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Published on: December 12, 2014
Measuring rotational diffusion of MHC class I on live cells by polarized FPR
David R Fooksman1, Michael Edidin, B George Barisas
1Department of Chemistry, Colorado State University, Ft. Collins, CO 80523, USA. fooksman@saturn.med.nyu.edu
Abstract:
Clustering of membrane proteins is a dynamic process which can regulate cellular function and signaling. The size of receptor and other membrane protein clusters can in principle be measured in terms of their rotational diffusion. However, in practice, measuring rotation of membrane proteins of live cells has been difficult, largely because of the difficulty of rigidly attaching reporter groups to the molecules of interest. Here we show that polarized photobleaching recovery can detect rotation of membrane proteins genetically tagged with yellow fluorescent protein, YFP. MHC class I molecules were engineered with a rigid, in-sequence, YFP tag followed at the C-terminus by a pair of crosslinkable domains. When crosslinker was added we could detect changes in rotational anisotropy decay consistent with clustering of the MHC molecules. This result points the way to use of engineered fluorescent fusion proteins to measure rotational diffusion in native cell membranes.
Insights
Researchers developed a new method to measure membrane protein rotation in live cells using genetically tagged yellow fluorescent protein (YFP). This technique, polarized photobleaching recovery, successfully detected clustering of MHC class I molecules, advancing membrane protein dynamics research.
Area of Science:
- Cellular Biology
- Biophysics
- Membrane Protein Dynamics
Background:
- Membrane protein clustering regulates cellular function and signaling.
- Measuring rotational diffusion of membrane proteins in live cells is challenging due to difficulties in attaching reporter groups.
- Rotational diffusion is a key parameter for understanding the size and dynamics of membrane protein clusters.
Purpose of the Study:
- To develop a method for measuring the rotational diffusion of membrane proteins in live cells.
- To demonstrate the utility of genetically engineered fluorescent fusion proteins for studying membrane protein dynamics.
- To investigate the clustering of MHC class I molecules using a novel rotational diffusion measurement technique.
Main Methods:
- Utilized polarized photobleaching recovery to detect membrane protein rotation.
- Engineered MHC class I molecules with an in-sequence yellow fluorescent protein (YFP) tag.
- Incorporated crosslinkable domains at the C-terminus of engineered MHC molecules to induce clustering.
Main Results:
- Successfully detected rotation of membrane proteins genetically tagged with YFP.
- Observed changes in rotational anisotropy decay upon addition of a crosslinker, indicating molecular clustering.
- Demonstrated that engineered fluorescent fusion proteins can measure rotational diffusion in native cell membranes.
Conclusions:
- Polarized photobleaching recovery is a viable method for measuring membrane protein rotation in live cells.
- Genetically engineered fluorescent fusion proteins offer a robust approach to study membrane protein dynamics and clustering.
- This technique provides new insights into the regulation of cellular function and signaling by membrane protein clusters.
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01:24Protein Diffusion in the Membrane
01:19Protein Dynamics in Living Cells
Fluorescent recovery after photobleaching (FRAP) is a fluorescent-protein-based detection technique used to quantify protein movement rates within the cell. This method exposes a small portion of the cell to an intense laser beam. The laser beam causes permanent photobleaching of the fluorophore-tagged proteins in the exposed region. As the bleached...