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Super-resolution Imaging of the Natural Killer Cell Immunological Synapse on a Glass-supported Planar Lipid Bilayer
Published on: February 11, 2015
Super-resolution imaging of the natural killer cell immunological synapse on a glass-supported planar lipid bilayer
Peilin Zheng1, Grant Bertolet2, Yuhui Chen1
1Center for Human Immunobiology, Texas Children's Hospital; Department of Pediatrics, Baylor College of Medicine.
Insights
This study details a protocol for creating supported lipid bilayers to image natural killer (NK) cell synapses using super-resolution microscopy. The technique visualizes key structures like perforin and actin within the NK cell immunological synapse.
Area of Science:
- Cell Biology
- Immunology
- Biophysics
Background:
- Glass-supported planar lipid bilayers mimic cell surfaces for studying cell interactions.
- Super-resolution microscopy enhances visualization of cellular structures.
- Natural killer (NK) cell synapses are crucial for immune responses.
Purpose of the Study:
- To provide a protocol for constructing supported lipid bilayers for NK cell synapse studies.
- To utilize stimulated emission depletion (STED) super-resolution microscopy to analyze NK cell synapses.
- To investigate the distribution of perforin and filamentous actin at NK cell synapses.
Main Methods:
- Preparation of synthetic phospholipids for bilayer synthesis.
- Quantification of protein density on bilayer attachment sites.
- Construction of lipid bilayers with anti-CD16 antibodies.
- Imaging human NK cells using STED microscopy.
Main Results:
- Successful construction of functional supported lipid bilayers.
- Demonstration of STED microscopy's utility in visualizing NK cell synapses.
- Detailed visualization of perforin and filamentous actin distribution at the synapse.
Conclusions:
- The combined supported lipid bilayer and STED microscopy system is feasible for studying NK cell synapses.
- This approach provides high-resolution insights into the molecular organization of the NK cell immunological synapse.
Abstract:
The glass-supported planar lipid bilayer system has been utilized in a variety of disciplines. One of the most useful applications of this technique has been in the study of immunological synapse formation, due to the ability of the glass-supported planar lipid bilayers to mimic the surface of a target cell while forming a horizontal interface. The recent advances in super-resolution imaging have further allowed scientists to better view the fine details of synapse structure. In this study, one of these advanced techniques, stimulated emission depletion (STED), is utilized to study the structure of natural killer (NK) cell synapses on the supported lipid bilayer. Provided herein is an easy-to-follow protocol detailing: how to prepare raw synthetic phospholipids for use in synthesizing glass-supported bilayers; how to determine how densely protein of a given concentration occupies the bilayer's attachment sites; how to construct a supported lipid bilayer containing antibodies against NK cell activating receptor CD16; and finally, how to image human NK cells on this bilayer using STED super-resolution microscopy, with a focus on distribution of perforin positive lytic granules and filamentous actin at NK synapses. Thus, combining the glass-supported planar lipid bilayer system with STED technique, we demonstrate the feasibility and application of this combined technique, as well as intracellular structures at NK immunological synapse with super-resolution.
