The endoplasmic reticulum (ER) is both the major source of intracellular Ca2+ for cell signaling and the organelle that forms the most extensive contacts with the plasma membrane and other organelles. Lysosomes fulfill important roles in degrading cellular materials and in cholesterol handling, but they also contribute to Ca2+ signaling by both releasing and sequestering Ca2+. Interactions between ER and other Ca2+-transporting membranes, notably mitochondria and the plasma membrane, often occur at sites where the two membranes are closely apposed, allowing local Ca2+ signaling between them. These interactions are often facilitated by scaffold proteins. Recent evidence suggests similar local interactions between ER and lysosomes. We describe simple fluorescence-based methods that allow the interplay between Ca2+ signals, the ER, and lysosomes to be examined.
