Phase Separation of a PKA Regulatory Subunit Controls cAMP Compartmentation and Oncogenic Signaling.

The fidelity of intracellular signaling hinges on the organization of dynamic activity architectures. Spatial compartmentation was first proposed over 30 years ago to explain how diverse G protein-coupled receptors achieve specificity despite converging on a ubiquitous messenger, cyclic adenosine monophosphate (cAMP). However, the mechanisms responsible for spatially constraining this diffusible messenger remain elusive. Here, we reveal that the type I regulatory subunit of cAMP-dependent protein kinase (PKA), RIα, undergoes liquid-liquid phase separation (LLPS) as a function of cAMP signaling to form biomolecular condensates enriched in cAMP and PKA activity, critical for effective cAMP compartmentation. We further show that a PKA fusion oncoprotein associated with an atypical liver cancer potently blocks RIα LLPS and induces aberrant cAMP signaling. Loss of RIα LLPS in normal cells increases cell proliferation and induces cell transformation. Our work reveals LLPS as a principal organizer of signaling compartments and highlights the pathological consequences of dysregulating this activity architecture.

Cell-Penetrating Peptide Mediates Intracellular Membrane Passage of Human Papillomavirus L2 Protein to Trigger Retrograde Trafficking.

Cell-penetrating peptides (CPPs) are short protein segments that can transport cargos into cells. Although CPPs are widely studied as potential drug delivery tools, their role in normal cell physiology is poorly understood. Early during infection, the L2 capsid protein of human papillomaviruses binds retromer, a cytoplasmic trafficking factor required for delivery of the incoming non-enveloped virus into the retrograde transport pathway. Here, we show that the C terminus of HPV L2 proteins contains a conserved cationic CPP that drives passage of a segment of the L2 protein through the endosomal membrane into the cytoplasm, where it binds retromer, thereby sorting the virus into the retrograde pathway for transport to the trans-Golgi network. These experiments define the cell-autonomous biological role of a CPP in its natural context and reveal how a luminal viral protein engages an essential cytoplasmic entry factor.

Tripartite Split-GFP for High Throughput Screening of Small Molecules: A Powerful Strategy for Targeting Transient/Labile Interactors like E2-E3 Ubiquitination Enzymes.

The search for inhibitors of the Ubiquitin Proteasome System (UPS) is an expanding area, due to the crucial role of UPS enzymes in several diseases. The complexity of the UPS and the multiple protein-protein interactions (PPIs) involved, either between UPS proteins themselves or between UPS components and theirs targets, offer an incredibly wide field for the development of chemical compounds for specifically modulating or inhibiting metabolic pathways. However, numerous UPS PPIs are transient/labile, due the processivity of the system (Ubiquitin [Ub] chain elongation, Ub transfer, etc.). Among the different strategies that can be used either for deciphering UPS PPI or for identifying/characterizing small compounds inhibitors, the split-GFP approach offers several advantages notably for high throughput screening of drugs. Split-GFP is based on the principle of protein-fragment complementation assay (PCA). PCA allows addressing PPIs by coupling each protein of interest (POI) to fragments of a reporter protein whose reconstitution is linked to the interaction of the POI. Here, we review the evolution of the split-GFP approach from bipartite to tripartite Split-GFP and its recent applicability for screening chemical compounds targeting the UPS.

Tracking of Human Parvovirus B19 Virus-Like Particles Using Short Peptide Tags Reveals a Membrane-Associated Extracellular Release of These Particles.

