Expansion Revealing: Decrowding Proteins to Unmask Invisible Brain Nanostructures

Abstract

Expansion microscopy1,2 is an increasingly widespread technology for nanoimaging, because its precise physical magnification of biological specimens enables ordinary microscopes to achieve nanoscale effective resolutions. Fluorescent labels such as antibodies can be applied either before or after expansion, with the latter offering the potential for better access to proteins within densely packed environments3–7. We here assess this possibility of epitope decrowding through physical expansion of proteins away from each other, using a 20x expansion protocol that we call expansion revealing (ExR), by labeling, within intact brain circuits, the same set of synaptic proteins both pre- and post-expansion. This comparison shows that post-expansion labeling introduces minimal spatial error and off-target staining relative to pre-expansion staining, while revealing the presence of proteins that are invisible when stained pre-expansion. Using ExR, we show in intact brain tissue the alignment of presynaptic calcium channels with postsynaptic machinery in nanocolumns, which may facilitate precision synaptic transmission, as well as the existence of periodic amyloid-containing nanoclusters containing ion channel proteins in Alzheimer’s model mice, which may help generate novel hypotheses for Alzheimer’s pathology and neural excitability. Thus, the decrowding power of ExR is able to reveal novel nanostructures within intact brain circuitry, and may find broad use in biology and medicine for unmasking nanostructures of importance in normal functions and disease.

Expansion microscopy facilitates quantitative super-resolution studies of cytoskeletal structures in kinetoplastid parasites

“…This likely reflects an increased accessibility of their epitopes in the axoneme due its expansion. Such a positive effect of de-crowding due to ExM was previously described in other systems (Sarkar et al, 2020;Tillberg et al, 2016). On the other hand, we observed that two tested antibodies, BBA4 staining the BBs and pBBs in T. brucei (Woods et al, 1989) and mAb62 staining the flagella connector (Abeywickrema et al, 2019) (see also Suppl.…”

Nanoscale Imaging of Biomolecules using Molecule Anchorable Gel-enabled Nanoscale In-situ Fluorescence Microscopy

“…2o; Supplementary Video 1). Additionally, we observed that conventional antigen retrieval protocol is not needed to achieve such an improvement, suggesting expansion may lead to improved accessibility of antibodies to epitopes, which is consistent with a previous report 32 (Fig. 2p-q; Supplementary Fig.…”

Shortened tethering filaments stabilize presynaptic vesicles in support of elevated release probability during LTP in rat hippocampus

“…Other proteins that might comprise the priming filaments include Munc13-1, Munc18-1, and the CAPS family of proteins, all of which stabilize vesicles in the primed state (29,37,38). Rab3-interacting molecule (RIM) mediates the interaction of synaptic vesicles with calcium channels embedded in the presynaptic membrane and has also been identified as a tethering filament (18,39). In all cases, the shortening or zippering of these candidate tethering molecules could enhance the probability of neurotransmitter release.…”