Super-resolution imaging of synaptic and Extra-synaptic AMPA receptors with different-sized fluorescent probes

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 min 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 min, and do not accumulate in extrasynaptic membranes.

Pin1 Binding to Phosphorylated PSD-95 Regulates the Number of Functional Excitatory Synapses

“…FIONA on neurons expressing EGFP or Pin1 and EGFP (Figure 3E) as in Yildiz et al (2003), Cai et al (2014), Lee et al (2017). Pin1 overexpression did not alter the size of GluA2 nanodomains, the area, or the number of nanodomains (Figures 3F-H).…”

“…In brief, on 12-13 days in vitro (DIV), neurons were co-transfected with control, Pin1 O.E. or KD plasmids, GluA2-AP (1 µg/coverslip), and BirA-ER (1 µg/coverslip) by using Lipofectamine 2000 transfection reagent as in Lee et al (2017). At 24 ∼ 72 h after transfection, the coverslips were transferred to warm imaging buffer (HBSS supplemented with 10 mM Hepes, 1 mM MgCl 2 , 1.2 mM CaCl 2 , and 2 mM D-glucose) for 5 min incubation and mounted onto an imaging dish (Warner RC-40LP).…”

“…To evaluate if Pin1 affects the nano-organization of PSD-95 in post-synaptic spines, we performed superresolution microscopy of endogenous PSD-95 in cultured neurons transfected with EGFP, Pin1 or the C113S mutant (Figure 3A). Previous studies have shown that AMPARs and PSD-95 lie in nanodomains, small clusters of about 70 nm in diameter (Nair et al, 2013;Cai et al, 2014;Constals et al, 2015;Li et al, 2016;Lee et al, 2017). It is hypothesized that the nanodomain organization of synaptic proteins plays a key role in excitatory synaptic transmission.…”

nanoPaint: Dynamic Imaging of Nanoscopic Structural Plasticity of the Plasma Membrane

“…demonstrated that slightly larger QD bioconjugates (QD-streptavidin bound to a secondary plus primary antibody) could be detected in the synaptic cleft, where they possessed smaller coefficients of diffusion as compared to the extra-synaptic QDs (Dahan et al, 2003). On the other hand, a recent study comparing different sizes of nanoconstructs recognizing the postsynaptic AMPA receptor, which has an extracellular domain of 12 nm, showed that steric impairment hampers the accessibility and diffusion into the synaptic cleft for large QDstreptavidin nanoconstructs (>20 nm in diameter) (Lee et al, 2017). Recognizing the relatively small extracellular domain of CB1 receptor, our QDs appear to be small enough to access and effectively map the entire topography of presynaptic boutons in mature synapses.…”

“…This limitation may be lifted using FRET-based 3 probes (Auer et al, 2017), though at the expense of adding two fluorophores, complex antibody-DNA constructs and finely-tuned complementary strands. Noteworthy, this technique has so far only been demonstrated for fixed cells (Legant et al, 2016;Lee et al, 2017;Deußner-Helfmann et al, 2018).…”