B19 virus (B19V) is a pathogenic human parvovirus that infects erythroid progenitor cells. Because there are limited in vitro culture systems to propagate this virus, little is known about the molecular mechanisms by which it propagates in cells. In this study, we introduced a HiBiT peptide tag into various loops of VP2 located on the surface of B19V particles and evaluated their ability to form virus-like particles (VLPs). Three independent sites were identified as permissive sites for peptide tag insertion without affecting VLP formation. When the HiBiT tag was introduced into B19V clones (pB19-M20) and transfected into a semipermissive erythroleukemia cell line (UT7/Epo-S1), HiBiT-dependent luciferase activities (HiBiT activities) increased depending on helicase activity of viral NS1. Furthermore, we used a GFP11 tag-split system to visualize VLPs in the GFP1-10-expressing live cells. Time-lapse imaging of green fluorescent protein (GFP)-labeled VLPs revealed that nuclear VLPs were translocated into the cytoplasm only after cell division, suggesting that the breakdown of the nuclear envelope during mitosis contributes to VLP nuclear export. Moreover, HiBiT activities of culture supernatants were dependent on the presence of a detergent, and the released VLPs were associated with extracellular vesicles, as observed under electron microscopy. Treatment with an antimitotic agent (nocodazole) enhanced the release of VLPs. These results suggest that the virions accumulated in the cytoplasm are constitutively released from the cell as membrane-coated vesicles. These properties are likely responsible for viral escape from host immune responses and enhance membrane fusion-mediated transmission. IMPORTANCE Parvovirus particles are expected to be applied as nanoparticles in drug delivery systems. However, little is known about how nuclear-assembled B19 virus (B19V) virions are released from host cells. This study provides evidence of mitosis-dependent nuclear export of B19V and extracellular vesicle-mediated virion release. Moreover, this study provides methods for modifying particle surfaces with various exogenous factors and contributes to the development of fine nanoparticles with novel valuable functions. The pB19-M20 plasmid expressing HiBiT-tagged VP2 is a novel tool to easily quantify VP2 expression. Furthermore, this system can be applied in high-throughput screening of reagents that affect VP2 expression, which might be associated with viral propagation.

Quantitative measurement of cell-surface displayed proteins based on split-GFP assembly.

BACKGROUND: Microbial cell surface display technology allows immobilizing proteins on the cell surface by fusing them to anchoring motifs, thereby endowing the cells with diverse functionalities. However, the assessment of successful protein display and the quantification of displayed proteins remain challenging. The green fluorescent protein (GFP) can be split into two non-fluorescent fragments, while they spontaneously assemble and emit fluorescence when brought together through complementation. Based on split-GFP assembly, we aim to: (1) confirm the success display of passenger proteins, (2) quantify the number of passenger proteins displayed on individual cells. RESULTS: In this study, we propose two innovative methods based on split-green fluorescent protein (split-GFP), named GFP1-10/GFP11 and GFP1-9/GFP10-11 assembly, for the purpose of confirming successful display and quantifying the number of proteins displayed on individual cells. We evaluated the display efficiency of SUMO and ubiquitin using different anchor proteins to demonstrate the feasibility of the two split-GFP assembly systems. To measure the display efficiency of functional proteins, laccase expression was measured using the split-GFP assembly system by co-displaying GFP11 or GFP10-11 tags, respectively. CONCLUSIONS: Our study provides two split-GFP based methods that enable qualitative and quantitative analyses of individual cell display efficiency with a simple workflow, thus facilitating further comprehensive investigations into microbial cell surface display technology. Both split-GFP assembly systems offer a one-step procedure with minimal cost, simplifying the fluorescence analysis of surface-displaying cells.

Rapid prototyping enzyme homologs to improve titer of nicotinamide mononucleotide using a strategy combining cell-free protein synthesis with split GFP.

