TY - JOUR T1 - Visualizing Long-Term Single-Molecule Dynamics <em>in vivo</em> by Stochastic Protein Labeling JF - bioRxiv DO - 10.1101/116186 SP - 116186 AU - Hui Liu AU - Peng Dong AU - Maria S. Ioannou AU - Li Li AU - Jamien Shea AU - H. Amalia Pasolli AU - Jonathan Grimm AU - Pat Rivlin AU - Luke D. Lavis AU - Minoru Koyama AU - Zhe Liu Y1 - 2017/01/01 UR - http://biorxiv.org/content/early/2017/06/12/116186.abstract N2 - Our ability to unambiguously image and track individual molecules in live cells is limited by packing of multiple copies of labeled molecules within the resolution limit. Here we devise a universal genetic strategy to precisely control copy number of fluorescently labeled molecules in a cell. This system has a dynamic titration range of &gt;10,000 fold, enabling sparse labeling of proteins expressed at different abundance levels. Combined with photostable labels, this system extends the duration of automated single-molecule tracking by 2 orders of magnitude. We demonstrate long-term imaging of synaptic vesicle dynamics in cultured neurons as well as in intact zebrafish. We found axon initial segment utilizes a ‘waterfall’ mechanism gating synaptic vesicle transport polarity by promoting anterograde transport processivity. Long-time observation also reveals that transcription factor hops between clustered binding sites in spatially-restricted sub-nuclear regions, suggesting that topological structures in the nucleus shape local gene activities by a sequestering mechanism. This strategy thus greatly expands the spatiotemporal length scales of live-cell single-molecule measurements, enabling new experiments to quantitatively understand complex control of molecular dynamics in vivo. ER -