RT Journal Article
SR Electronic
T1 Super-resolution Imaging of Synaptic and Extra-synaptic Pools of AMPA Receptors with Different-sized Fluorescent Probes
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 096966
DO 10.1101/096966
A1 Sang Hak Lee
A1 Chaoyi Jin
A1 En Cai
A1 Pinghua Ge
A1 Yuji Ishitsuka
A1 Kai Wen Teng
A1 Andre A. de Thomaz
A1 Duncan Nall
A1 Murat Baday
A1 Okunola Jeyifous
A1 Daniel Demonte
A1 Christopher M. Dundas
A1 Sheldon Park
A1 William N. Green
A1 Paul R. Selvin
YR 2016
UL http://biorxiv.org/content/early/2016/12/27/096966.abstract
AB Whether AMPA receptors (AMPARs) enter into neuronal synapses, by exocytosis from an internal pool, or by diffusion from an external membrane-bound pool, is hotly contested. 3D super-resolution fluorescent nanoscopy to measure the dynamics and placement of AMPAR is a powerful method for addressing this issue. However, probe size and accessibility to tightly packed spaces can be limiting. We have therefore labeled AMPARs with differently sized fluorophores: small organic fluorescent dyes (~ 4 nm), small quantum dots (sQD, ~10 nm in diameter), or big (commercial) quantum dots (bQD, ~ 20 nm in diameter). We then compared their diffusion rate, trajectories, and placement with respect to a postsynaptic density (PSD) protein, Homer 1c. Labeled with the small probes of sQDs or organic fluorophores, we find that AMPARs are located largely within PSDs (~73-93%), and generally reside in “nanodomains” with constrained diffusion. In contrast, when labeled with bQDs, only 5-10% of AMPARs are within PSDs. The results can be explained by relatively free access, or lack thereof, to synaptic clefts of the AMPARs when labeled with small or big probes, respectively. This implies that AMPARs primarily enter PSDs soon after their exocytosis and not from a large diffusive pool of extrasynaptic AMPARs.