Photobleaching methods to study golgi complex dynamics in living cells

Erik Lee Snapp

Research output: Contribution to journalArticle

1 Citation (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)195-216
Number of pages22
JournalMethods in Cell Biology
Volume118
DOIs
StatePublished - 2013

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Photobleaching
Golgi Apparatus
Protein Transport
Proteins
Secretory Pathway
Endoplasmic Reticulum
Organelles
Lasers
Research Personnel
Cell Membrane
Lipids

Keywords

  • Diffusion
  • FLIP
  • FRAP
  • Membrane
  • Superfolder GFP

ASJC Scopus subject areas

  • Cell Biology

Cite this

Photobleaching methods to study golgi complex dynamics in living cells. / Snapp, Erik Lee.

In: Methods in Cell Biology, Vol. 118, 2013, p. 195-216.

Research output: Contribution to journalArticle

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