Imaging of Organelle Membrane Systems and Membrane Traffic in Living Cells
This protocol was adapted from “Imaging of Organelle Membrane Systems and Membrane Traffic in Living Cells,” Chapter 32, in Live Cell Imaging: A Laboratory Manual (eds. Goldman and Spector). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2005.INTRODUCTION
Eukaryotic cells are composed of an intricate system of internal membranes that are organized into different compartments, including the endoplasmic reticulum, the nucleus, the Golgi apparatus, lysosomes, endosomes, mitochondria, and peroxisomes, with specialized roles within the cell. Only recently have the localization and dynamics of these organelles been studied in the context of the living cell because of the availability of green fluorescent protein (GFP) fusion proteins and recent advances in fluorescent microscope imaging systems.
In this protocol, a confocal laser-scanning microscope (CLSM) and GFP fusion proteins are used in time-lapse imaging to visualize the behavior of organelles and to track membrane-bound transport intermediates that bud off from organelles. Practical issues related to the construction and expression of GFP fusion proteins are also discussed. 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.
