AMBROGGIO ERNESTO
Capítulos de libros
Título:
DIRECT VISUALIZATION OF THE INTERACTION OF ACTIVE-MEMBRANE PEPTIDES WITH MODEL MEMBRANES
Autor/es:
AMBROGGIO ERNESTO; BAGATOLLI LUIS A.
Libro:
Membrane-active peptides: methods and results on structure and function
Editorial:
FIRST
Referencias:
Año: 2010; p. 179 - 200
Resumen:
Biological membranes form the scaffolding that defines cells and their
organelles and separates them from the surrounding environment. These
membranes are composed of hundreds of different lipid species and are
the framework for membrane proteins, arranged in different ways, which
have a close interaction with this lipid milieu. This symbiosis between
lipids and proteins conforms an exquisite matrix with specific chemical
and physical properties that serves as a selective permeability barrier for
cells and a perfect medium for cellular events like enzymatic reactions;
signal reception and propagation, transport; movement;1?11defense; and
toxicity mediated by proteins and peptides,12?23 where all of them are
directly related to lipid?protein (or peptide) interactions. The conven-
tional model for this matrix dates back to 1972 and was outlined as
a fluid mosaic in which proteins freely diffuse in the lateral plane of
a fluid lipid bilayer.24This hypothesis has been refined, particularly in
the last few years when thermodynamics aspects and other features like
membrane lateral heterogeneity were included in the model.25?27Visual
data about membrane lateral heterogeneity is now, particularly in the last
decade, understood to be an important piece of information.28
Nonrandom lateral organization of lipid membranes, proposed more
than three decades ago,29,30was acknowledged by the biology field when
the raft hypothesis was postulated ten years ago.31?34Although its role in
membrane structure is now accepted, the physical bases of this phenom-
enon (including potential links with membrane function) remain
obscure. For example, from experimental information obtained in model
systems, lateral heterogeneity in biological membranes can be rational-
ized simply as local (transient) compositional fluctuations or phase-
separation phenomena.27 Proper identification of these phenomena in
biologically relevant membranes will become extremely important for
understanding the in-plane distribution of lipid and proteins.
The combination of giant unilamellar vesicles35,36(GUVs) with fluo-
rescence microscopy28 is a promising new experimental approach. The
sensitivity and flexibility of a microscope with the addition of fluores-
cence spectroscopy allows collection of spatially resolved information,
bridging membrane morphology with dynamic and structural information
obtained at the molecular level using fluorescence spectroscopy (lipid or
protein mobility, hydration, etc.). For instance, many papers have
appeared in recent years describing the use of GUVs to study  different
physical aspects of membranes (lateral structure as well as mechanical
properties), mostly lipid?lipid interactions but also to a  lesser extent
lipid?DNA, lipid?protein, and lipid?peptide interactions.28 The most
popular studies using GUVs focus on changes in mechanical properties
of membranes (bending elasticity, changes in GUV shape) in the pres-
ence of particular peptides.37?41Experiments not based on fluorescence
microscopy use the so-called micropipette aspiration technique,42,43
which uses phase-contrast microscopy (or Hoffman illumination) to
observe changes in GUVs due to peptide interaction with the lipid mem-
brane. Few studies using fluorescence microscopy techniques have been
done on the interaction of peptides with GUVs (composed of different
lipids or lipid mixtures). Studies have mainly focused on detecting leak-
age of fluorophores as a result of a peptide-induced membrane destabi-
lization44?47 by using wide-field or confocal fluorescence microscopy,
diffusion of fluorescently labeled peptides upon membrane interaction
by using fluorescence correlation  spectroscopy,48and effect of peptide?
lipid interactions on the lateral structure of lipid mixtures by using mul-
tiphoton excitation fluorescence microscopy.49
In this chapter, we describe some technical details of fluorescence-
microscopy-based experiments related to GUVs and peptide?lipid inter-
actions. The direct microscopic observation of GUVs and peptide?lipid
interactions has potential for extensive study of mechanisms of peptides
interacting with lipid membranes. Specific aspects of the GUV model
system will also be discussed, including a detailed description of prepara-
tion protocols. Finally, potential combinations of the different approaches
will be discussed.