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

2 Citations (Scopus)

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

Fingerprint

Phagosomes
rho GTP-Binding Proteins
Fluorescence Resonance Energy Transfer
Biosensing Techniques
Phagocytosis
Actin Cytoskeleton
Macrophages

Keywords

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

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

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

Using fluorescence resonance energy transfer-based biosensors to probe Rho GTPase activation during phagocytosis. / Miskolci, Veronika; Hodgson, Louis; Cox, Dianne.

Methods in Molecular Biology. Vol. 1519 Humana Press Inc., 2017. p. 125-143 (Methods in Molecular Biology; Vol. 1519).

Research output: Chapter in Book/Report/Conference proceedingChapter

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, Methods in Molecular Biology, vol. 1519, Humana Press Inc., pp. 125-143. https://doi.org/10.1007/978-1-4939-6581-6_9
Miskolci V, Hodgson L, Cox D. Using fluorescence resonance energy transfer-based biosensors to probe Rho GTPase activation during phagocytosis. In Methods in Molecular Biology. Vol. 1519. Humana Press Inc. 2017. p. 125-143. (Methods in Molecular Biology). https://doi.org/10.1007/978-1-4939-6581-6_9
Miskolci, Veronika ; Hodgson, Louis ; Cox, Dianne. / Using fluorescence resonance energy transfer-based biosensors to probe Rho GTPase activation during phagocytosis. Methods in Molecular Biology. Vol. 1519 Humana Press Inc., 2017. pp. 125-143 (Methods in Molecular Biology).
@inbook{d2a44a3aa14c41508a4beec62846bd5b,
title = "Using fluorescence resonance energy transfer-based biosensors to probe Rho GTPase activation during phagocytosis",
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.",
keywords = "Biosensors, Deconvolution, FRET, Macrophages, Phagosome, Ratiometric imaging, Z-stack",
author = "Veronika Miskolci and Louis Hodgson and Dianne Cox",
year = "2017",
doi = "10.1007/978-1-4939-6581-6_9",
language = "English (US)",
volume = "1519",
series = "Methods in Molecular Biology",
publisher = "Humana Press Inc.",
pages = "125--143",
booktitle = "Methods in Molecular Biology",

}

TY - CHAP

T1 - Using fluorescence resonance energy transfer-based biosensors to probe Rho GTPase activation during phagocytosis

AU - Miskolci, Veronika

AU - Hodgson, Louis

AU - Cox, Dianne

PY - 2017

Y1 - 2017

N2 - 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.

AB - 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.

KW - Biosensors

KW - Deconvolution

KW - FRET

KW - Macrophages

KW - Phagosome

KW - Ratiometric imaging

KW - Z-stack

UR - http://www.scopus.com/inward/record.url?scp=84994504506&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84994504506&partnerID=8YFLogxK

U2 - 10.1007/978-1-4939-6581-6_9

DO - 10.1007/978-1-4939-6581-6_9

M3 - Chapter

VL - 1519

T3 - Methods in Molecular Biology

SP - 125

EP - 143

BT - Methods in Molecular Biology

PB - Humana Press Inc.

ER -