Super-resolution imaging of glutamate receptors trafficking at live synapses

AMPA Receptor;super-resolution imaging;Photoactivated localization microscopy (PALM);single particle tracking (SPT)

Cai, En

关键词: AMPA Receptor;    super-resolution imaging;    Photoactivated localization microscopy (PALM);    single particle tracking (SPT);   

Others  :  https://www.ideals.illinois.edu/bitstream/handle/2142/50429/En_Cai.pdf?sequence=1&isAllowed=y

美国|英语

来源: The Illinois Digital Environment for Access to Learning and Scholarship

【 摘 要 】

Neurons are the basic building blocks in the nervous system. They communicate with each other via synapses. The synapse is the fundamental structure for signal transmission, which is composed of presynapse, postsynapse and a 20 nm synaptic cleft in between. Located on the tip of postsynapse is the postsynaptic density (PSD) that receives signals (neurotransmitters) from presynapse via glutamate receptors, mainly AMPA receptors and NMDA receptors. Trafficking of AMPA receptors in and out of synapses affect synaptic plasticity, which underlies the molecular mechanism of learning and memory. Lateral diffusion of AMPA receptors near synapses has been extensively studied in the past decade. However, most previous studies were based on two-dimensional single particle tracking; lacking information on receptor movement in the z direction. Further more, the postsynapses were imaged with conventional fluorescent microscopy with diffraction-limited resolution. As a result, the detail localization of AMPA diffusion sites on the synapse cannot be resolved. To address these problems, we employed high-precision three-dimensional single particle tracking (SPT) to image receptor diffusion; and super-resolution imaging technique Photoactivated localization microscopy (PALM) to localize the PSD. Combining PALM and SPT, we were able to examine the AMPA receptor diffusion behavior around synapse in great detail. It was discovered that very few of the AMPA receptors labeled with commercial QDs (15-35 nm) could diffuse into the synapse. To improve the accessibility of the synaptic cleft by QD labeled AMPA receptors, we developed a novel method to coat quantum dot cores, producing a smaller streptavidin-functionalized sQDs (8.9 ± 0.2 nm in diameter) than commercial QDs. These sQDs enabled specific labeling of AMPA receptors and allowed much greater access to the synaptic region than commercial QDs. Further more, we directly observed sQD labeled AMPA receptors stabilized on the surface of postsynapse within confined regions, also known as AMPA receptor nanodomains. The photostability of sQD enable the observation of slow migration of a single AMPA receptor inside a nanodomain.Even boarder range of application of sQD were achieved by extending of functional group of sQDs beyond streptavidin, to biotin and GBP. Notably, sQD functionalized with GBP can successfully label NMDA receptors with extracellular GFP tags, which reveal different diffusion pattern from AMPA receptors.