Using fluorescence resonance energy transfer-based biosensors to probe Rho GTPase activation during phagocytosis

Veronika Miskolci, Louis Hodgson, Dianne Cox

Research output: Chapter in Book/Report/Conference proceedingChapter

3 Scopus citations

Abstract

The p21-family members of Rho GTPases are important for the control of actin cytoskeleton dynamics, and are critical regulators of phagocytosis. The three-dimensional structure of phagosomes and the highly compartmentalized nature of the signaling mechanisms during phagocytosis require high-resolution imaging using ratiometric biosensors to decipher Rho GTPase activities regulating phagosome formation and function. Here we describe methods for the expression and ratiometric imaging of FRET-based Rho GTPase biosensors in macrophages during phagocytosis. As an example, we show Cdc42 activity at the phagosome over Z-serial planes. In addition, we demonstrate the usage of a new, fast, and user-friendly deconvolution package that delivers significant improvements in the attainable details of Rho GTPase activity in phagosome structures.

Original languageEnglish (US)
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Pages125-143
Number of pages19
Volume1519
DOIs
StatePublished - 2017

Publication series

NameMethods in Molecular Biology
Volume1519
ISSN (Print)10643745

Keywords

  • Biosensors
  • Deconvolution
  • FRET
  • Macrophages
  • Phagosome
  • Ratiometric imaging
  • Z-stack

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

Fingerprint Dive into the research topics of 'Using fluorescence resonance energy transfer-based biosensors to probe Rho GTPase activation during phagocytosis'. Together they form a unique fingerprint.

  • Cite this

    Miskolci, V., Hodgson, L., & Cox, D. (2017). Using fluorescence resonance energy transfer-based biosensors to probe Rho GTPase activation during phagocytosis. In Methods in Molecular Biology (Vol. 1519, pp. 125-143). (Methods in Molecular Biology; Vol. 1519). Humana Press Inc.. https://doi.org/10.1007/978-1-4939-6581-6_9