PUBLICATION
Visualizing long-term single-molecule dynamics in vivo by stochastic protein labeling
- Authors
- Liu, H., Dong, P., Ioannou, M.S., Li, L., Shea, J., Pasolli, H.A., Grimm, J.B., Rivlin, P.K., Lavis, L.D., Koyama, M., Liu, Z.
- ID
- ZDB-PUB-171230-2
- Date
- 2017
- Source
- Proceedings of the National Academy of Sciences of the United States of America 115(2): 343-348 (Journal)
- Registered Authors
- Keywords
- long-term single-molecule imaging, stochastic labeling, synaptic vesicle transport, transcription factor dynamics, translational readthrough
- MeSH Terms
-
- Animals
- Binding Sites
- Cell Line, Tumor
- Cell Tracking/methods*
- Cells, Cultured
- Humans
- Kinetics
- Neurons/cytology
- Neurons/metabolism*
- Synaptic Vesicles/metabolism*
- Time-Lapse Imaging/methods
- Transcription Factors/metabolism*
- Zebrafish
- PubMed
- 29284749 Full text @ Proc. Natl. Acad. Sci. USA
Citation
Liu, H., Dong, P., Ioannou, M.S., Li, L., Shea, J., Pasolli, H.A., Grimm, J.B., Rivlin, P.K., Lavis, L.D., Koyama, M., Liu, Z. (2017) Visualizing long-term single-molecule dynamics in vivo by stochastic protein labeling. Proceedings of the National Academy of Sciences of the United States of America. 115(2):343-348.
Abstract
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 range of ∼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 two 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 subnuclear 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.
Genes / Markers
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Orthology
Engineered Foreign Genes
Mapping