Engineering biological systems to test new pathway variants containing different enzyme homologs is laborious and time-consuming. To tackle this challenge, a strategy was developed for rapidly prototyping enzyme homologs by combining cell-free protein synthesis (CFPS) with split green fluorescent protein (GFP). This strategy featured two main advantages: (1) dozens of enzyme homologs were parallelly produced by CFPS within hours, and (2) the expression level and activity of each homolog was determined simultaneously by using the split GFP assay. As a model, this strategy was applied to optimize a 3-step pathway for nicotinamide mononucleotide (NMN) synthesis. Ten enzyme homologs from different organisms were selected for each step. Here, the most productive homolog of each step was identified within 24 h rather than weeks or months. Finally, the titer of NMN was increased to 1213 mg/L by improving physiochemical conditions, tuning enzyme ratios and cofactor concentrations, and decreasing the feedback inhibition, which was a more than 12-fold improvement over the initial setup. This strategy would provide a promising way to accelerate design-build-test cycles for metabolic engineering to improve the production of desired products.

Biosensor-guided rapid screening for improved recombinant protein secretion in Pichia pastoris.

Pichia pastoris (Komagataella phaffii) is widely used for industrial production of heterologous proteins due to high secretory capabilities but selection of highly productive engineered strains remains a limiting step. Despite availability of a comprehensive molecular toolbox for construct design and gene integration, there is high clonal variability among transformants due to frequent multi-copy and off-target random integration. Therefore, functional screening of several hundreds of transformant clones is essential to identify the best protein production strains. Screening methods are commonly based on deep-well plate cultures with analysis by immunoblotting or enzyme activity assays of post-induction samples, and each heterologous protein produced may require development of bespoke assays with multiple sample processing steps. In this work, we developed a generic system based on a P. pastoris strain that uses a protein-based biosensor to identify highly productive protein secretion clones from a heterogeneous set of transformants. The biosensor uses a split green fluorescent protein where the large GFP fragment (GFP1-10) is fused to a sequence-specific protease from Tobacco Etch Virus (TEV) and is targeted to the endoplasmic reticulum. Recombinant proteins targeted for secretion are tagged with the small fragment of the split GFP (GFP11). Recombinant protein production can be measured by monitoring GFP fluorescence, which is dependent on interaction between the large and small GFP fragments. The reconstituted GFP is cleaved from the target protein by TEV protease, allowing for secretion of the untagged protein of interest and intracellular retention of the mature GFP. We demonstrate this technology with four recombinant proteins (phytase, laccase, ß-casein and ß-lactoglobulin) and show that the biosensor directly reports protein production levels that correlate with traditional assays. Our results confirm that the split GFP biosensor can be used for facile, generic, and rapid screening of P. pastoris clones to identify those with the highest production levels.

Analysis of protein secretion in Bacillus subtilis by combining a secretion stress biosensor strain with an in vivo split GFP assay.

BACKGROUND: Bacillus subtilis is one of the workhorses in industrial biotechnology and well known for its secretion potential. Efficient secretion of recombinant proteins still requires extensive optimization campaigns and screening with activity-based methods. However, not every protein can be detected by activity-based screening. We therefore developed a combined online monitoring system, consisting of an in vivo split GFP assay for activity-independent target detection and an mCherry-based secretion stress biosensor. The split GFP assay is based on the fusion of a target protein to the eleventh ß-sheet of sfGFP, which can complement a truncated sfGFP that lacks this ß-sheet named GFP1-10. The secretion stress biosensor makes use of the CssRS two component quality control system, which upregulates expression of mCherry in the htrA locus thereby allowing a fluorescence readout of secretion stress. RESULTS: The biosensor strain B. subtilis PAL5 was successfully constructed by exchanging the protease encoding gene htrA with mCherry via CRISPR/Cas9. The Fusarium solani pisi cutinase Cut fused to the GFP11 tag (Cut11) was used as a model enzyme to determine the stress response upon secretion mediated by signal peptides SPPel, SPEpr and SPBsn obtained from naturally secreted proteins of B. subtilis. An in vivo split GFP assay was developed, where purified GFP1-10 is added to the culture broth. By combining both methods, an activity-independent high-throughput method was created, that allowed optimization of Cut11 secretion. Using the split GFP-based detection assay, we demonstrated a good correlation between the amount of secreted cutinase and the enzymatic activity. Additionally, we screened a signal peptide library and identified new signal peptide variants that led to improved secretion while maintaining low stress levels. CONCLUSION: Our results demonstrate that the combination of a split GFP-based detection assay for secreted proteins with a secretion stress biosensor strain enables both, online detection of extracellular target proteins and identification of bottlenecks during protein secretion in B. subtilis. In general, the system described here will also enable to monitor the secretion stress response provoked by using inducible promoters governing the expression of different enzymes.

