Super-resolution imaging of synaptic and extra-synaptic AMPA receptors with different-sized fluorescent probes
- University of Illinois at Urbana-Champaign, United States
- University of California, San Francisco, United States
- University of Illinois at Urbana Champaign, United States
- University of Campinas, Brazil
- University of Chicago, United States
- State University of New York, United States
- University of Texas, United States
Abstract
Previous studies tracking AMPA receptor (AMPAR) diffusion at synapses observed a large mobile extrasynaptic AMPAR pool. Using super-resolution microscopy, we examined how fluorophore size and photostability affected AMPAR trafficking outside of, and within, post-synaptic densities (PSDs) from rats. Organic fluorescent dyes (≈4 nm), quantum dots, either small (≈10 nm diameter; sQDs) or big (>20 nm; bQDs), were coupled to AMPARs via different-sized linkers. We find that >90% of AMPARs labeled with fluorescent dyes or sQDs were diffusing in confined nanodomains in PSDs, which were stable for 15 minutes or longer. Less than 10% of sQD-AMPARs were extrasynaptic and highly mobile. In contrast, 5–10% of bQD-AMPARs were in PSDs and 90-95% were extrasynaptic as previously observed. Contrary to the hypothesis that AMPAR entry is limited by the occupancy of open PSD "slots", our findings suggest that AMPARs rapidly enter stable "nanodomains" in PSDs with lifetime ≥15 minutes, and do not accumulate in extrasynaptic membranes.
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Author details
Funding
National Institutes of Health (NIH NS090903)
- William N Green
- Paul R Selvin
National Science Foundation (PHY-1430124)
- Paul R Selvin
National Science Foundation (CBET-1264051)
- Sheldon Park
National Institutes of Health (NIH NS100019)
- William N Green
- Paul R Selvin
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Ethics
Animal experimentation: Primary hippocampal cultures were prepared from E18 rats according to UIUC guidelines. All rats were handled according to approved institutional animal care and use committee (IACUC) protocols (#15254) of UIUC.
Copyright
© 2017, Lee et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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Super-resolution imaging of synaptic and extra-synaptic AMPA receptors with different-sized fluorescent probes
eLife 6:e27744.
https://doi.org/10.7554/eLife.27744
Further reading
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- Biochemistry and Chemical Biology
- Structural Biology and Molecular Biophysics
The two identical motor domains (heads) of dimeric kinesin-1 move in a hand-over-hand process along a microtubule, coordinating their ATPase cycles such that each ATP hydrolysis is tightly coupled to a step and enabling the motor to take many steps without dissociating. The neck linker, a structural element that connects the two heads, has been shown to be essential for head–head coordination; however, which kinetic step(s) in the chemomechanical cycle is ‘gated’ by the neck linker remains unresolved. Here, we employed pre-steady-state kinetics and single-molecule assays to investigate how the neck-linker conformation affects kinesin’s motility cycle. We show that the backward-pointing configuration of the neck linker in the front kinesin head confers higher affinity for microtubule, but does not change ATP binding and dissociation rates. In contrast, the forward-pointing configuration of the neck linker in the rear kinesin head decreases the ATP dissociation rate but has little effect on microtubule dissociation. In combination, these conformation-specific effects of the neck linker favor ATP hydrolysis and dissociation of the rear head prior to microtubule detachment of the front head, thereby providing a kinetic explanation for the coordinated walking mechanism of dimeric kinesin.
