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Published on: August 7, 2016
Multicolor and electron microscopic imaging of connexin trafficking
Guido Gaietta1, Thomas J Deerinck, Stephen R Adams
1National Center for Microscopy and Imaging Research, Department of Neurosciences, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA.
Science (New York, N.Y.)
|April 20, 2002
Summary
Newly synthesized connexin43 proteins travel in vesicles to gap junctions, while older proteins are removed. This study visualizes protein age and trafficking in living cells using multicolor fluorescence and electron microscopy.
Area of Science:
- Cell biology
- Biochemistry
- Microscopy
Background:
- Connexin43 forms gap junctions crucial for cell communication.
- Understanding protein trafficking is vital for cellular processes.
Purpose of the Study:
- To visualize the age-dependent trafficking and degradation of connexin43 in living cells.
- To distinguish newly synthesized from older connexin43 molecules using advanced microscopy techniques.
Main Methods:
- Utilized recombinant proteins with tetracysteine tags for sequential labeling with biarsenical fluorophores.
- Employed correlated optical and electron microscopy to track protein molecules of known ages.
- Investigated the transport of connexin43 in vesicles and its incorporation/removal at gap junctions.
Main Results:
- Newly synthesized connexin43 was observed in 100- to 150-nanometer vesicles, moving to the plasma membrane and integrating into existing gap junctions.
- Older connexin43 molecules were removed from the center of gap junction plaques and packaged into pleiomorphic vesicles of various sizes.
- Demonstrated the ability to distinguish protein age and localization using multicolor fluorescence and electron microscopy.
Conclusions:
- The study provides a novel method for visualizing age-specific protein dynamics in situ.
- Revealed distinct trafficking pathways for newly synthesized and older connexin43 proteins.
- Highlights the potential of correlated microscopy for elucidating fundamental protein trafficking and turnover mechanisms.
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