A Split-Cre system designed to detect simultaneous expression of two genes based on SpyTag/SpyCatcher conjugation and Split-GFP dimerization.

The Split-Cre system is a powerful tool for genetic manipulation and can be used to spatiotemporally control gene expression in vivo. However, the low activity of the reconstituted NCre/CCre recombinase in the Split-Cre system limits its application as an indicator of the simultaneous expression of a pair of genes of interest. Here, we describe two approaches for improving the activity of the Split-Cre system after Cre reconstitution based on self-associating split GFP (Split-GFP) and SpyTag/SpyCatcher conjugation. First, we created the Split-GFP-Cre system by constructing fusion proteins of NCre and CCre with the N-terminal and C-terminal subunits of GFP, respectively. Reconstitution of Cre by GFP-mediated dimerization of the two fusion proteins resulted in recombinase activity approaching that of full-length Cre in living cells. Second, to further increase recombinase activity at low levels of Split-Cre expression, the Split-Spy-GCre system was established by incorporating the sequences for SpyTag and SpyCatcher into the components of the Split-GFP-Cre system. As anticipated, covalent conjugation of the SpyTag and SpyCatcher segments improved Split-GFP dimerization to further increase Cre recombinase activity in living cells. The increased efficiency and robustness of this dual-split system (Split-Cre and Split-GFP) minimize the problems of incomplete double gene-specific KO or low labeling efficiency due to poor NCre/CCre recombinase activity. Thus, this Split-Spy-GCre system allows more precise gene manipulation of cell subpopulations, which will provide advanced analysis of genes and cell functions in complex tissue such as the immune system.

Accelerated strain construction and characterization of C. glutamicum protein secretion by laboratory automation.

Secretion of bacterial proteins into the culture medium simplifies downstream processing by avoiding cell disruption for target protein purification. However, a suitable signal peptide for efficient secretion needs to be identified, and currently, there are no tools available to predict optimal combinations of signal peptides and target proteins. The selection of such a combination is influenced by several factors, including protein biosynthesis efficiency and cultivation conditions, which both can have a significant impact on secretion performance. As a result, a large number of combinations must be tested. Therefore, we have developed automated workflows allowing for targeted strain construction and secretion screening using two platforms. Key advantages of this experimental setup include lowered hands-on time and increased throughput. In this study, the automated workflows were established for the heterologous production of Fusarium solani f. sp. pisi cutinase in Corynebacterium glutamicum. The target protein was monitored in culture supernatants via enzymatic activity and split GFP assay. Varying spacer lengths between the Shine-Dalgarno sequence and the start codon of Bacillus subtilis signal peptides were tested. Consistent with previous work on the secretory cutinase production in B. subtilis, a ribosome binding site with extended spacer length to up to 12 nt, which likely slows down translation initiation, does not necessarily lead to poorer cutinase secretion by C. glutamicum. The best performing signal peptides for cutinase secretion with a standard spacer length were identified in a signal peptide screening. Additional insights into the secretion process were gained by monitoring secretion stress using the C. glutamicum K9 biosensor strain. KEY POINTS: • Automated workflows for strain construction and screening of protein secretion • Comparison of spacer, signal peptide, and host combinations for cutinase secretion • Signal peptide screening for secretion by C. glutamicum using the split GFP assay.

Targeting specificity of nuclear-encoded organelle proteins with a self-assembling split-fluorescent protein toolkit.

