Multi-color Localization Microscopy of Single Membrane Proteins in Organelles of Live Mammalian Cells

DC FieldValueLanguage
dc.contributor.authorAppelhans, Timo
dc.contributor.authorBeinlich, Felix R. M.
dc.contributor.authorRichter, Christian P.
dc.contributor.authorKurre, Rainer
dc.contributor.authorBusch, Karin B.
dc.date.accessioned2021-12-23T16:09:16Z-
dc.date.available2021-12-23T16:09:16Z-
dc.date.issued2018
dc.identifier.issn1940087X
dc.identifier.urihttps://osnascholar.ub.uni-osnabrueck.de/handle/unios/8706-
dc.description.abstractKnowledge about the localization of proteins in cellular subcompartments is crucial to understand their specific function. Here, we present a super-resolution technique that allows for the determination of the microcompartments that are accessible for proteins by generating localization and tracking maps of these proteins. Moreover, by multi-color localization microscopy, the localization and tracking profiles of proteins in different subcompartments are obtained simultaneously. The technique is specific for live cells and is based on the repetitive imaging of single mobile membrane proteins. Proteins of interest are genetically fused with specific, so-called self-labeling tags. These tags are enzymes that react with a substrate in a covalent manner. Conjugated to these substrates are fluorescent dyes. Reaction of the enzyme-tagged proteins with the fluorescence labeled substrates results in labeled proteins. Here, Tetramethylrhodamine (TMR) and Silicon Rhodamine (SiR) are used as fluorescent dyes attached to the substrates of the enzymes. By using substrate concentrations in the pM to nM range, sub-stoichiometric labeling is achieved that results in distinct signals. These signals are localized with similar to 15-27 nm precision. The technique allows for multi-color imaging of single molecules, whereby the number of colors is limited by the available membrane-permeable dyes and the repertoire of selflabeling enzymes. We show the feasibility of the technique by determining the localization of the quality control enzyme (Pten)-induced kinase 1 (PINK1) in different mitochondria! compartments during its processing in relation to other membrane proteins. The test for true physical interactions between differently labeled single proteins by single molecule FRET or co-tracking is restricted, though, because the low labeling degrees decrease the probability for having two adjacent proteins labeled at the same time. While the technique is strong for imaging proteins in membrane compartments, in most cases it is not appropriate to determine the localization of highly mobile soluble proteins.
dc.description.sponsorship[SFB 944]; The authors would like to thank the Biophysics group and Jacob Piehler at the University of Osnabruck for continuous support, Wladislaw Kohl for technical assistance and preparation of material, and the CellNanOs board for providing microscopes for use. The project was funded by the SFB 944.
dc.language.isoen
dc.publisherJOURNAL OF VISUALIZED EXPERIMENTS
dc.relation.ispartofJOVE-JOURNAL OF VISUALIZED EXPERIMENTS
dc.subjectBiology
dc.subjectdual-color super-resolution microscopy
dc.subjectdynamics of membrane proteins
dc.subjectFRAP
dc.subjectIssue 136
dc.subjectlive cell imaging
dc.subjectLIVING CELLS
dc.subjectMITOCHONDRIA
dc.subjectMOLECULE TRACKING
dc.subjectMultidisciplinary Sciences
dc.subjectORGANIZATION
dc.subjectPINK1
dc.subjectPROBES
dc.subjectrhodamine dyes
dc.subjectScience & Technology - Other Topics
dc.subjectSELF-LABELING ENZYMES
dc.subjectsingle molecule localization
dc.subjectSingle molecule tracking
dc.subjectSUPERRESOLUTION MICROSCOPY
dc.titleMulti-color Localization Microscopy of Single Membrane Proteins in Organelles of Live Mammalian Cells
dc.typejournal article
dc.identifier.doi10.3791/57690
dc.identifier.isiISI:000444752100091
dc.description.issue136
dc.contributor.orcid0000-0003-0525-0191
dc.contributor.orcid0000-0002-6411-0909
dc.contributor.researcheridABH-8594-2020
dc.contributor.researcheridAAM-8374-2021
dc.publisher.place1 ALEWIFE CENTER, STE 200, CAMBRIDGE, MA 02140 USA
dcterms.isPartOf.abbreviationJ. Vis. Exp.
dcterms.oaStatusGreen Published
crisitem.author.deptFB 05 - Biologie/Chemie-
crisitem.author.deptidfb05-
crisitem.author.orcid0000-0002-6872-6567-
crisitem.author.parentorgUniversität Osnabrück-
crisitem.author.netidKuRa617-
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