Biology Reference
In-Depth Information
Abstract
Lipid rafts, cell membrane domains with unique composition and properties, mod-
ulate the membrane distribution of receptors and signaling molecules facilitating the
assembly of active signaling platforms. However, the underlying mechanisms that
link signal transduction and lipid rafts are not fully understood, mainly because of
the transient nature of these membrane assemblies. Several methods have been used
to study the association of membrane receptors with lipid rafts. In the first part of this
chapter, a description of how biochemical methods such as raft disruption by cho-
lesterol depletion agents are useful in qualitatively establishing protein association
with lipid rafts is presented. The second part of this chapter is dedicated to imaging
techniques used to study membrane receptor organization and lipid rafts. We cover
conventional approaches such as confocal microscopy to advanced imaging tech-
niques such as homo-FRET microscopy and superresolution methods. For each tech-
nique described, their advantages and drawbacks are discussed.
INTRODUCTION AND RATIONALE
Cell membranes are presently described to be dynamically compartmentalized into
physically, compositionally, and functionally distinct regions, which, according to
their lipid and protein content, fulfill specific cellular roles (
Simons & Gerl,
2010
). These regions, called membrane domains, are formed due to cooperative be-
havior between certain membrane components. A special type of membrane do-
mains, which has been associated with a multitude of cellular processes such as
endocytosis, intracellular trafficking, lipid and protein sorting, and cell signaling,
is lipid rafts (
Simons & Gerl, 2010
). These structures are defined as heterogeneous
and highly dynamic nanoscale assemblies (5-200 nm) enriched in sphingolipids
and certain sterols such as cholesterol or ergosterol, which present average lifetimes
in subsecond timescales (
Pike, 2006
). These small and transient domains can be sta-
bilized by interactions with particular lipids, proteins, and actin cytoskeleton, result-
ing in long-lived (ms timescales) micrometer-sized raft regions (
Pike, 2006; Simons
&Gerl, 2010
). Due to their unique content in high temperature melting sphingolipids
and cholesterol, lipid rafts are more ordered than the bulk membrane (
Owen,
Williamson, Magenau, & Gaus, 2012
), imposing the localization or exclusion of
specific membrane proteins to these domains. Moreover, protein diffusion is reduced
in these regions (
Lenne et al., 2006
), promoting and stabilizing protein-protein
interactions. Adding up, these features make lipid rafts particularly suitable for bio-
chemical processes requiring a specific compartmentalization of macromolecular
complexes, such as those occurring in cell signaling. In this context, it has been pos-
tulated that rafts spatially organize and concentrate signaling molecules at particular
membrane regions while excluding inhibitor molecules. In this way, the favorable
interactions between signaling molecules necessary for signal transduction are
