A method to rapidly induce organelle-specific molecular activities and membrane tethering

Toru Komatsu, Takanari Inoue

Research output: Contribution to journalArticle

Abstract

In this chapter we describe a technique for rapid protein targeting to individual intracellular organelles. This method enables a real-time imaging-based study of cellular behavior in response to controlled induction of signaling events in a specifically targeted cellular compartment. We provide rationales and a step-by-step protocol for probe design and imaging of protein targeting along with two different applications of this technique. One application involves organelle-specific activation of small GTPases, while the other application involves membrane tethering of two different organelles. In the former case, we activate Rac1 and Ras at distinct intracellular locations in order to study compartmentalization of their signaling pathways, and in the latter example, we induce membrane tethering of the endoplasmic reticulum and mitochondria in order to study organelle-organelle communication. The described technique allows to rapidly perturb molecular activities and organelle-organelle communications at precise locations with specified timing and represents a powerful strategy to dissect spatiotemporally complex biological processes.

Original languageEnglish (US)
Pages (from-to)231-245
Number of pages15
JournalMethods in Molecular Biology
Volume1174
DOIs
StatePublished - Jan 1 2014
Externally publishedYes

Keywords

  • Cell signaling
  • Endoplasmic reticulum
  • Fluorescence imaging
  • Membrane lipid signaling
  • Organelle-organelle interaction
  • Small GTPases
  • Synthetic biology

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

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