Abstract
Optogenetic methods for switching molecular states in cells are increasingly prominent tools in life sciences. Förster Resonance Energy Transfer (FRET)-based sensors can provide quantitative and sensitive readouts of altered cellular biochemistry, e.g. from optogenetics. However, most of the light-inducible domains respond to the same wavelength as is required for excitation of popular CFP/YFP-based FRET pairs, rendering the techniques incompatible with each other. In order to overcome this limitation, we red-shifted an existing CFP/YFP-based OP18 FRET sensor (COPY) by employing an sYFP2 donor and mScarlet-I acceptor. Their favorable quantum yield and brightness result in a red-shifted FRET pair with an optimized dynamic range, which could be further enhanced by an R125I point mutation that stimulates intramolecular interactions. The new sensor was named ROPY and it visualizes the interaction between the microtubule regulator stathmin/OP18 and free tubulin heterodimers. We show that through phosphorylation of the ROPY sensor, its tubulin sequestering ability can be locally regulated by photo-activatable Rac1 (PARac1), independent of the FRET readout. Together, ROPY and PARac1 provide spatiotemporal control over free tubulin levels. ROPY/PARac1-based optogenetic regulation of free tubulin levels allowed us to demonstrate that depletion of free tubulin prevents the formation of pioneer microtubules, while local upregulation of tubulin concentration allows localized microtubule extensions to support the lamellipodia.
Highlights
Optogenetic methods for switching molecular states in cells are increasingly prominent tools in life sciences
We decided to focus on fluorescent protein (FP) pairs which can elicit the maximum theoretical Förster Resonance Energy Transfer (FRET) potential in the desired excitation range, i.e. a donor FP with high quantum yield with optimal excitation at 514 nm and acceptor with high brightness and efficient maturation6,8. mScarlet-I was chosen as the best FRET acceptor currently available in the red spectrum[7] and we combined it with yellow FPs either Ypet[23] or sYFP224
Some have stated that single FP-based reporters might be the future of optogenetic readouts[33], there are far less of these reporters currently available than FRET sensors, and most single FP-based sensors provide qualitative rather than quantitative readout
Summary
Optogenetic methods for switching molecular states in cells are increasingly prominent tools in life sciences. In order to overcome this limitation, we red-shifted an existing CFP/YFP-based OP18 FRET sensor (COPY) by employing an sYFP2 donor and mScarlet-I acceptor Their favorable quantum yield and brightness result in a red-shifted FRET pair with an optimized dynamic range, which could be further enhanced by an R125I point mutation that stimulates intramolecular interactions. In this study we aimed to generate a new optogenetic application with FRET readout to allow simultaneous local control of microtubule dynamics and monitoring of local free tubulin concentrations. To this end we created a novel red-shifted stathmin sensor we termed ROPY. We show the compatibility of the ROPY FRET sensor with blue light responsive optogenetics and demonstrate that localized release of tubulin can stimulate microtubule growth in that area
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