title: Probing endoplasmic reticulum dynamics using fluorescence imaging and photobleaching techniques.
abstract: This unit describes approaches and tools for studying the dynamics and organization of endoplasmic reticulum (ER) membranes and proteins in living cells using fluorescence microscopy. The ER plays a key role in secretory protein biogenesis, calcium regulation, and lipid synthesis. However, study of these processes has often been restricted to biochemical assays that average millions of lysed cells or imaging of static fixed cells. With new fluorescent protein (FP) reporter tools, sensitive commercial microscopes, and photobleaching techniques, investigators can interrogate the behaviors of ER proteins, membranes, and stress pathways in single live cells. Solutions are described for imaging challenges relevant to the ER, including the mobility of ER membranes, a range of ER structures, and the influence of post-translational modifications on FP reporters. Considerations for performing photobleaching assays for ER proteins are discussed. Finally, reporters and drugs for studying misfolded secretory protein stress and the unfolded protein response are described.
authors: Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, New York.
authors: Lindsey
authors: L
authors: Costantini
authors: Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, New York.
authors: Erik
authors: E
authors: Snapp
mesh_terms: D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D004285:Dogs; D004721:Endoplasmic Reticulum / Q000378:metabolism* / Q000648:ultrastructure; D049452:Green Fluorescent Proteins / Q000032:analysis; D061985:Madin Darby Canine Kidney Cells; D061848:Optical Imaging / Q000379:methods*; D038761:Photobleaching; D012441:Saccharomyces cerevisiae / Q000648:ultrastructure; D014026:Nicotiana / Q000648:ultrastructure
chemical_list: D049452:Green Fluorescent Proteins
keywords: FLIP; FRAP; confocal microscopy; diffusion; live cell imaging; membrane; microtubule; superfolder green fluorescent protein; tubule