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

Autor(en): Appelhans, Timo
Beinlich, Felix R. M.
Richter, Christian P.
Kurre, Rainer 
Busch, Karin B.
Stichwörter: Biology; dual-color super-resolution microscopy; dynamics of membrane proteins; FRAP; Issue 136; live cell imaging; LIVING CELLS; MITOCHONDRIA; MOLECULE TRACKING; Multidisciplinary Sciences; ORGANIZATION; PINK1; PROBES; rhodamine dyes; Science & Technology - Other Topics; SELF-LABELING ENZYMES; single molecule localization; Single molecule tracking; SUPERRESOLUTION MICROSCOPY
Erscheinungsdatum: 2018
Herausgeber: JOURNAL OF VISUALIZED EXPERIMENTS
Journal: JOVE-JOURNAL OF VISUALIZED EXPERIMENTS
Ausgabe: 136
Zusammenfassung: 
Knowledge 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.
ISSN: 1940087X
DOI: 10.3791/57690

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