Periodic Reporting for period 1 - PhotoXLink (Development of biomolecular tools for the spatiotemporal photocontrol of AMPA receptors mobility)
Período documentado: 2019-07-01 hasta 2021-06-30
The understanding of learning and memory in our brain at cellular and molecular levels, and specifically, the role of AMPA receptors’ mobility regulation at the synaptic site in neurons is of special interest. The regulation of AMPAR trafficking is crucial to its synaptic function. Indeed, the widely accepted model at present is that AMPARs are in dynamic equilibrium between synaptic, extrasynaptic and intracellular compartments. The changes in this equilibrium, affects AMPAR numbers at the synaptic sites what underlies long term plasticity of the efficacy of synaptic transmission, such as LTP (long term potentiation) and LTD (long term depression). This dynamic regulation is the basis of current molecular theories of learning and memory. Despite being a core locus of molecular information storage, the nature of this regulation is not yet completely understood because of the lack in technical tools. The principal aim of this project was to develop innovative methods to control AMPAR mobility, and to monitor the resulting effects with unique spatial and temporal resolution. This addressed technical limitations and allowed investigating with a unique spatiotemporal resolution the impact of AMPARs mobility in short and long term plasticity in the brain function. More precisely, we aimed to design tools that provide a control of endogenous or genetically modified AMPARs mobility by way of light-controlled crosslinking. This project consisted on developing innovative methods to photoreversibly control receptor clustering/stabilization in order to be able to investigate how the dynamics of AMPAR is linked to the functional properties of synaptic transmission in normal and pathological brain states. I used a two-step strategy to achieve our goals, where we first focused on the photosensitive domain optimization and then implemented our work to more efficient binders. The project was organized around the following three objectives: (1) Design and production of a first generation of photocrosslinkers, (2) Validate and characterize the system in cellular environments and (3) Expand the photocrosslinking systems to minimally invasive epitope/binder pairs (2nd generation photocrosslinkers).
- I engineered 1st generation of photocrosslinkers: It consisted on the rational and structural-based design of the photosensitive domains fused to the model binders. It included the strategy by molecular biology first for generating a construct that was used for the protein expression in bacteria. They were efficiently produced in E.Coli and purified by His-tag purification columns. Moreover, I characterized the ligands and verify that their oligomerization state and their affinity for their target were consistent with expectations by biophysical techniques.
- I validated and characterized them in cell environments: In order to do the validation of the approach in cellular environment, I performed FRET-FLIM experiments. For that aim, I first generated a novel red shifted-FRET pair and validated it in cells. For that validation I created new molecular biology constructs and generated the required control conditions. Moreover, I ensured that the FRET-pair could be used for optogenetics and more specifically with Dronpa145N. For this aim, I used Dronpa to control protein-protein interactions (article in preparation) and monitor the interaction by using the FRET-pair. The 1st generation Photocrosslinkers were validated by FRET-FLIM in heterologous cells (COS-7 cells) and I determining the best conditions of use and their limits. I determined the specific amount of energy for the photoactivation and the crosslinking effect by using a system based on pDisplay. We have also checked the effect of the crosslinkers on AMPAR localization by u-PAINT technique.
- I engineered 2nd generation of photocrosslinkers (expanded photocrosslinkers): After selecting the most suitable photosensitive domains for the photocrosslinkers, I implemented the strategy developed that is described above to minimally invasive epitopes such as biotin. The same methods as the ones described above were used and the work essentially consisted on producing fully functional fusions of the photosensitive domains to the improved binders.
