Dissecting the molecular dynamics of cell surface receptors in immune cells using state-of-the-art fluorescence-based single molecule and fluctuation techniques

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Experimental Biomolecular physics

KTH (Royal Institute of Technology)

Applied Physics, KTH

AlbaNova University Center

Karolinska Institute

Hokkaido University

Olympus company

Project Description

The investigations to be performed within the project will largely be based on Fluorescence Correlation Spectroscopy (FCS) and related techniques. FCS is a technique based on the analysis of intensity fluctuations of fluorescent molecules excited by a focused laser beam. The technique offers information about molecular dynamic processes at the single molecular level, that takes place in the nanosecond time range and longer and manifests itself as a change in fluorescence intensity. This project implements a broad combined expertise in experimental and theoretical physics, fluorescence spectroscopy, cellular and molecular biology, biochemistry and molecular medicine. The objective is to explore and develop FCS and related techniques for studies of molecular dynamics at immune cell surfaces. A particular focus will be studies of natural killer (NK) lymphocytes, critical mediators of anti-viral immunity and protectors against cancer spread. Our studies will provide new and essential knowledge pertinent to molecular dynamics in NK cells and other immune cells, at a level of resolution that is currently unexplored. However, the experience gained from these investigations will also be used to advance the use of FCS and variants thereof for studies of molecular interactions in cell membranes in general.

The specific aims of this project are:

1. To explore and further develop FCS and related techniques as tools to study molecular interactions at immune cell surfaces, using proteins with known interaction partners, such as tetraspanins and protein linked to glycosylphosphatidylinositol (GPI) as model systems.
2. To quantitatively determine the absolute numbers and relative mobilities of MHC class I, Ly49 and KIR molecules present in NK cell membranes and to investigate how these parameters change when NK cells undergo immune synapse formation with other cells.

If successful, this project will significantly develop the art and procedures involved in the FCS-based characterization of cell surface molecules, of benefit for molecular dynamic and interaction studies at cell surfaces in general. In addition, it will provide novel and critical information pertinent to molecular dynamics of NK cells receptors that has proven difficult to extract by other experimental means.