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Method to Visualize and Analyze Membrane Interacting Proteins by Transmission Electron Microscopy
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Low copy numbers of DC-SIGN in cell membrane microdomains: implications for structure and function
Ping Liu1, Xiang Wang, Michelle S Itano
1Department of Cell Biology and Physiology, University of North Carolina at Chapel Hill, Chapel Hill, NC, 27599-7090, USA.
Traffic (Copenhagen, Denmark)
|December 10, 2013
Summary
This study quantifies C-type lectin (DC-SIGN) molecules in cell membrane microdomains using TIRFM imaging. Small assemblies of 4-8 DC-SIGN molecules are sufficient for dengue virus uptake and infection.
Area of Science:
- Cell Biology
- Immunology
- Microscopy
Background:
- Estimating molecule numbers in membrane domains is challenging.
- DC-SIGN (C-type lectin) plays a role in cellular interactions and pathogen recognition.
Purpose of the Study:
- To quantify the number of DC-SIGN molecules within membrane microdomains.
- To investigate the functional significance of these microdomain assemblies in pathogen internalization.
Main Methods:
- Total Internal Reflection Fluorescence Microscopy (TIRFM) for imaging.
- Quantification by comparing microdomain fluorescence intensity to single fluorophores.
- Labeling of DC-SIGN using monoclonal antibodies or GFP fusion proteins in dendritic cells and NIH3T3 cells.
Main Results:
- Microdomain dimensions vary from the diffraction limit to over 1 µm.
- Occupancy of DC-SIGN molecules per microdomain ranges from a few to over 20.
- Small microdomains (diffraction limit) contain 4-8 DC-SIGN molecules, sufficient for dengue virus binding and internalization.
Conclusions:
- Small molecular assemblies of DC-SIGN in membrane microdomains are functionally relevant.
- These microdomains facilitate the internalization of pathogens like dengue virus, leading to host cell infection.
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