Given the large size (larger than the limit of resolution of light microscopy) of membrane heterogeneities observed in artificial model membranes, one would expect to observe them in biological membranes with reasonable ease. However, even with the spectrum of protein- and lipid-related techniques available, ranging from electron microscopy, optical tweezers and single molecule studies to biochemical detection, the direct visualization of lipid rafts has been elusive. An association of proteins expected to be present in lipid rafts or DRMs is not detectable by using biochemical procedures such as SDS-PAGE, which indicates that an association between raft constituents - if present - is weak, noncovalent, and/or transient.
Within the resolution of light microscopy, DRM constituents such as GPI-an-chored proteins appear to be diffusely distributed , indicating that if segregated "raft" structures exist they must be smaller than the resolution of light microscopy (at best 200 nm). Studying the distribution of GPI-anchored proteins by electron microscopy (EM), which has the correct scale of detection, also failed to detect any clustering of lipid raft constituents [31-33]. Elaborate fixation procedures and the efficiency of labeling with electron-dense tags might be possible causes of this loss of detection sensitivity . As each technique is scrutinized for its suitability for raft detection, it has become clear that these structures - if they exist - are extremely difficult to visualize and/or detect in direct manner.
In the absence of any other option, indirect methods remain in widespread use to define the constituents and structure of lipid rafts; biophysical studies hold the promise of understanding lipid rafts. In the following sections a variety of different biophysical techniques used to study lipid rafts are discussed, together with an idea of their impact on our understanding of "raft" structures in living cell membranes.
The biophysical techniques used to examine the organization of lipid-based components may be divided into two types:
• those based on studying the diffusion characteristics of membrane components, based on the assumptions that raft-association will be detected as a change in local membrane viscosity, or will generate large-sized entities that should show deviations from diffusive behavior attributable to monomer diffusion; and • those based on detecting enhanced proximity between raft-associated molecules.
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