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

Authors

  • Federico Iseppon Neuroscience Area - International School for Advanced Studies
  • Luisa MR Napolitano Neuroscience Area - International School for Advanced Studies
  • Vincent Torre Neuroscience Area - International School for Advanced Studies
  • Dan Cojoc Optical Manipulation Lab, CNR-IOM, the National Research Council of Italy - Institute Of Materials

DOI:

https://doi.org/10.14440/jbm.2017.159

Keywords:

FRET, growth cones, guidance molecules, local stimulation, Optical Tweezers.

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.

Author Biography

Federico Iseppon, Neuroscience Area - International School for Advanced Studies

PhD Student - Neurobiology Department

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Published

2017-03-07

How to Cite

1.
Iseppon F, Napolitano LM, Torre V, Cojoc D. Combining FRET and optical tweezers to study RhoGTPases spatio-temporal dynamics upon local stimulation. J Biol Methods [Internet]. 2017Mar.7 [cited 2021Oct.15];4(1):e65. Available from: https://jbmethods.org/jbm/article/view/159

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Section

Protocols