Self-labelling enzymes as universal tags for fluorescence microscopy, super-resolution microscopy and electron microscopy

DC FieldValueLanguage
dc.contributor.authorLiss, Viktoria
dc.contributor.authorBarlag, Britta
dc.contributor.authorNietschke, Monika
dc.contributor.authorHensel, Michael
dc.date.accessioned2021-12-23T16:03:12Z-
dc.date.available2021-12-23T16:03:12Z-
dc.date.issued2015
dc.identifier.issn20452322
dc.identifier.urihttps://osnascholar.ub.uni-osnabrueck.de/handle/unios/5862-
dc.description.abstractResearch in cell biology demands advanced microscopy techniques such as confocal fluorescence microscopy (FM), super-resolution microscopy (SRM) and transmission electron microscopy (TEM). Correlative light and electron microscopy (CLEM) is an approach to combine data on the dynamics of proteins or protein complexes in living cells with the ultrastructural details in the low nanometre scale. To correlate both data sets, markers functional in FM, SRM and TEM are required. Genetically encoded markers such as fluorescent proteins or self-labelling enzyme tags allow observations in living cells. Various genetically encoded tags are available for FM and SRM, but only few tags are suitable for CLEM. Here, we describe the red fluorescent dye tetramethylrhodamine (TMR) as a multimodal marker for CLEM. TMR is used as fluorochrome coupled to ligands of genetically encoded self-labelling enzyme tags HaloTag, SNAP-tag and CLIP-tag in FM and SRM. We demonstrate that TMR can additionally photooxidize diaminobenzidine (DAB) to an osmiophilic polymer visible on TEM sections, thus being a marker suitable for FM, SRM and TEM. We evaluated various organelle markers with enzymatic tags in mammalian cells labelled with TMR-coupled ligands and demonstrate the use as efficient and versatile DAB photooxidizer for CLEM approaches.
dc.description.sponsorshipMinistry for Science and Culture of Lower Saxony; Deutsche ForschungsgemeinschaftGerman Research Foundation (DFG) [P4, SFB 944]; This work was supported by the Ministry for Science and Culture of Lower Saxony and the Deutsche Forschungsgemeinschaft through grants P4 and Z in SFB 944. The plasmids pPalmitoyl-mTurquoise2 and pmTurqouise2-Golgi were a gift from Dorus Gadella (Addgene plasmids #36209, #36205). The stably transfected cell line HeLa LifeAct-HaloTag-meGFP was kindly provided by Jacob Piehler. Plasmids encoding Mito-HaloTag or ER-FRP-HaloTag were kindly provided by Karin Busch and Joost Holthuis, respectively. We thank Christian P. Richter for providing the MatLab-based Software SLimFast.
dc.language.isoen
dc.publisherNATURE PUBLISHING GROUP
dc.relation.ispartofSCIENTIFIC REPORTS
dc.subjectCORRELATED LIGHT
dc.subjectDIAMINOBENZIDINE
dc.subjectFUSION PROTEINS
dc.subjectHORSERADISH-PEROXIDASE
dc.subjectIDENTIFICATION
dc.subjectLIVING CELLS
dc.subjectMARKERS
dc.subjectMultidisciplinary Sciences
dc.subjectPHOTOCONVERSION
dc.subjectPHOTOOXIDATION
dc.subjectREPORTER GENE
dc.subjectScience & Technology - Other Topics
dc.titleSelf-labelling enzymes as universal tags for fluorescence microscopy, super-resolution microscopy and electron microscopy
dc.typejournal article
dc.identifier.doi10.1038/srep17740
dc.identifier.isiISI:000365959800001
dc.description.volume5
dc.contributor.orcid0000-0002-1304-2512
dc.contributor.orcid0000-0001-6604-6253
dc.publisher.placeMACMILLAN BUILDING, 4 CRINAN ST, LONDON N1 9XW, ENGLAND
dcterms.isPartOf.abbreviationSci Rep
dcterms.oaStatusgold, Green Published
crisitem.author.deptFB 05 - Biologie/Chemie-
crisitem.author.deptidfb05-
crisitem.author.orcid0000-0001-6604-6253-
crisitem.author.parentorgUniversität Osnabrück-
crisitem.author.netidHeMi480-
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