Synaptic Complex Formation Research Articles

Temporal dynamics and stoichiometry in human Notch signaling from Notch synaptic complex formation to nuclear entry of the Notch intracellular domain

Mammalian Notch signaling occurs when the binding of Delta or Jagged to Notch stimulates the proteolytic release of the Notch intracellular domain (NICD), which enters the nucleus to control target gene expression. To determine the temporal dynamics of events associated with Notch signaling under native conditions, we fluorescently tagged Notch and Delta at their endogenous genomic loci and visualized them upon pairing of receiver (Notch) and sender (Delta) cells as a function of time after cell contact. At contact sites, Notch and Delta immediately accumulated at 1:1 stoichiometry in synapses, which resolved by 15-20min after contact. Synapse formation preceded the entrance of the Notch extracellular domain into the sender cell and accumulation of NICD in the nucleus of the receiver cell, which approached a maximum after ∼45min and was prevented by chemical and genetic inhibitors of signaling. These findings directly link Notch-Delta synapse dynamics to NICD production with spatiotemporal precision.

Human RAD52 stimulates the RAD51-mediated homology search

Homologous recombination (HR) is a DNA repair mechanism of double-strand breaks and blocked replication forks, involving a process of homology search leading to the formation of synaptic intermediates that are regulated to ensure genome integrity. RAD51 recombinase plays a central role in this mechanism, supported by its RAD52 and BRCA2 partners. If the mediator function of BRCA2 to load RAD51 on RPA-ssDNA is well established, the role of RAD52 in HR is still far from understood. We used transmission electron microscopy combined with biochemistry to characterize the sequential participation of RPA, RAD52, and BRCA2 in the assembly of the RAD51 filament and its activity. Although our results confirm that RAD52 lacks a mediator activity, RAD52 can tightly bind to RPA-coated ssDNA, inhibit the mediator activity of BRCA2, and form shorter RAD51-RAD52 mixed filaments that are more efficient in the formation of synaptic complexes and D-loops, resulting in more frequent multi-invasions as well. We confirm the in situ interaction between RAD51 and RAD52 after double-strand break induction in vivo. This study provides new molecular insights into the formation and regulation of presynaptic and synaptic intermediates by BRCA2 and RAD52 during human HR.

A spinal muscular atrophy modifier implicates the SMN protein in SNARE complex assembly at neuromuscular synapses

Reduced survival motor neuron (SMN) protein triggers the motor neuron disease, spinal muscular atrophy (SMA). Restoring SMN prevents disease, but it is not known how neuromuscular function is preserved. We used model mice to map and identify an Hspa8G470R synaptic chaperone variant, which suppressed SMA. Expression of the variant in the severely affected mutant mice increased lifespan >10-fold, improved motor performance, and mitigated neuromuscular pathology. Mechanistically, Hspa8G470R altered SMN2 splicing and simultaneously stimulated formation of a tripartite chaperone complex, critical for synaptic homeostasis, by augmenting its interaction with other complex members. Concomitantly, synaptic vesicular SNARE complex formation, which relies on chaperone activity for sustained neuromuscular synaptic transmission, was found perturbed in SMA mice and patient-derived motor neurons and was restored in modified mutants. Identification of the Hspa8G470R SMA modifier implicates SMN in SNARE complex assembly and casts new light on how deficiency of the ubiquitous protein causes motor neuron disease.

Site-search process for SfiI restriction enzyme characterized by time-lapse AFM

The interaction between two distant sites on the DNA underlies various genetic processes. These interactions are controlled by specialized proteins able to form site-specific protein-DNA synaptic complexes. Little is known about the molecular mechanisms of synaptic complex formation and how proteins search the DNA for specific sites during the formation of synaptosomes. Notably, type II DNA restriction enzymes form synaptic complexes to complete site-specific DNA cleavage. Seminal theoretical analysis presented by Berg, von Hippel, and Winter established a simple framework for the search processes of single-site search, including sliding, hopping/jumping, and intersegmental transfers.

Control of the Serine Integrase Reaction: Roles of the Coiled-Coil and Helix E Regions in DNA Site Synapsis and Recombination.

Bacteriophage serine integrases catalyze highly specific recombination reactions between defined DNA segments called att sites. These reactions are reversible depending upon the presence of a second phage-encoded directionality factor. The bipartite C-terminal DNA-binding region of integrases includes a recombinase domain (RD) connected to a zinc-binding domain (ZD), which contains a long flexible coiled-coil (CC) motif that extends away from the bound DNA. We directly show that the identities of the phage A118 integrase att sites are specified by the DNA spacing between the RD and ZD DNA recognition determinants, which in turn directs the relative trajectories of the CC motifs on each subunit of the att-bound integrase dimer. Recombination between compatible dimer-bound att sites requires minimal-length CC motifs and 14 residues surrounding the tip where the pairing of CC motifs between synapsing dimers occurs. Our alanine-scanning data suggest that molecular interactions between CC motif tips may differ in integrative (attP × attB) and excisive (attL × attR) recombination reactions. We identify mutations in 5 residues within the integrase oligomerization helix that control the remodeling of dimers into tetramers during synaptic complex formation. Whereas most of these gain-of-function mutants still require the CC motifs for synapsis, one mutant efficiently, but indiscriminately, forms synaptic complexes without the CC motifs. However, the CC motifs are still required for recombination, suggesting a function for the CC motifs after the initial assembly of the integrase synaptic tetramer. IMPORTANCE The robust and exquisitely regulated site-specific recombination reactions promoted by serine integrases are integral to the life cycle of temperate bacteriophage and, in the case of the A118 prophage, are an important virulence factor of Listeria monocytogenes. The properties of these recombinases have led to their repurposing into tools for genetic engineering and synthetic biology. In this report, we identify determinants regulating synaptic complex formation between correct DNA sites, including the DNA architecture responsible for specifying the identity of recombination sites, features of the unique coiled-coil structure on the integrase that are required to initiate synapsis, and amino acid residues on the integrase oligomerization helix that control the remodeling of synapsing dimers into a tetramer active for DNA strand exchange.

