2010_Dieterich_Nature Neuroscience

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1、teChnICalrepOrtSInsituvisualizationanddynamicsofnewlysynthesizedproteinsinrathippocampalneuronsDanielaCDieterich1,2,JenniferJLHodas1,GéraldineGouzer3,IlyaYShadrin1,JohnTNgo4,AntoineTriller3,DavidATirrell4&ErinMSchuman1,5Proteintranslationhasbeenimplicatedindifferentformsdendriticprotei

2、nsynthesisisusedtoallowneuronalsynapsestoofsynapticplasticity,butdirectinsituvisualizationofnewresponddynamicallyandspecificallytothestimulationpatternsproteinsislimitedtooneortwoproteinsatatime.Herewethatdrivetheestablishment,maintenanceandplasticityofsynapticdescribeametaboliclabelin

3、gapproachbasedonincorporationconnections3–6.Moreover,severalstudieshavedemonstratedthatofnoncanonicalaminoacidsintoproteinsfollowedbyvariousbiochemicalfractionslackingsomaticproteinsynthesischemoselectivefluorescencetaggingbymeansof‘clickmachineryarenonethelesscapableofsynthesizingprot

4、eins(seechemistry’.Afterabriefincubationwithazidohomoalaninerefs.7,8forreview),andseveralproteomicsstudieshaveidenti-orhomopropargylglycine,arobustfluorescentsignalwasfiedproteinsbelongingtodifferentfunctionalclasses9–11.Despitedetectedinsomataanddendrites.Pulse-chaseapplicationoverwhe

5、lmingsupportforthegeneralnotionofdendriticproteinofazidohomoalanineandhomopropargylglycineallowedtranslationinplasticityandsynapticfunction,localproteintransla-visualizationofproteinssynthesizedintwosequentialtimetionhasbeenmonitoredsofarforonlyafewcandidateproteins.periods.Thistechniq

6、uecanbeusedtodetectchangesVisualizationofnewlysynthesizedproteinsinneuronshaslargelyinproteinsynthesisandtoevaluatethefateofproteinsreliedontheuseofgreenfluorescentprotein(GFP)—eitherasasynthesizedindifferentcellularcompartments.Moreover,fusionwiththeproteinofinterestorwiththecodingseq

7、uenceforusingstrain-promotedcycloaddition,weexploredthedynamicsGFPflankedbythe5′and3′untranslatedregions(toconferregulationofnewlysynthesizedmembraneproteinsusingsingle-particleofmRNAtranslation)oftheproteinofinterest12–14—oraprotease-trackingandquantumdots.Thenewlysynthesizedprotein