Ever since their introduction two decades ago, single-molecule (SM) fluorescence methods have matured and branched out to address numerous biological questions, which were inaccessible via ensemble measurements. Among the current arsenal, SM fluorescence techniques have capabilities of probing the dynamic interactions of nucleic acids and proteins via Förster (fluorescence) resonance energy transfer (FRET), tracking single particles over microns of distances, and deciphering the rotational motion of multisubunit systems. In this exciting era of transitioning from in vitro to in vivo and in situ conditions, it is anticipated that SM fluorescence methodology will become a common tool of molecular biology.

Proteins perform most cellular functions in macromolecular complexes. The same protein often participates in different complexes to exhibit diverse functionality. Current ensemble approaches of identifying cellular protein interactions cannot reveal physiological permutations of these interactions. Here, we describe a single molecule pull-down (SiMPull) assay that combines the principles of conventional pull-down assay with single molecule fluorescence microscopy and enables direct visualization of individual cellular protein complexes. SiMPull can reveal how many proteins and of which kinds are present in the in vivo complex, as we show using protein kinase A. We then demonstrate a wide applicability to various signaling proteins found in cytosol, membrane, and cellular organelles, and to endogenous protein complexes from animal tissue extracts. The pulled down proteins are functional and are used, without further processing, for single molecule biochemical studies. SiMPull should provide a rapid, sensitive and robust platform for analyzing protein assemblies in biological pathways.

The antimicrobial peptide protegrin-1 (PG-1) interacts with membranes in a manner that strongly depends on membrane lipid composition. In this research we use an approach representing the outer layers of bacterial and red blood cell membranes with lipid monolayers and using a combination of insertion assay, epifluorescence microscopy, and surface x-ray scattering to gain a better understanding of antimicrobial peptide's mechanism of action. We find that PG-1 inserts readily into anionic dipalmitoyl-phosphatidylglycerol, palmitoyl-oleoyl-phosphatidylglycerol, and lipid A films, but significantly less so into zwitterionic dipalmitoyl-phosphatidylcholine, palmitoyl-oleoyl-phosphatidylcholine, and dipalmitoyl-phosphatidylethanolamine monolayers under similar experimental conditions. Epifluorescence microscopy shows that the insertion of PG-1 into the lipid layer results in the disordering of lipid packing; this disordering effect is corroborated by grazing incidence x-ray diffraction data. X-ray reflectivity measurements further point to the location of the peptide in the lipid matrix. In a pathologically relevant example we show that PG-1 completely destabilizes monolayer composed of lipid A, the major component in the outer membrane of Gram-negative bacteria, which is likely to be the mechanism by which PG-1 disrupts the outer membrane, thus allowing it to reach the target inner membrane.

Interaction of the human antimicrobial peptide LL-37 with lipid monolayers has been investigated by a range of complementary techniques including pressure-area isotherms, insertion assay, epifluorescence microscopy, and synchrotron x-ray scattering, to analyze its mechanism of action. Lipid monolayers were formed at the air-liquid interface to mimic the surface of the bacterial cell wall and the outer leaflet of erythrocyte cell membrane by using phosphatidylglycerol (DPPG), phosphatidylcholine (DPPC), and phosphatidylethanolamine (DPPE) lipids. LL-37 is found to readily insert into DPPG monolayers, disrupting their structure and thus indicating bactericidal action. In contrast, DPPC and DPPE monolayers remained virtually unaffected by LL-37, demonstrating its nonhemolytic activity and lipid discrimination. Specular x-ray reflectivity data yielded considerable differences in layer thickness and electron-density profile after addition of the peptide to DPPG monolayers, but little change was seen after peptide injection when probing monolayers composed of DPPC and DPPE. Grazing incidence x-ray diffraction demonstrated significant peptide insertion and lateral packing order disruption of the DPPG monolayer by LL-37 insertion. Epifluorescence microscopy data support these findings.

