Journal ArticleDOI
Multicolor and Electron Microscopic Imaging of Connexin Trafficking
Guido M. Gaietta,Thomas J. Deerinck,Stephen R. Adams,James C. Bouwer,Oded Tour,Dale W. Laird,Gina E. Sosinsky,Roger Y. Tsien,Mark H. Ellisman +8 more
TLDR
This approach was used to show that newly synthesized connexin43 was transported predominantly in 100- to 150-nanometer vesicles to the plasma membrane and incorporated at the periphery of existing gap junctions, whereas older connexins were removed from the center of the plaques into pleiomorphic vesicle of widely varying sizes.Abstract:
Recombinant proteins containing tetracysteine tags can be successively labeled in living cells with different colors of biarsenical fluorophores so that older and younger protein molecules can be sharply distinguished by both fluorescence and electron microscopy. Here we used this approach to show that newly synthesized connexin43 was transported predominantly in 100- to 150-nanometer vesicles to the plasma membrane and incorporated at the periphery of existing gap junctions, whereas older connexins were removed from the center of the plaques into pleiomorphic vesicles of widely varying sizes. Selective imaging by correlated optical and electron microscopy of protein molecules of known ages will clarify fundamental processes of protein trafficking in situ.read more
Citations
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Journal ArticleDOI
Imaging intracellular fluorescent proteins at nanometer resolution.
Eric Betzig,George H. Patterson,Rachid Sougrat,O. Wolf Lindwasser,Scott G. Olenych,Juan S. Bonifacino,Michael W. Davidson,Jennifer Lippincott-Schwartz,Harald F. Hess +8 more
TL;DR: This work introduced a method for optically imaging intracellular proteins at nanometer spatial resolution and used this method to image specific target proteins in thin sections of lysosomes and mitochondria and in fixed whole cells to image retroviral protein Gag at the plasma membrane.
Journal ArticleDOI
The fluorescent toolbox for assessing protein location and function
TL;DR: The focus is on protein detection in live versus fixed cells: determination of protein expression, localization, activity state, and the possibility for combination of fluorescent light microscopy with electron microscopy.
Journal ArticleDOI
Bioorthogonal Chemistry: Fishing for Selectivity in a Sea of Functionality
TL;DR: The bioorthogonal chemical reactions developed to date are described and how they can be used to study biomolecules.
Journal ArticleDOI
Creating new fluorescent probes for cell biology.
TL;DR: Advances include the continued development of 'passive' markers for the measurement of biomolecule expression and localization in live cells, and 'active' indicators for monitoring more complex cellular processes such as small-molecule-messenger dynamics, enzyme activation and protein–protein interactions.
Journal ArticleDOI
A general method for the covalent labeling of fusion proteins with small molecules in vivo
TL;DR: A general method for the covalent labeling of fusion proteins in vivo that complements existing methods for noncovalentlabeling of proteins and that may open up new ways of studying proteins in living cells is described.
References
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Journal ArticleDOI
Evidence for recycling of synaptic vesicle membrane during transmitter release at the frog neuromuscular junction
John E. Heuser,Thomas S. Reese +1 more
TL;DR: During stimulation the intracellular compartments of this synapse change shape and take up extracellular protein in a manner which indicates that synaptic vesicle membrane added to the surface during exocytosis is retrieved by coated vesicles and recycled into new synaptic vESicles by way of intermediate cisternae.
Journal ArticleDOI
Specific Covalent Labeling of Recombinant Protein Molecules Inside Live Cells
TL;DR: This system provides a recipe for slightly modifying a target protein so that it can be singled out from the many other proteins inside live cells and fluorescently stained by small nonfluorescent dye molecules added from outside the cells.
Journal ArticleDOI
Studying protein dynamics in living cells.
TL;DR: Live cell imaging, in combination with photobleaching, energy transfer or fluorescence correlation spectroscopy are providing unprecedented insights into the movement of proteins and their interactions with cellular components.
Journal ArticleDOI
Connexins, connexons, and intercellular communication
TL;DR: Structural-function studies provide a molecular understanding of the significance of connexin diversity and demonstrate the unique regulation of Connexins by tyrosine kinases and oncogenes.
Journal ArticleDOI
"Fluorescent timer": protein that changes color with time.
Alexey V. Terskikh,Arkady F. Fradkov,Galina V. Ermakova,Andrey G. Zaraisky,Patrick Tan,Andrey V. Kajava,Xiaoning Zhao,Sergey Lukyanov,Mikhail V. Matz,Stuart K. Kim,Irving L. Weissman,Paul D. Siebert +11 more
TL;DR: In vivo labeling with E5 is used to measure expression from the heat shock-dependent promoter in Caenorhabditis elegans and from the Otx-2 promoter in developing Xenopus embryos to monitor both activation and down-regulation of target promoters on the whole-organism scale.