Session 4: Technology: what do we need to know and how can we measure it?
What techniques (both fluorescence- and non-fluorescence-based) can help us detect membrane microdomains and measure affinities of protein-lipid and protein-protein interactions within membranes?
Daniel Axelrod
The term 'membrane microdomains' in general can refer to many kinds of heterogeneities; both biochemical non-uniformities in the two-dimensional 'plane' of the membrane and local geometric deviations from planarity. Microdomains can be important in mediating various membrane processes, including: translocation of proteins and lipids; local clustering of proteins; control of kinetic rates of reactions, both within the membrane and between membranes and reversibly bound species from the adjacent solution; formation of putative lipid rafts; involvement in exo- and endocytosis; and anchoring of cytoskeletal structures. However, microdomains can be hard to detect because they are often of a size smaller than light microscope resolution, rapidly transient, or of a nature that does not affect the refraction, diffraction or scattering of light. A variety of fluorescence techniques and several non-fluorescent ones can overcome many of these obstacles.
Fluorescence techniques are particularly useful for studies of living cells. Modern fluorescence image-analysis approaches based on digital detectors can significantly improve effective lateral resolution beyond the standard Raleigh optical resolution limit. Fluorescence redistribution after photobleaching (or after photoactivation; FRAP), especially in combination with microscopes using automated focused beam or stage motion (such as confocal or multiphoton systems), can be used to qualitatively and quantitatively visualize transport among cellular and membrane compartments. Fluorescence correlation spectroscopy (FCS) can (at least in principle) measure not only some transport rates but also absolute concentrations and degrees of aggregation in the membrane. Fluorescence resonance energy transfer (FRET) can detect transient associations on a distance scale at least an order of magnitude smaller than the microscope resolution. Total internal reflection fluorescence (TIRF) can visualize deviations from membrane planarity on the scale of 5 nm (at least two orders of magnitude better than optical resolution). In combination with the polarization properties of some fluorophores, TIRF can detect submicroscopic localized curvature in membranes. Many of these techniques can be mixed with each other for special purposes; for example, TIRF and FRAP can be combined to measure spatially resolved local binding/unbinding rates at the membranes of living cells. The ability to use multiple spectrally distinct labels in many of the above techniques allows one to study the relative distributions, and possibly correlated motions, of two or more membrane components simultaneously.
Non-fluorescence techniques, such as electron microscopy, X-ray diffraction and atomic force microscopy, can have even better spatial resolution than the best fluorescence techniques, and different contrast mechanisms. Despite their relative slowness and limitation to analysing non-living samples, they have produced spectacular examples of microdomain structure.
Further reading
Axelrod, D. Total internal reflection fluorescence microscopy in cell biology. Methods Enzymol. 361, 1-33 (2003)
Lawrence, J. C., Saslowsky, D. E., Edwardson, J. M. & Henderson, R. M. Real-time analysis of the effects of cholesterol on lipid raft behavior using atomic force microscopy. Biophys. J. 84, 1827-1832 (2003)
Lippincott-Schwartz, J., Snapp, E. & Kenworthy, A. Studying protein dynamics in living cells. Nature Rev. Mol. Cell Biol. 2, 444-456 (2001)
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