Dynamin microscopy

Wittrup and Belting have in several studies on CPP-mediated DNA delivery established protocols for fluorescence assisted cell sorting (FACS) analysis to obtain reliable, quantitative data on CPP-DNA complex uptake in cultured cancer cells (9). Also, procedures for co-localization studies with known markers of various endocytotic pathways using confocal microscopy are described as well as the expression of dominant negative dynamin (GTPase deficient dynamin-2) to evaluate the dynamin dependence of the uptake mechanism. The use of various drugs commonly used to disrupt endocytosis is discussed, especially with regard to their limited specificity (9). 

In this chapter, we provide protocols to determine the ability of a peptide to mediate DNA internalization in cultured human tumor cells. Fluorescence-assisted cell sorting (FACS) analysis is used to obtain quantitative data on the time and temperature dependence of macromolecular delivery. Confocal microscopy is used to study the subcellular localization in both fixed and live cells. Fluorescently labeled transferrin and dextran are used to label the clathrin-dependent (15) and the non-clathrin, non-caveolar (16) endocytic compartments, respectively. Expression of a caveolin-l-YFP fusion protein is used to label cell surface caveolae and intracellular caveosomes (17). Finally a protocol, for the overexpression of dominant-negative dynamin [GTPase deficient dynamin-2 containing the amino acid substitution K44A (18)] is provided to evaluate the dynamin dependence of the uptake mechanism. 

Fluorescence Lifetime Imaging Microscopy of Auxilin-Dynamin Interactions... 

Our previous approaches to detect endogenous complexes of dynamin and auxilin using co-immunoprecipitation approaches were unsuccessful, so we turned to fluorescence lifetime imaging microscopy (FLIM). While fluorescence microscopy provides two- or three-dimensional information about fiuorophore concentration, FLIM can reveal spatial differences in fluorophore population lifetimes that are independent of concentration. Besides being useful in fiuorophore identification, which transcends issues of spectral overlap, FLIM inherently observes lifetime truncations on a pixel by pixel basis that are induced by fluorescence resonance energy... 

---