Fusion pore formation and expansion, crucial steps for neurotransmitter release and vesicle recycling in soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-dependent vesicle fusion, have not been well studied in vitro due to the lack of a reliable content-mixing fusion assay. Using methods detecting the intervesicular mixing of small and large cargoes at a single-vesicle level, we found that the neuronal SNARE complexes have the capacity to drive membrane hemifusion. However, efficient fusion pore formation and expansion require synaptotagmin 1 and Ca 2+ . Real-time measurements show that pore expansion detected by content mixing of large DNA cargoes occurs much slower than initial pore formation that transmits small cargoes. Slow pore expansion perhaps provides a time window for vesicles to escape the full collapse fusion pathway via alternative mechanisms such as kissand-run. The results also show that complexin 1 stimulates pore expansion significantly, which could put bias between two pathways of vesicle recycling. S oluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) mediate intracellular vesicle fusion in a wide variety of cellular activities such as neurotransmitter release. The fast synaptic vesicle fusion for neurotransmitter release is regulated with precision by various proteins including synaptotagmins, complexins, and SM proteins (1, 2). During this process, an initial fusion pore between two membranes can either close back or expand to a larger pore. Fusion pore expansion to the point where the vesicle membrane flattens on the plasma membrane surface, leading to the complete luminal contents release, is thought to be the final step in the fusion process (3, 4). SNAREs and accessary proteins may then be recycled to make fresh vesicles through endocytosis. Without pore expansion, however, the vesicles may be used again through the mechanism known as "kiss-and-run" (5). Therefore, pore expansion is an important event that determines how synaptic vesicles are regenerated.To dissect the SNARE-mediated membrane fusion process, we and others developed in vitro single-vesicle assays based on lipid mixing of proteoliposomes reconstituted with SNARE proteins and content mixing of small cargoes (6-10). However, these assays are blind to the expansion of the fusion pore and therefore unable to tell how the regulatory proteins are involved in this final step of the full-collapse fusion pathway, in which the small opening of the pore continues to expand to a large pore.To monitor fusion pore expansion, we developed a singlemolecule/vesicle content-mixing assay based on vesicle-encapsulated DNA molecules (11,12). This assay can detect expansion of the fusion pore that is large enough to pass ∼11-kDa DNA probes between two apposed proteoliposomes. With this method, we showed that yeast SNAREs alone can efficiently drive expansion of the fusion pore (12). In this work, we systematically dissect lipid mixing, fusion pore opening, and fusion pore expansion steps i...

SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins are a highly regulated class of membrane proteins that drive the efficient merger of two distinct lipid bilayers into one interconnected structure. This protocol describes our fluorescence resonance energy transfer (FRET)-based single vesicle-vesicle fusion assays for SNAREs and accessory proteins. Both lipid-mixing (with FRET pairs acting as lipophilic dyes in the membranes) and content-mixing assays (with FRET pairs present on a DNA hairpin that becomes linear via hybridization to a complementary DNA) are described. These assays can be used to detect substages such as docking, hemifusion, and pore expansion and full fusion. The details of flow cell preparation, protein-reconstituted vesicle preparation, data acquisition and analysis are described. These assays can be used to study the roles of various SNARE proteins, accessory proteins and effects of different lipid compositions on specific fusion steps. The total time required to finish one round of this protocol is 3–6 d.

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.

The in vitro studies of membrane fusion mediated by soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) have primarily been performed by following the mixing of the lipids. However, the formation a of fusion pore and its expansion has been difficult to detect directly due to the leakiness of proteoliposomes, vesicle aggregation and rupture that often complicate the interpretation of ensemble fusion experiments. Fusion pore expansion is an essential step for full collapse fusion and recycling of the fusion machineries. Here, we demonstrate a method to detect the inter-vesicular mixing of large cargoes at the single molecule and vesicle level. The change in FRET signal when a DNA hairpin encapsulated in a surface-tethered vesicle encounters a complementary DNA strand from another vesicle indicates content mixing. We found that that the yeast SNARE complex alone without any accessory proteins can expand the fusion pore large enough to transmit ~ 11 kD cargoes.