A large number of nuclear-encoded proteins are targeted to the organelles of endosymbiotic origin, namely mitochondria and plastids. To determine the targeting specificity of these proteins, fluorescent protein tagging is a popular approach. However, ectopic expression of fluorescent protein fusions commonly results in considerable background signals and often suffers from the large size and robust folding of the reporter protein, which may perturb membrane transport. Among the alternative approaches that have been developed in recent years, the self-assembling split-fluorescent protein (sasplit-FP) technology appears particularly promising to analyze protein targeting specificity in vivo Here, we improved the sensitivity of this technology and systematically evaluated its utilization to determine protein targeting to plastids and mitochondria. Furthermore, to facilitate high-throughput screening of candidate proteins we developed a Golden Gate-based vector toolkit (PlaMinGo). As a result of these improvements, dual targeting could be detected for a number of proteins that had earlier been characterized as being targeted to a single organelle only. These results were independently confirmed with a plant phenotype complementation approach based on the immutans mutant.This article has an associated First Person interview with the first author of the paper.

Scaling production of GFP1-10 detector protein in E. coli for secretion screening by split GFP assay.

BACKGROUND: The split GFP assay is a well-known technology for activity-independent screening of target proteins. A superfolder GFP is split into two non-fluorescent parts, GFP11 which is fused to the target protein and GFP1-10. In the presence of both, GFP1-10 and the GFP11-tag are self-assembled and a functional chromophore is formed. However, it relies on the availability and quality of GFP1-10 detector protein to develop fluorescence by assembly with the GFP11-tag connected to the target protein. GFP1-10 detector protein is often produced in small scale shake flask cultivation and purified from inclusion bodies. RESULTS: The production of GFP1-10 in inclusion bodies and purification was comprehensively studied based on Escherichia coli as host. Cultivation in complex and defined medium as well as different feed strategies were tested in laboratory-scale bioreactor cultivation and a standardized process was developed providing high quantity of GFP1-10 detector protein with suitable quality. Split GFP assay was standardized to obtain robust and reliable assay results from cutinase secretion strains of Corynebacterium glutamicum with Bacillus subtilis Sec signal peptides NprE and Pel. Influencing factors from environmental conditions, such as pH and temperature were thoroughly investigated. CONCLUSIONS: GFP1-10 detector protein production could be successfully scaled from shake flask to laboratory scale bioreactor. A single run yielded sufficient material for up to 385 96-well plate screening runs. The application study with cutinase secretory strains showed very high correlation between measured cutinase activity to split GFP fluorescence signal proofing applicability for larger screening studies.

A novel fluorescent reporter detects plastic remodeling of mitochondria-ER contact sites.

Mitochondria-ER contact sites (MERCs) enable communication between the ER and mitochondria and serve as platforms for many cellular events, including autophagy. Nonetheless, the molecular organization of MERCs is not known, and there is no bona fide marker of these contact sites in mammalian cells. In this study, we designed a genetically encoded reporter using split GFP protein for labeling MERCs. We subsequently analyzed its distribution and dynamics during the cell cycle and under stressful cellular conditions such as starvation, apoptosis and ER stress. We found that MERCs are dynamic structures that undergo remodeling within minutes. Mitochondrial morphology, but not ER morphology, affected the distribution of MERCs. We also found that carbonyl cyanidem-chlorophenyl hydrazone (CCCP) and oligomycin A treatment enhanced MERC formation. The stimulations that led to apoptosis or autophagy increased the MERC signal. By contrast, increasing cellular lipid droplet load did not change the pattern of MERCs.

High-content tripartite split-GFP cell-based assays to screen for modulators of small GTPase activation.

