Journal Article

Direct observation of mobility state transitions in RNA trajectories by sensitive single molecule feedback tracking

Jan-Hendrik Spille, Tim P. Kaminski, Katharina Scherer, Jennifer S. Rinne, Alexander Heckel and Ulrich Kubitscheck

in Nucleic Acids Research

Volume 43, issue 2, pages e14-e14
Published in print January 2015 | ISSN: 0305-1048
Published online November 2014 | e-ISSN: 1362-4962 | DOI: https://dx.doi.org/10.1093/nar/gku1194

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  • Molecular and Cell Biology
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Observation and tracking of fluorescently labeled molecules and particles in living cells reveals detailed information about intracellular processes on the molecular level. Whereas light microscopic particle observation is usually limited to two-dimensional projections of short trajectory segments, we report here image-based real-time three-dimensional single particle tracking in an active feedback loop with single molecule sensitivity. We tracked particles carrying only 1–3 fluorophores deep inside living tissue with high spatio-temporal resolution. Using this approach, we succeeded to acquire trajectories containing several hundred localizations. We present statistical methods to find significant deviations from random Brownian motion in such trajectories. The analysis allowed us to directly observe transitions in the mobility of ribosomal (r)RNA and Balbiani ring (BR) messenger (m)RNA particles in living Chironomus tentans salivary gland cell nuclei. We found that BR mRNA particles displayed phases of reduced mobility, while rRNA particles showed distinct binding events in and near nucleoli.

Journal Article.  8663 words.  Illustrated.

Subjects: Molecular and Cell Biology ; Research Methods in Life Sciences

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