Analyzing dynamic properties of intermediate filaments

Intermediate filaments (IF) have been considered to be the most stable and consequently the least dynamic of the various cytoskeletal systems within eukaryotic cells. This widely held view of IF is related to numerous factors, including their relative insolubility in vitro over a wide range of reasonably physiological conditions, and the unavailability of a specific reversible in vivo disrupting agent such as colchicine for microtubules and cytochalasin for microfilaments. Over the past 10–12 years, techniques have been developed for studying dynamic changes in the cytoplasmic architecture of cells. These employ the use of “labeled” cytoskeletal proteins in microinjection studies carried out on live cells. These types of studies have highlighted the dynamic aspects of the microfilament and microtubule-based cytoskeletal systems with regard to protein subunit exchange. In theory, these methods also should be useful in studies of the IF system. However, purifying and maintaining IF structural proteins in a soluble state under nondenaturing conditions is difficult. Another complicating factor in designing experiments aimed at determining the dynamic nature of the IF system is the fact that each major cell type contains IF composed of different structural proteins. This chapter presents different methods, which use fluorescent labeled desmin and neurofilament protein to analyze the properties of intermediate filaments.