The human Ras superfamily of small GTPases controls essential cellular processes such as gene expression and cell proliferation. As their deregulation is widely associated with human cancer, small GTPases and their regulatory proteins have become increasingly attractive for the development of novel therapeutics. Classical methods to monitor GTPase activation include pulldown assays that limit the analysis of GTP-bound form of proteins from cell lysates. Alternatively, live-cell FRET biosensors may be used to study GTPase activation dynamics in response to stimuli, but these sensors often require further optimization for high-throughput applications. Here, we describe a cell-based approach that is suitable to monitor the modulation of small GTPase activity in a high-content analysis. The assay relies on a genetically encoded tripartite split-GFP (triSFP) system that we integrated in an optimized cellular model to monitor modulation of RhoA and RhoB GTPases. Our results indicate the robust response of the reporter, allowing the interrogation of inhibition and stimulation of Rho activity, and highlight potential applications of this method to discover novel modulators and regulators of small GTPases and related protein-binding domains.

Mechanism and bottlenecks in strand photodissociation of split green fluorescent proteins (GFPs).

Split GFPs have been widely applied for monitoring protein-protein interactions by expressing GFPs as two or more constituent parts linked to separate proteins that only fluoresce on complementing with one another. Although this complementation is typically irreversible, it has been shown previously that light accelerates dissociation of a noncovalently attached ß-strand from a circularly permuted split GFP, allowing the interaction to be reversible. Reversible complementation is desirable, but photodissociation has too low of an efficiency (quantum yield <1%) to be useful as an optogenetic tool. Understanding the physical origins of this low efficiency can provide strategies to improve it. We elucidated the mechanism of strand photodissociation by measuring the dependence of its rate on light intensity and point mutations. The results show that strand photodissociation is a two-step process involving light-activated cis-trans isomerization of the chromophore followed by light-independent strand dissociation. The dependence of the rate on temperature was then used to establish a potential energy surface (PES) diagram along the photodissociation reaction coordinate. The resulting energetics-function model reveals the rate-limiting process to be the transition from the electronic excited-state to the ground-state PES accompanying cis-trans isomerization. Comparisons between split GFPs and other photosensory proteins, like photoactive yellow protein and rhodopsin, provide potential strategies for improving the photodissociation quantum yield.

ER-Mitochondria Contact Sites Reporters: Strengths and Weaknesses of the Available Approaches.

Organelle intercommunication represents a wide area of interest. Over the last few decades, increasing evidence has highlighted the importance of organelle contact sites in many biological processes including Ca2+ signaling, lipid biosynthesis, apoptosis, and autophagy but also their involvement in pathological conditions. ER-mitochondria tethering is one of the most investigated inter-organelle communications and it is differently modulated in response to several cellular conditions including, but not limited to, starvation, Endoplasmic Reticulum (ER) stress, and mitochondrial shape modifications. Despite many studies aiming to understand their functions and how they are perturbed under different conditions, approaches to assess organelle proximity are still limited. Indeed, better visualization and characterization of contact sites remain a fascinating challenge. The aim of this review is to summarize strengths and weaknesses of the available methods to detect and quantify contact sites, with a main focus on ER-mitochondria tethering.

Detection of membrane protein-protein interaction in planta based on dual-intein-coupled tripartite split-GFP association.

Despite the great interest in identifying protein-protein interactions (PPIs) in biological systems, only a few attempts have been made at large-scale PPI screening in planta. Unlike biochemical assays, bimolecular fluorescence complementation allows visualization of transient and weak PPIs in vivo at subcellular resolution. However, when the non-fluorescent fragments are highly expressed, spontaneous and irreversible self-assembly of the split halves can easily generate false positives. The recently developed tripartite split-GFP system was shown to be a reliable PPI reporter in mammalian and yeast cells. In this study, we adapted this methodology, in combination with the ß-estradiol-inducible expression cassette, for the detection of membrane PPIs in planta. Using a transient expression assay by agroinfiltration of Nicotiana benthamiana leaves, we demonstrate the utility of the tripartite split-GFP association in plant cells and affirm that the tripartite split-GFP system yields no spurious background signal even with abundant fusion proteins readily accessible to the compartments of interaction. By validating a few of the Arabidopsis PPIs, including the membrane PPIs implicated in phosphate homeostasis, we proved the fidelity of this assay for detection of PPIs in various cellular compartments in planta. Moreover, the technique combining the tripartite split-GFP association and dual-intein-mediated cleavage of polyprotein precursor is feasible in stably transformed Arabidopsis plants. Our results provide a proof-of-concept implementation of the tripartite split-GFP system as a potential tool for membrane PPI screens in planta.

