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

Federico Iseppon, Luisa MR Napolitano, Vincent Torre, Dan Cojoc

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.

DOI

Versatile microsphere attachment of GFP-labeled motors and other tagged proteins with preserved functionality

Michael Bugiel, Horatiu Fantana, Volker Bormuth, Anastasiya Trushko, Frederic Schiemann, Jonathon Howard, Erik Schäffer, Anita Jannasch

Microspheres are often used as handles for protein purification or force spectroscopy. For example, optical tweezers apply forces on trapped particles to which motor proteins are attached. However, even though many attachment strategies exist, procedures are often limited to a particular biomolecule and prone to non-specific protein or surface attachment. Such interactions may lead to loss of protein functionality or microsphere clustering. Here, we describe a versatile coupling procedure for GFP-tagged proteins via a polyethylene glycol linker preserving the functionality of the coupled proteins. The procedure combines well-established protocols, is highly reproducible, reliable, and can be used for a large variety of proteins. The coupling is efficient and can be tuned to the desired microsphere-to-protein ratio. Moreover, microspheres hardly cluster or adhere to surfaces. Furthermore, the procedure can be adapted to different tags providing flexibility and a promising attachment strategy for any tagged protein.

DOI