Imaging of membrane systems and membrane traffic in living cells

Eukaryotic cells are composed of an intricate system of internal membranes that are organized into different compartments-including the endoplasmic reticulum (ER), the nuclear envelope, the Golgi complex (GC), lysosomes, endosomes, caveolae, mitochondria, and peroxisomes-that perform specialized tasks within the cell. The localization and dynamics of intracellular compartments are now being studied in living cells because of the availability of green fluorescent protein (GFP)- fusion proteins and recent advances in fluorescent microscope imaging systems. Results using these techniques are revealing how intracellular compartments maintain their steady-state organization and distributions, how they undergo growth and division, and how they transfer protein and lipid components between themselves through the formation and trafficking of membrane transport intermediates. This article describes methods using GFP-fusion proteins to visualize the behavior of organelles and to track membrane-bound transport intermediates moving between them. Practical issues related to the construction and expression of GFP-fusion proteins are discussed first. These are essential for optimizing the brightness and expression levels of GFP-fusion proteins so that intracellular membrane-bound structures containing these fusion proteins can be readily visualized. Next, techniques for performing time-lapse imaging using a confocal laser-scanning microscope (CLSM) are detailed, including the use of photobleaching to highlight organelles and transport intermediates. Methods for the acquisition and analysis of data are then discussed. Finally, commonly used and exciting new approaches for perturbing membrane traffic are outlined.