THE NUCLEUS & NUCLEOCYTOPLASMIC TRANSPORT1. Introductiona. The nucleusb. The nuclear envelopec. Transport cargoes2. The solid phase: the nuclear pore complex (NPC)3. Signalsa. Nuclear localization signals (NLSs)b. Nuclear export signals (NESs)4. The soluble phase: nuclear transport factorsa. Importins and exportinsb. The GTPase Ran5. Mechanism of transport through the NPC6. Segregation of nuclear components during cell divisiona. The Nuclear Envelope1. IntroductionmRNAssnRNAssnRNPstRNAssnRNPssnRNAsProteinsProteinsIonsSmall molecules(<60kD)Karyophilic proteins(NLS)RibosomalProteinsRibosomalSubunitsrRNAs5SrRNAsNuc le olusb. Transport Cargoes2. The solid phase: the nuclear pore complex (NPC)Cytoplasmic sideNuclear sideThe nuclear pore has a 9nm wide aqueous diffusion channel3. Signals: how were they identified?IMPORTINEXPORTINIMPORT EXPORTNUCLEUSCYTOPLASMNUCLEUSCYTOPLASMEXPORTINIMPORTINIMPORTCARGOIMPORTCARGOEXPORTCARGOEXPORTCARGO4. The soluble phase a. Importins and ExportinsRan-GTPRan-GTPH2OPiGTPGDPRanGAP 1RanGEF:RCC1CYTOPLASMRan-GDPRan-GDPNUCLEUS4. The soluble phase b. The GTPase RanIMPORTINEXPORTINIMPORT EXPORTRanGTP-binding and import cargo releaseRanGTP-hydrolysis and export cargo releaseNUCLEUSCYTOPLASMNUCLEUSCYTOPLASMEXPORTINGTPGTPGDPGDPIMPORTINIMPORTCARGOIMPORTCARGOEXPORTCARGOEXPORTCARGO Ran regulates substrate binding and release4. The soluble phase5. A model for translocation through the NPCExample: Reversible nuclear accumulation of NF-ATCompartmentalisation allows the regulation of gene expressionSUMMARYI. Signals target proteins into and out of the nucleusa. active transportb. signals are necessary and sufficientII. Transport occurs through the nuclear pore complex (NPC), a large multi-protein complexa. NPC has aqueous channelb. no unfolding of cargoes is requiredc. transport is bi-directionalIII. Signals are recognized by soluble receptors:Importins and exportinsa. bind specific classes of cargob. shuttle between cytoplasm and nucleusc. interact with nucleoporinsIV. Ran-GTP defines the nucleoplasm and the perichromatin spacea. Ran-GTP is asymmetrically distributedb. Ran regulates cargo binding and releaseVisualization of Transcription in Cells or Tissue Sections•3H-Uridine Incorporation•Br-UTP Incorporation•Fluorescence In Situ HybridizationVisualizing Gene Expression in Living CellsJanicki et al. (2004) Cell 116, 683-6982.5 hrs. post-transfection (-) DoxCFP-LacRepressorMS2 BindingProtein-YFPMergeJanicki et al. (2004) Cell 116, 683-698.CFP-Lac RepressorCFP-SKLMS2 BindingProtein-YFPMerge2.5 hrs. post-transfection + 2.5 hrs. after theaddition of DoxJanicki et al. (2004) Cell 116, 683-698.CFP-lac repressorCFP-SKLYFP-RNA pol II MergeThe RNA polymerase II large subunit isrecruited to the active locusJanicki et al. (2004) Cell 116, 683-698.Fluorescence Recovery after photobleaching (FRAP)FRAP analysis of a pre-mRNA splicing factorin the cell nucleusSF2/ASF has a half-time of fluorescence recovery of approximately1.8 (+/- 0.6) seconds.Bubulya et al. (2004) J. Cell Biol. 167, 51-63.SUMMARYI. Single Cell Imaging of Gene RegulationII. Development of in vivo fluorescent tagsIII. New ways to amplify gene loci and detecttranscriptsIV. Clever ways of using RNA binding proteins to localize RNA products V. Using laser to bleach and measure recovery of proteins to a locusVI. Fluorescence in situ hybridization (FISH) and multi-copy gene arrays to localize specific
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