A human pathogenic bacterium Shigella proliferates in plants through adoption of type III effectors for shigellosis.

Shigella, which infects primates, can be transmitted via fresh vegetables; however, its molecular interactions with plants have not been elucidated. Here, we show that four Shigella strains, Shigella boydii, Shigella sonnei, Shigella flexneri 2a, and S. flexneri 5a, proliferate at different levels in Arabidopsis thaliana. Microscopic studies revealed that these bacteria were present inside leaves and damaged plant cells. Green fluorescent protein (GFP)-tagged S. boydii and S. flexneri 5a colonized leaves only, whereas S. flexneri 2a colonized both leaves and roots. Using Shigella mutants lacking type III secretion systems (T3SSs), we found that T3SSs that regulate the pathogenesis of shigellosis in humans also play a central role in bacterial proliferation in Arabidopsis. Strikingly, the immunosuppressive activity of two T3S effectors, OspF and OspG, was required for proliferation of Shigella in Arabidopsis. Of note, delivery of OspF or OspG effectors inside plant cells upon Shigella inoculation was confirmed using a split GFP system. These findings demonstrate that the human pathogen Shigella can proliferate in plants by adapting immunosuppressive machinery used in the original host human.

Development and Applications of Superfolder and Split Fluorescent Protein Detection Systems in Biology.

Molecular engineering of the green fluorescent protein (GFP) into a robust and stable variant named Superfolder GFP (sfGFP) has revolutionized the field of biosensor development and the use of fluorescent markers in diverse area of biology. sfGFP-based self-associating bipartite split-FP systems have been widely exploited to monitor soluble expression in vitro, localization, and trafficking of proteins in cellulo. A more recent class of split-FP variants, named « tripartite ¼ split-FP, that rely on the self-assembly of three GFP fragments, is particularly well suited for the detection of protein-protein interactions. In this review, we describe the different steps and evolutions that have led to the diversification of superfolder and split-FP reporter systems, and we report an update of their applications in various areas of biology, from structural biology to cell biology.

Direct visualization of trans-synaptic neurexin-neuroligin interactions during synapse formation.

Neurexins and neuroligins are synaptic cell-adhesion molecules that are essential for normal synapse specification and function and are thought to bind to each other trans-synaptically, but such interactions have not been demonstrated directly. Here, we generated neurexin-1ß and neuroligin-1 and neuroligin-2 fusion proteins containing complementary "split" GFP fragments positioned such that binding of neurexin-1ß to neuroligin-1 or neuroligin-2 allowed GFP reconstitution without dramatically changing their binding affinities. GFP fluorescence was only reconstituted from split-GFP-modified neurexin-1ß and neuroligin-1 if and after neurexin-1ß bound to its neuroligin partner; reassociation of the split-GFP components with each other did not mediate binding. Using trans-cellular reconstitution of GFP fluorescence from split-GFP-modified neurexin-1ß and neuroligins as an assay, we demonstrate that trans-synaptic neurexin/neuroligin binding indeed occurred when mouse hippocampal neurons formed synapses onto non-neuronal COS-7 cells expressing neuroligins or when mouse hippocampal neurons formed synapses with each other. This visualization of synapses by neurexin/neuroligin binding prompted us to refer to this approach as "SynView." Our data demonstrate that neurexin-1ß forms a trans-synaptic complex with neuroligin-1 and neuroligin-2 and that this interaction can be used to label synapses in a specific fashion in vivo.