Fluorescent Protein-Based Biosensors pp 109-128

Part of the Methods in Molecular Biology book series (MIMB, volume 1071) | Cite as

Imaging the Activity of Ras Superfamily GTPase Proteins in Small Subcellular Compartments in Neurons

  • Ana F. Oliveira
  • Ryohei Yasuda
Protocol

Abstract

Resolving the spatiotemporal dynamics of intracellular signaling is important for understanding the molecular mechanisms of various cellular processes induced by extracellular signals. Two-photon fluorescence lifetime imaging microscopy (2pFLIM) in combination with a fluorescence resonance energy transfer (FRET)-based signaling sensors allows one to image signaling within small subcellular compartments, such as dendritic spines of neurons, with high sensitivity and spatiotemporal resolution. In this protocol, we describe the procedures and equipment required for imaging intracellular signaling activity, with a particular focus on signaling mediated by the Ras superfamily of small GTPase proteins.

Key words

FLIM FRET Lifetime Ras GTPase Ras RhoA Cdc42 

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Copyright information

© Springer Science+Business Media, LLC 2014

Authors and Affiliations

  • Ana F. Oliveira
    • 1
  • Ryohei Yasuda
    • 2
    • 3
  1. 1.Department of NeurobiologyDuke University Medical CenterDurhamUSA
  2. 2.Departments of Neurobiology, Cell Biology, and PhysicsDuke University Medical CenterDurhamUSA
  3. 3.Max-Planck Florida Institute for NeuroscienceJupiterUSA

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