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Combining FRET and optical tweezers to study RhoGTPases spatio-temporal dynamics upon local stimulation

Iseppon, F; Napolitano, LMR; Torre, V; Cojoc, D; (2017) Combining FRET and optical tweezers to study RhoGTPases spatio-temporal dynamics upon local stimulation. Journal of Biological Methods , 4 (1) , Article e65. 10.14440/jbm.2017.159. Green open access

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Abstract

Local stimulation with optical tweezers has been used to mimic natural stimuli that occur in biological processes such as cell migration or differentiation. Carriers (beads and lipid vesicles) with sizes down to 30 nm can be manipulated with a high spatial and temporal resolution: they are positioned with a sub-micrometric precision on a specific cell compartment and the beginning of the stimulation can be triggered with millisecond precision. RhoGTPases are a Ras-related family of proteins that regulate many different functions including cell polarity, microtubule dynamics and membrane transport pathways. Here we combine local stimulation with FRET microscopy to study RhoGTPases spatial and temporal activation following guidance cue local stimulation. We used two different vectors for local delivery: silica micro-beads and micro-sized lipid vesicles. The experimental methods associated with neuronal growth cone local stimulation are discussed in detail, as well as the analysis methods. Here we present a protocol that enables to study neuronal growth cone cytoskeleton rearrangements in response to a gradient of molecules in a way that better mimics physiological conditions, and it can be similarly applied to each secreted molecule involved in cell signaling.

Type: Article
Title: Combining FRET and optical tweezers to study RhoGTPases spatio-temporal dynamics upon local stimulation
Open access status: An open access version is available from UCL Discovery
DOI: 10.14440/jbm.2017.159
Publisher version: http://doi.org/10.14440/jbm.2017.159
Language: English
Additional information: This work is licensed under a Creative Commons Attribution 3.0 License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/3.0/
UCL classification: UCL
UCL > Provost and Vice Provost Offices
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Medical Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Medical Sciences > Div of Medicine
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Medical Sciences > Div of Medicine > Wolfson Inst for Biomedical Research
URI: https://discovery.ucl.ac.uk/id/eprint/1572500
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