title: Photohighlighting approaches to access membrane dynamics of the Golgi apparatus.
abstract: By providing quantitative, visual data of live cells, fluorescent protein-based microscopy techniques are furnishing novel insights into the complexities of membrane trafficking pathways and organelle dynamics. In this chapter, we describe experimental protocols employing fluorescent protein-based photohighlighting techniques to quantify protein movement into and out of the Golgi apparatus, an organelle that serves as the central sorting and processing station of the secretory pathway. The methods allow kinetic characteristics of Golgi-associated protein trafficking to be deciphered, which can help clarify how the Golgi maintains itself as a steady-state structure despite a continuous flux of secretory cargo passing into and out of this organelle. The guidelines presented in this chapter can also be applied to examine the dynamics of other intracellular organelle systems, elucidating mechanisms for how proteins are maintained in specific organelles and/or circulated to other destinations within the cell.
authors.affiliation: The Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland, USA.
authors.forename: Prabuddha
authors.initials: P
authors.lastname: Sengupta
authors.forename: Jennifer
authors.initials: J
authors.lastname: Lippincott-Schwartz
mesh_terms: D000818:Animals; D016466:CHO Cells; D019556:COS Cells; D002522:Chlorocebus aethiops; D006224:Cricetinae; D003412:Cricetulus; D036681:Fluorescence Recovery After Photobleaching; D006056:Golgi Apparatus / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D008562:Membrane Glycoproteins / Q000096:biosynthesis / Q000235:genetics; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D055571:Secretory Pathway; D014759:Viral Envelope Proteins / Q000096:biosynthesis / Q000235:genetics
chemical_list: C028391:G protein, vesicular stomatitis virus; D008562:Membrane Glycoproteins; D011993:Recombinant Fusion Proteins; D014759:Viral Envelope Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins
keywords: Golgi; Organelle dynamics; Photoactivation; Photobleaching; Protein trafficking; Secretory pathway