Using SPRINT to study GLUT4 trafficking and dispersal 

My name is Angéline Geiser. I am a PhD student at the University of Strathclyde focusing on the study of GLUT4, the primary glucose transporter found in adipose and muscle tissues. Unlike other glucose transporters, GLUT4 has specialised insulin- and/or exercise-dependent regulatory mechanisms, which involve GLUT4 trafficking between intracellular storage compartments and the plasma membrane (Bryant et al., 2002). In 2010, Stenkula et al. showed that insulin also had a fundamental impact on the spatial distribution of GLUT4 within the plasma membrane in adipose cells. It was the emergence of high-resolution live cell microscopy techniques, as well as the development of dual-coloured probes to follow GLUT4 trafficking and dispersal in the membrane, that highlighted the existence of GLUT4 molecules as stationary clusters or monomers at the cell surface (Jiang et al., 2008; Fazakerley et al., 2009; Stenkula et al., 2010; Lizunov et al., 2012).

 

The objective of my PhD is to further elucidate such molecular processes by using 3T3-L1 adipocytes transfected with HA-GLUT4-GFP, a construct extensively known and used in the GLUT4 research community. In this construct, GLUT4 proteins possess a hemagglutinin (HA)-tag attached to their exofacial loop and a green fluorescent protein (GFP)-tag fused to their carboxyl-terminal (see Figure 1). While the fluorescence emitted by GFP is directly proportional to the total amount of GLUT4 expressed by the specimen, the HA-tag becomes accessible via fluorescent staining when GLUT4 is embedded in the membrane. Our aim is therefore to develop a microscopy-based assay to study and quantify GLUT4 dispersal dynamics at the plasma membrane using the present PicoQuant MicroTime 200 microscope.

 

Firstly I used fluorescence correlation spectroscopy (FCS) to observe fluctuations of both the concentration and diffusion coefficient of fluorescent particles (see below). The HA-tag was stained using a DNA-PAINT approach with Atto655-labelled DNA strands, and this was studied at the cell surface under basal and insulin-stimulated conditions. The MicroTime 200 can also be used to perform stimulated emission depletion (STED)-FCS. This STED-FCS approach leads to a reduced observation volume for FCS and an enhanced resolution in images on the order of 50 nm, which allows to nicely reveal GLUT4 as stationary clusters or monomers. Such ability to track individual glucose receptors in the membrane, which opens up the possibility to probe diabetes models, would not be possible without this equipment.

 

My fundamental expertise is in biology and understanding to use the MicroTime 200 has required some training, but this experience has spurred on a strong interest in bioimaging and a drive to expand my knowledge about optical microscopy. During my training, PicoQuant offered great customer service and advice, showing me the best way to use this intuitive and powerful system. Over the years, the demand for high-resolution pictures showing well-defined biological structures has gradually been replaced by a need for quantitative data. The Picoquant MicroTime 200 at the University of Strathclyde offers the opportunity to obtain both.