BBSRC NLD Doctoral Training Partnership: iGly. Development and characterisation of novel genetically-encoded glycine indicators for imaging inhibitory neurotransmission

In the central nervous system (CNS), glutamate and glycine are pivotal excitatory and inhibitory neurotransmitters, respectively. Optical sensors for tracking neurotransmitters in space and time can provide important information on neurotransmission and signal processing. As an example, the development of the indicator iGluSnFR for the neurotransmitter glutamate, has led to ground-breaking advances in excitatory neurotransmission, particularly on synaptic spillover and exocytic vesicle fusion. However, there is no equivalent biosensor to monitor glycine homeostasis and therefore information on inhibitory neurotransmission remains limited. Visualising glycine dynamics in vivo using fluorescent sensors would represent a significant new approach that will greatly contribute to understanding the processes mediated by this major neurotransmitter in health and disease. In order to achieve this long-term goal, new glycine biosensors are urgently required.

Considering the high demand for such biosensors, we propose an innovative project intended to fill this major experimental gap by developing a “toolbox” of entirely new genetically-encoded indicators that will allow in vitro, ex vivo and in vivo studies of inhibitory glycine neurotransmission. Based on our track record in developing protein-based fluorescent sensors, we aim to create a unique palette of genetically-encoded glycine indicators (iGly) that will allow direct, real-time, visualisation of glycine dynamics in tissues or living animals.

Our objectives are to:

1. Generate first generation glycine biosensors based on circularly permutated EGFP (cpEGFP) and NR1 S1S2 loops from NMDA receptor.

2. Quantify biosensor responses to glycine in vitro and in cells. We will obtain a detailed biophysical characterisation of the biosensors (fluorescence dynamic range, affinity, selectivity, quantum yield, pH-dependence, binding kinetics) using spectrofluorimetry and stopped-flow kinetics.

This project will combine molecular biology, protein biochemistry, advanced protein biophysics and imaging techniques to develop and characterise intensometric glycine indicators with high signal-to-noise ratio and compatible with multi-colour imaging. These novel tools will facilitate the elucidation of important information on inhibitory neurotransmission and will revolutionise our understanding of the role of glycine in the CNS. iGly will allow researchers to address questions that are unapproachable with the currently available techniques and enable advances in scientific disciplines such as neurophysiology and healthy ageing.

HOW TO APPLY:

Applications should be made by emailing bbsrcdtp@liverpool.ac.uk with:

• a CV (including contact details of at least two academic (or other relevant) referees);
• a covering letter – clearly stating your first-choice project, and optionally 2nd ranked project, as well as including whatever additional information you feel is pertinent to your application; you may wish to indicate, for example, why you are particularly interested in the selected project(s) and at the selected University;
• copies of your relevant undergraduate degree transcripts and certificates;
• a copy of your passport (photo page).

A GUIDE TO THE FORMAT REQUIRED FOR THE APPLICATION DOCUMENTS IS AVAILABLE AT https://www.nld-dtp.org.uk/how-apply. Applications not meeting these criteria may be rejected.

In addition to the above items, please email a completed copy of the Additional Details Form (as a Word document) to bbsrcdtp@liverpool.ac.uk. A blank copy of this form can be found at: https://www.nld-dtp.org.uk/how-apply.

Informal enquiries may be made to nhelassa@liverpool.ac.uk

The deadline for all applications is 12noon on Monday 22nd July 2024.

 

Part-Time Study Options

All NLD DTP PhDs are available as part time or full time, with part time being a minimum of 50% of full time. Please discuss potential part time arrangements with the primary supervisor before applying to the programme.

Project CASE Status

This project is not a CASE project. While individual applicant quality is our overriding criterion for selection, the NLD DTP has a commitment to fund 8 CASE projects per year – as such, CASE projects may be favoured in shortlisting applicants when candidates are otherwise deemed to be equal or a consensus on student quality cannot be reached.