellular localization of p65 wai munafhorescence intensity dymami d by fractionation and W blotting (Figure 4D and glutamate-stimulated cultures compared with Inock tran 250 of ble protein from ytopla extract fected, gutamatestiraulated neurons, For fuructional atalysis, vie imuruunoblotted for p65 protein. with the evel In agreement used a T porter genc assay to finther cvahiatr the cffect of dymcim above experime treat inhibition on NF rcBe transport by dynamitin overexpresio result ed n reduced relocation of p55 from ytopla to the PLOS ONE. July 2007 Issue 7 589 Rww.plosnne.orgExplanation / Answer
It is said that NFkB has nuclear localisation signal and it is transported to nucleus by binding of p65 to dynenin/dynactin assembly directs to nucleus transport. If dynamitin is expressed more then the transport to nucleus is inhibited. So for the transport of NFkB in to the nucleus , it is detected using luciferase assay. In this assay the luciferase reporter gene is combined with the neuronal expression gene. When the neuronal gene is expressed then the intensity of the reportor gene will be increased and decreased if the neuronal gene not expressed. So from the experiment it is said that unstimulated neurons have NFkB located in the cytosol. Glutamate stimulation localises NFkB to nucleus. When dynamitin is over expressed the translocation of NFkB is impaired even in the presence of glutamate. In p65 fluroscence you can see the glutamate stimulated neurons have increased fluroscence compared to normal neuron . Similarly the intensity of lumiscence produced in luciferase assay (as luciferase enzyme is responsible for fluroscent colouring during the reaction) is directly proportion to the NFkB transcription. So NFkB is transcribed more in case of glutamate stimulation an decreased expression in neurons with glutamate and over expression of dynamitin.
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