Photobleaching Methods to Study Golgi Complex Dynamics in Living Cells

Abstract

The Golgi complex (GC) is a highly dynamic organelle that constantly receives and exports proteins and lipids from both the endoplasmic reticulum and the plasma membrane. While protein trafficking can be monitored with traditional biochemical methods, these approaches average the behaviors of millions of cells, provide modest temporal information and no spatial information. Photobleaching methods enable investigators to monitor protein trafficking in single cells or even single GC stacks with subsecond precision. Furthermore, photobleaching can be exploited to monitor the behaviors of resident GC proteins to provide insight into mechanisms of retention and trafficking. In this chapter, general photobleaching approaches with laser scanning confocal microscopes are described. Importantly, the problems associated with many fluorescent proteins (FPs) and their uses in the secretory pathway are discussed and appropriate choices are suggested. For example, Enhanced Green Fluorescent Protein (EGFP) and most red FPs are extremely problematic. Finally, options for data analyses are described.

A Selective Autophagy Pathway for Phase-Separated Endocytic Protein Deposits

“…Fitting of normalized FRAP curves was performed using a single exponential equation with the software Origin (version 2019b). FLIP analysis was performed as described before (Snapp, 2013).…”

1270 nm near-infrared light as a novel vaccine adjuvant acts on mitochondrial photoreception in intradermal vaccines

“…Confluent P-815 cells were rinsed and resuspended in RPMI 1640 Medium, no phenol red (Thermo Fisher Scientific, MA, USA) and seeded at 1.8 × 10 5 cells/well in 48-well plates (IWAKI, Shizuoka, Japan). We used RPMI 1640 medium without phenol red because phenol red absorbs light ( 17 ). 2′,7′-Dichlorodihydrofluorescein diacetate (H2DCFDA; Thermo Fisher Scientific, MA, USA) was added at a final concentration of 1 µM; the plate temperature was kept constant at 37°C using a hot plate (TOKAI HIT, Shizuoka, Japan) and the cells were irradiated with 1270 nm CW laser at 200 or 300 mW/cm 2 for 1 minute.…”