MemCage · Plasma membrane probes for photo-controlled release of signaling molecules

Plasma membrane probes for photo-controlled release of signaling molecules

Introduction and the state-of-the art: A primary boundary between the extracellular environment and the cytoplasm, the plasma membrane (PM) of eukaryotic cells is a highly dynamic structure involves in diverse biological processes such as cellular uptake, neural communication, muscle contraction, and cell trafficking and signaling. The PM delimits the cell surface, therefore its shape and dynamics reflect the cell status including cell division and cell death processes. More importantly, the PMs thus play essential role in communication between cells, especially between neurons. In particular, the exocytosis process leads to liberation of neurotransmitters and neuromodulators. On the other hand, it is a platform for receptors that convert the recognition of neuroactive molecules into ion flux and/or action potential. Therefore, to modulate and monitor the activity of neurons, an attractive approach is to design molecular tools that target cell PMs. To modulate neuron activity, various photoremovable groups (photocages/cages) protected neurotransmitters and receptor’s agonists/antagonists have been used successfully in the cultured neurons/brain slices or in vivo models. Additionally, caged-membrane derived lipids were also reported to modulate neuron activity. However, to the best of our knowledge, the direction on caged neurotransmitters localized at the neuronal cell PMs have not been explored to date. In this direction, Klymchenko’s group has already developed fluorescent lipid membrane probes based on linkers bearing negatively charged sulfonate or zwitterionic head group and the alkyl chain of varied lengths. They target selectively the outer cell membrane leaflet without flip-flop to the inner leaflet, and, therefore, localize at the PM. Probes with longer alkyl chain strongly bind with the PM in an irreversible manner while the short chain probe exhibits the reversible binding with the PM and thus enables on-off switching required for super-resolution imaging. Recently, the group developed cyanine-based MemBright probes allowed the identification of neurons in brain tissues/neuromuscular junctions, and revealed that endogenous glutamate receptors aggregated at the axonal-dendritic contact site. These results really motivated me to think that the incorporation of a caging group (photocage) in the membrane probes could allow both visualization of neuronal organization, and selective photo-controlled release of the signaling molecules at the PM. In this regard, we planned to integrate three different ideas 1) use of the fluorescent caging groups tethered with the membrane targeting linkers for the selective labelling of the PM, 2) use of signaling molecule covalently masked with a fluorescent caging group to achieve the precise spatial and temporal control over neuronal modulation upon photoactivation of signaling molecule, and 3) use of two fluorescent photocages with independent absorption wavelengths to achieve the bidirectional photocontrol on signaling behavior in neurons by orthogonal two-color uncaging of two different signaling molecules at the same location. Research Objectives. General objective. Design and synthesis of membrane-targeting fluorescent probes for imaging of neurons and control of their activity by photorelease of signaling molecules namely dopamine and glutamate. Specific objective 1. Design and synthesis of the PM-targeting caged-signaling molecules. I proposed to design and synthesize chemical tools that implement the following functions: 1) selective labelling of the cellular PM, neuronal one in particular, 2) spatial and temporal photocontrol over the activity of signaling molecules and 3) bidirectional photocontrol on signaling behavior in neuronal cells. To realize these functions, I chose BODIPY- (λabs ~650 nm) and Coumarin-based (λabs ~450 nm) photocages bearing lipid membrane-targeting linkers. Specific objective 2. Application of the probes in cellular systems. First, I planned to confirm the localization of Memcaged-probes in HEK293 and neuronal cells. 1) HEK293 cells expressing the Dopamine D1 receptors will be employed to check the photorelease of dopamine. As the read out of dopamine photorelease, increase in Ca2+ level upon activation of D1 receptors will be measured via the response of Ca2+ probe (Fluo4) or genetically encoded Ca2+ sensor GCaMP7. Bidirectional photocontrol on the dopamine signaling will be evaluated by performing two-color uncaging of dopamine and its antagonist. After establishing the proof of principle in the HEK293 cells, photostimulation will be performed in the cultured hippocampal and primary dissociated cortical neurons. 2) Memcaged-Glutamate probes will be directly applied to the cultured hippocampal astrocytes and neurons. One-photon uncaging of the suitable probes will be performed to study neuronal signaling. Host group has already shown that AMPA Glutamate receptors are aggregated at the axon-dendritic junction. Therefore, Glutamate induced Ca2+ elevation via activation of AMPA Glutamate receptors will be monitored by imaging Fluo4 or genetically encoded GCaMP7. Specific objective 3. In vivo evaluation of Memcage probes: Further validation of our strategy for the release of neurotransmitters from the proposed Memcages in neurons and the deeper tissues of the brain of live mice. In these experiments, Memcaged-neurotransmitters will be injected into the brain of live mice and the photorelease will be done using the one-/two-photon setup, specially adapted for mice brain imaging. As the readout of photorelease of neurotransmitters, Ca2+ transients in neurons of mice brain expressing GCaMP7 will be measured to see the stimulation effect.

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