- Dissemination: Scientific journals. Two papers are in preparation (paper 1: “A red-shifted FRETpair for the engineering of optogenetic protein-protein interaction modulators” and paper 2: “Photoreversible crosslinkers for fine tuning of AMPA receptors mobility” and expected to be published in 2022. The authors of the manuscript will send a communication to the Project Officer upon submission. Conferences, congresses, and other outreach activities. The knowledge generated by PhotoXLink was exploited and disseminated by different activities, including: (i) Oral communications: I presented my results during the whole granted period (24 months + 4 months due to COVID extension) at local seminars (once per month at DOFS team) and one talk at IINS meeting in June 2020. I took part in “Women in Photonics” congress by an oral presentation in November 2020. I was also invited by Bordeaux NeuroCampus to give a “Tech´Talk” in April 2021 about the main imaging technique I used for PhotoXLink project for the validation of the tools developed. (ii) Outreach activities: I took part in 4 outreach activities events: European Researchers´ Night in Bordeaux (2019 and 2020)) and during October and November 2021, I participated in 2 outreach activities organized by Declics organization and are supported by the Cercle FSER. (ii) I also created “VPhotoXLink” Twitter Account to share the latest news about the project including the scientific progress of the project, congresses I have assisted and outreach activities in which I have taken part and share latest news about the optogenetic tools
- I validated and characterized them in cell environments: In order to do the validation of the approach in cellular environment, I performed FRET-FLIM experiments. For that aim, I first generated a novel red shifted-FRET pair and validated it in cells. For that validation I created new molecular biology constructs and generated the required control conditions. Moreover, I ensured that the FRET-pair could be used for optogenetics and more specifically with Dronpa145N. For this aim, I used Dronpa to control protein-protein interactions (article in preparation) and monitor the interaction by using the FRET-pair. The 1st generation Photocrosslinkers were validated by FRET-FLIM in heterologous cells (COS-7 cells) and I determining the best conditions of use and their limits. I determined the specific amount of energy for the photoactivation and the crosslinking effect by using a system based on pDisplay. We have also checked the effect of the crosslinkers on AMPAR localization by u-PAINT technique.
- I engineered 2nd generation of photocrosslinkers (expanded photocrosslinkers): After selecting the most suitable photosensitive domains for the photocrosslinkers, I implemented the strategy developed that is described above to minimally invasive epitopes such as biotin. The same methods as the ones described above were used and the work essentially consisted on producing fully functional fusions of the photosensitive domains to the improved binders.
- Dissemination: Scientific journals. Two papers are in preparation (paper 1: “A red-shifted FRETpair for the engineering of optogenetic protein-protein interaction modulators” and paper 2: “Photoreversible crosslinkers for fine tuning of AMPA receptors mobility” and expected to be published in 2022. The authors of the manuscript will send a communication to the Project Officer upon submission. Conferences, congresses, and other outreach activities. The knowledge generated by PhotoXLink was exploited and disseminated by different activities, including: (i) Oral communications: I presented my results during the whole granted period (24 months + 4 months due to COVID extension) at local seminars (once per month at DOFS team) and one talk at IINS meeting in June 2020. I took part in “Women in Photonics” congress by an oral presentation in November 2020. I was also invited by Bordeaux NeuroCampus to give a “Tech´Talk” in April 2021 about the main imaging technique I used for PhotoXLink project for the validation of the tools developed. (ii) Outreach activities: I took part in 4 outreach activities events: European Researchers´ Night in Bordeaux (2019 and 2020)) and during October and November 2021, I participated in 2 outreach activities organized by Declics organization and are supported by the Cercle FSER. (ii) I also created “VPhotoXLink” Twitter Account to share the latest news about the project including the scientific progress of the project, congresses I have assisted and outreach activities in which I have taken part and share latest news about the optogenetic tools
We have developed for the first time reversible crosslinkers. More precisely, we have generated tools that provide a control of endogenous or genetically modified AMPARs mobility by way of light-controlled crosslinking. The main advantage of these crosslinkers is that we can now monitor the resulting effects with unique spatial and temporal resolution. The “optogenetic” tools we designed stood out from the current applications of light sensitive domains in the sense that we aimed to target them via specific binders to endogenous proteins and thereby alleviated all repetitive genetic manipulation of the proteins of interest. Additionally, in our strategies we produced rather than expressed them (as in standard optogenetic applications), which made them comparable to pharmaceutical agents. With the new tools we achieved to overcome previous technical limitations to allow people from my current team to start investigating with a unique spatiotemporal resolution the impact of AMPARs mobility in short and long term plasticity in the brain function. The validation of the tools developed by the project here presented applied to a specific set of receptors (AMPAr) could also benefit a large scientific community interested in protein studies via implementation to other systems or set of proteins, engaging organisations or individuals inside and outside academia. In this regard, a new collaboration has been created within IINS with the “Spatio-Temporal and Mechanical Control of Motile Structures” research team that are applying the Photocrosslinkers to study motile structures.