Interrogating Synaptic Architecture: Approaches for Labeling Organelles and Cytoskeleton Components

Abstract

Synaptic transmission has been studied for decades, as a fundamental step in brain function. The structure of the synapse, and its changes during activity, turned out to be key aspects not only in the transfer of information between neurons, but also in cognitive processes such as learning and memory. The overall synaptic morphology has traditionally been studied by electron microscopy, which enables the visualization of synaptic structure in great detail. The changes in the organization of easily identified structures, such as the presynaptic active zone, or the postsynaptic density, are optimally studied via electron microscopy. However, few reliable methods are available for labeling individual organelles or protein complexes in electron microscopy. For such targets one typically relies either on combination of electron and fluorescence microscopy, or on super-resolution fluorescence microscopy. This review focuses on approaches and techniques used to specifically reveal synaptic organelles and protein complexes, such as cytoskeletal assemblies. We place the strongest emphasis on methods detecting the targets of interest by affinity binding, and we discuss the advantages and limitations of each method.

Keywords: actin; cytoskeleton; nanoscopy; super-resolution; synapse; vesicles.

FIGURE 1

Synaptic organelles and specificity of probes directed toward recycling membranes. (A) Schematic representation of main organelles present at the synapses. The pH level of endosomal components is visualized with different shades of blue. The different pHs aid in differential labeling by membrane probes, as shown in (B). (B) Membranes that are labeled by different membrane-labeling tools highlighted in green, the identity of the organelles preserved from (A).

FIGURE 2

Tools for visualizing recycling vesicles. (A) The mechanism of styryl dyes action. Colored and gray shapes represent fluorescent and non-fluorescent FM molecules, respectively. Upon addition to cellular medium, FM dye incorporates into the outer leaflet of the PM and becomes fluorescent. Following endocytosis, these fluorescent molecules are trapped in recycling vesicles, while unspecific signal from the PM can be eliminated by washing the cells. Since FM dye incorporation into the membrane is reversible, after the vesicle is fused with the PM during exocytosis, the fluorescent signal is lost again. (B) Chemical structures of FM 1–43, FM 1–43FX, and mCLING.

FIGURE 3

CypHer5E as a tool to visualize the synaptic vesicle cycle. CypHer5E is a pH-sensitive fluorophore that can be coupled to antibodies against the luminal domain of synaptic vesicle proteins. Following exocytosis, the luminal domains of the antibodies are exposed to the extracellular medium and the antibodies can bind them. At the neutral pH of the extracellular medium, the fluorophore is quenched. When the luminal pH is lowered after endocytosis, the fluorophores bound to the synaptic vesicle proteins through respective antibodies become fluorescent and allow visualization of internalized vesicles. When such a vesicle fuses with the plasma membrane during synaptic activity, the fluorescence is lost again.

FIGURE 4

Comparison of the size of quantum dots with synaptic structures and other probes used to label the plasma membrane, recycling membranes, or membrane receptors in the synapse. Realistic sizes are presented for all labels. For quantum dots we assume that their core is covered by a streptavidin layer, to which antibodies are then attached.

FIGURE 5

Commonly used actin probes. Green shapes represent fluorescent moieties; all molecules shown in different shades of gray are non-fluorescent and hence are invisible under fluorescent microscope. Size differences of the shapes approximately represent size differences of the molecules. (A) Ectopically expressed actin-GFP partially incorporates into actin filaments but also contributes to background fluorescence from monomeric actin-GFP molecules and increases the concentration of the monomers. (B) Due to small size of dyes, chemically labeled actin has higher polymerization ability, however, still displays significant background fluorescence from monomers and also increases the concentration of actin monomers. (C) Density of antibody labeling depends on epitope accessibility and is significantly restricted by large size of antibody molecules, which also introduces large linkage errors; background fluorescence observed from antibodies bound to actin monomers in solution. (D) Comparison of fluorophore displacement from targeted epitope caused by common immunostaining procedures with resolution abilities of modern super resolution methods. When a combination of primary and secondary antibodies is used to image an actin filament, fluorophores of antibodies that recognize neighboring actin subunits might be located more than 50 nm apart (10 times larger distance then a thickness of an actin filament). Modern microscopy techniques can resolve objects that are as close as few nanometers apart, so usage of such large probes leads to significant loss of advantages super-resolution methods can offer. (E) Phalloidin is a small chemical that binds exclusively F-actin with high specificity and affinity, shows high density of labeling and low background signal. (F) Genetically encoded actin binders (F-tractin illustrated as an example) fused to GFP bind F-actin in vivo. Unbound molecules contribute to background fluorescence, but the concentration of actin monomers is not changed. (G) Low affinity of Lifeact binding to F-actin can be used for certain types of super resolution microscopy. Here Lifeact coupled to a dye acts as an exchangeable probe. Multiple frames are collected with Lifeact molecules having different locations in different frames. Post-imaging processing allows reconstructing F-actin architecture from all individual Lifeact locations. (H) SiR-actin is cell membrane-permeable, specifically labels F-actin and additionally has low fluorescence when not bound to F-actin (off state) but cannot be fixed by aldehydes.