Targeting ion sensors to synaptic vesicles using a Nanobody against luminal Synaptotagmin 1
by Rashi Goel
Date of Examination:2021-11-02
Date of issue:2022-02-24
Advisor:Prof. Dr. Reinhard Jahn
Referee:Prof. Dr. Reinhard Jahn
Referee:Prof. Dr. Silvio Rizzoli
Referee:Prof. Dr. Claudia Steinem
Referee:Prof. Dr. Ulf Diederichsen
Referee:Prof. Dr. Tiago Fleming Outeiro
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Name:Rashi Goel_Final PhD Thesis_Sept 2021_FINAL.pdf
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EnglishThe synapse is a specialized nanostructure that connects two neurons where the ionic composition in each of its nano compartments is the driving process for synaptic transmission. Synaptic transmission relies on the repetitive fusion of neurotransmitter-containing synaptic vesicles (SVs) with the plasma membrane (PM) of the presynaptic neuron, to release the neurotransmitter molecules into the synaptic cleft that bind to the receptors present at the postsynaptic neuron. The amount of neurotransmitters stored in an SV directly modulates the strength of the synaptic transmission. SVs are filled with neurotransmitters through the neurotransmitter transporters (NTs) at the expense of the energy derived from the electrochemical gradient across the SV membrane. The electrochemical gradient comprises a pH gradient ΔpH and an electrical gradient Δψ, both of which are regulated by different ion fluxes across the vesicle membrane. The ionic composition inside the SV constantly changes during the recycling of SVs. SVs are recycled in the exo-endocytosis cycle, where an SV fuses with the PM, and its lumen gets exposed to the extracellular fluid followed by its retrieval through endocytosis. During endocytosis, the SV encapsulates the ionic content from the extracellular fluid. An SV then exchanges its ionic content with the neurotransmitter molecules and stores the neurotransmitters inside its lumen until the further round of release. The type and quantity of ions residing in an SV are critical in regulating the activity of NTs by either allosteric binding of the ion with the NT or by a coupled exchange of ions with the neurotransmitter molecules via the NTs. This makes it especially critical for an SV to maintain the appropriate ionic stoichiometry to allow an efficient filling with the neurotransmitters. How do the neurotransmitter transporters remain operational when the solute composition inside the SV changes dramatically? To study this, there is a need to have ion sensors targeted to the SV lumen to understand how the neurotransmitter transporters operate to fill the SV with thousands of neurotransmitter molecules within seconds. The project aims to develop a method to target fluorescence-based ion-sensitive probes and measure the concentration of various ions present inside the SVs. To this aim, we have generated a nanobody (Nb) against the luminal domain of an SV protein, Synaptotagmin 1 (Syt1). By fusing the Nb with a HaloTag protein (Nb-Halo), we have developed a flexible system, where the Nb-Halo can bind with an ion probe conjugated to a Halo ligand. In this method, the ion probe serves as a sensing module that can be delivered into the lumen of the SV using the Nb as a targeting module. Thus, the Nb probes can be used to target the sensor to the lumen of SVs during the endocytosis process. As there are Syt1 molecules stranded at the PM from the rounds of Exo and endocytosis, the Nb-Halo fluorescent probe binds with them and makes it difficult to distinguish the fluorophore signal of SV from that of the PM. To remove nonspecific fluorescence generated from probes at the PM, a TEV protease cleavage site has been engineered in the Nb. Thus, it enables the removal of the sensors from the Nb at the PM and helps us to obtain a clean vesicular signal. Using the above method, we have targeted novel pH and Cl- sensors and measured the luminal resting pH of Glutamatergic and GABAergic SVs in cultured hippocampal neurons. We have measured a resting pH of ~5.1 in both Glutamatergic and GABAergic SVs, which is much lower than what has been reported earlier. I hope that our method of targeting specific sensors into SVs will open new avenues to measure various others ion concentrations and will allow mapping out the ionic composition of an SV.
Keywords: Synapse; Neurotransmitter transporters; Glutamate and GABA; ion-sensors; Nanobody