Quantifying spatial and temporal variations of the cell membrane ultra-structure by bimFCS.
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abstract
It has been long recognized that the cell membrane is heterogeneous on scales ranging from a couple of molecules to micrometers in size and hence diffusion of receptors is length scale dependent. This heterogeneity modulates many cell-membrane-associated processes requiring transient spatiotemporal separation of components. The transient increase in local concentration of interacting signal components enables robust signaling in an otherwise thermally noisy system. Understanding how lipids and proteins self-organize and interact with the cell cortex requires quantifying the motion of the components. Multi-length scale diffusion measurements by single particle tracking, fluorescence correlation spectroscopy (FCS) or related techniques are able to identify components being transiently trapped in nanodomains, from freely moving one and from ones with reduced long-scale diffusion due to interaction with the cell cortex. One particular implementation of multi-length scale diffusion measurements is the combination of FCS with a spatially resolved detector, such as a camera and two-dimensional extended excitation profile. The main advantages of this approach are that all length scales are interrogated simultaneously, uniquely permits quantifying changes to the membrane structure caused by extrenal or internal perturbations. Here, we review how combining total internal reflection microscopy (TIRF) with FC resolves the membrane organization in living cells. We show how to implement the method, which requires only a few seconds of data acquisition to quantify membrane nanodomains, or the spacing of membrane fences caused by the actin cortex. The choice of diffusing fluorescent probe determines which membrane heterogeneity is detected. We review the instrument, sample preparation, experimental and computational requirements to perform such measurements, and discuss the potential and limitations. The discussion includes examples of spatial and temporal comparisons of the membrane structure in response to perturbations demonstrating the complex cell physiology.
