Abstract
The mobility of proteins and lipids within the cell, sculpted oftentimes by the organization of the membrane, reveals a great wealth of information on the function and interaction of these molecules as well as the membrane itself. Single particle tracking has proven to be a vital tool to study the mobility of individual molecules and unravel details of their behavior. Interferometric scattering (iSCAT) microscopy is an emerging technique well-suited for visualizing the diffusion of gold nanoparticle-labeled membrane proteins to a spatial and temporal resolution beyond the means of traditional fluorescent labels. We discuss the applicability of interferometric single particle tracking (iSPT) microscopy to investigate the minutia in the motion of a protein through measurements visualizing the mobility of the epidermal growth factor receptor in various biological scenarios on the live cell.
Highlights
All cells are enclosed by an outer plasma membrane and, in addition, eukaryotic cells are commonly compartmentalized by internal membranes into cell organelles, to generate specialized functional entities
We show how Interferometric scattering (iSCAT) microscopy is highly suited for investigating the mobility of a transmembrane signaling protein to fine detail within the live cell membrane
We introduce a scanning confocal fluorescence channel from above the sample, shown in Figure 1A, to provide a macroscopic overview image of the live cell sample, as shown in Figure 1B. iSCAT microscopy may be performed in parallel to confocal fluorescence imaging, and Figure 1C presents an image of a portion of the lower cell membrane viewed with iSCAT microscopy
Summary
All cells are enclosed by an outer plasma membrane and, in addition, eukaryotic cells are commonly compartmentalized by internal membranes into cell organelles, to generate specialized functional entities. The plasma membrane acts as a barrier, transport, and communication platform between the cell and its environment that separates the interior of the cell from its surroundings, controls the flux of ions and nutrients and mediates sending and sensing of signals to and from the cell. These multiple functions of the membrane are enabled by its complex composition of phospholipids, sphingo- and glycolipids, cholesterol, and proteins.
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