Confocal-FRAP Analysis of ECM Molecular Interactions - Methods in Molecular Biology: Extracellular Matrix Protocols - page 79

8
Confocal-FRAP Analysis
of ECM Molecular Interactions
Timothy Hardingham and Philip Gribbon
1. Introduction
Extracellular matrices (ECM) contain a mixture of fibrillar and nonfibrillar
macromolecular components, which together form a composite structure
(1-3) . It is the ECM that defines the architecture, the form, and the biome-
chanical properties of different tissues (4 , 5) . Among the nonfibrillar macro-
molecules, the highly charged proteoglycans and hyaluronan are major
components that occur at high concentration and greatly influence the move-
ment of solutes and water between the tissue and the circulation, and control
the access to cells of nutrients, metabolites, growth factors, and chemokines
(6 , 7) . This local environmental regulation may have important conse-
quences on cellular functions, especially in tissues with large dense ECMs,
such as articular cartilage.
Studies on the physical properties of proteoglycans and glycosaminogly-
cans have commonly included rheological, light scattering, and ultracentrifu-
gation analyses. These are carried out under “ideal” conditions, which are at
low concentration in order to minimize the effects of intermolecular interac-
tions (8-11) . However, to understand their properties at the high concentra-
tions found physiologically, these methods are of limited application. An
alternative experimental approach has been developed using fluorescence
recovery after photobleaching (FRAP) (12) . It was originally used for investi-
gating cell membrane dynamics, but in an adapted procedure with a confocal
microscope, (13-15) , it has now been applied to study intermolecular interac-
tions and network formation of biopolymers in solution (16) . It was used to
characterize the intermolecular interactions of aggrecan, the large aggregating
proteoglycan containing many chondroitin sulphate and keratan sulphate