Extreme C-terminal element of SprA serine integrase is a potential component of the "molecular toggle switch" which controls the recombination and its directionality.

In Bacillus subtilis, a sporulation-related gene, spsM, is disrupted by SPβ prophage, but reconstituted during sporulation through SPβ excision. The spsM reconstitution is catalyzed by a site-specific DNA recombinase, SprA, and its cognate recombination directionality factor, SprB. SprB interacts with SprA, directing the SprA-mediated recombination reaction from integration to excision; however, the details of the directionality control remains unclear. Here, we demonstrate the importance of the extreme C-terminal region (ECT) of SprA in the DNA recombination and directionality control. We created a series of SprA C-terminal deletants and examined their DNA-binding and recombination activities. Deletions in the ECT caused a loss of integration and excision activity, the magnitudes of which positively correlated with the deletion size. Gel shift study revealed that the loss of the integration activity was attributable to the failure of synaptic complex formation. The excision deficiency was caused by defective interaction with SprB. Moreover, alanine scanning analysis revealed that Phe532 is essential to interact with SprB. SprAF532A , therefore, showed almost no excision activity, while retaining the integration activity. Collectively, these results suggest that the ECT plays the crucial roles in the interaction of SprA with SprB and possibly in the directional control of the recombination.

XLF acts as a flexible connector during non-homologous end joining.

Non-homologous end joining (NHEJ) is the predominant pathway that repairs DNA double-strand breaks in vertebrates. During NHEJ DNA ends are held together by a multi-protein synaptic complex until they are ligated. Here, we use Xenopus laevis egg extract to investigate the role of the intrinsically disordered C-terminal tail of the XRCC4-like factor (XLF), a critical factor in end synapsis. We demonstrate that the XLF tail along with the Ku-binding motif (KBM) at the extreme C-terminus are required for end joining. Although the underlying sequence of the tail can be varied, a minimal tail length is required for NHEJ. Single-molecule FRET experiments that observe end synapsis in real-time show that this defect is due to a failure to closely align DNA ends. Our data supports a model in which a single C-terminal tail tethers XLF to Ku, while allowing XLF to form interactions with XRCC4 that enable synaptic complex formation.

Structural basis of SALM3 dimerization and synaptic adhesion complex formation with PTP\u03c3

Synaptic adhesion molecules play an important role in the formation, maintenance and refinement of neuronal connectivity. Recently, several leucine rich repeat (LRR) domain containing neuronal adhesion molecules have been characterized including netrin G-ligands, SLITRKs and the synaptic adhesion-like molecules (SALMs). Dysregulation of these adhesion molecules have been genetically and functionally linked to various neurological disorders. Here we investigated the molecular structure and mechanism of ligand interactions for the postsynaptic SALM3 adhesion protein with its presynaptic ligand, receptor protein tyrosine phosphatase σ (PTPσ). We solved the crystal structure of the dimerized LRR domain of SALM3, revealing the conserved structural features and mechanism of dimerization. Furthermore, we determined the complex structure of SALM3 with PTPσ using small angle X-ray scattering, revealing a 2:2 complex similar to that observed for SALM5. Solution studies unraveled additional flexibility for the complex structure, but validated the uniform mode of action for SALM3 and SALM5 to promote synapse formation. The relevance of the key interface residues was further confirmed by mutational analysis with cellular binding assays and artificial synapse formation assays. Collectively, our results suggest that SALM3 dimerization is a pre-requisite for the SALM3-PTPσ complex to exert synaptogenic activity.

Structural basis of seamless excision and specific targeting by piggyBac transposase

The piggyBac DNA transposon is used widely in genome engineering applications. Unlike other transposons, its excision site can be precisely repaired without leaving footprints and it integrates specifically at TTAA tetranucleotides. We present cryo-EM structures of piggyBac transpososomes: a synaptic complex with hairpin DNA intermediates and a strand transfer complex capturing the integration step. The results show that the excised TTAA hairpin intermediate and the TTAA target adopt essentially identical conformations, providing a mechanistic link connecting the two unique properties of piggyBac. The transposase forms an asymmetric dimer in which the two central domains synapse the ends while two C-terminal domains form a separate dimer that contacts only one transposon end. In the strand transfer structure, target DNA is severely bent and the TTAA target is unpaired. In-cell data suggest that asymmetry promotes synaptic complex formation, and modifying ends with additional transposase binding sites stimulates activity.

In vitro role of Rad54 in Rad51-ssDNA filament-dependent homology search and synaptic complexes formation

Homologous recombination (HR) uses a homologous template to accurately repair DNA double-strand breaks and stalled replication forks to maintain genome stability. During homology search, Rad51 nucleoprotein filaments probe and interact with dsDNA, forming the synaptic complex that is stabilized on a homologous sequence. Strand intertwining leads to the formation of a displacement-loop (D-loop). In yeast, Rad54 is essential for HR in vivo and required for D-loop formation in vitro, but its exact role remains to be fully elucidated. Using electron microscopy to visualize the DNA-protein complexes, here we find that Rad54 is crucial for Rad51-mediated synaptic complex formation and homology search. The Rad54−K341R ATPase-deficient mutant protein promotes formation of synaptic complexes but not D-loops and leads to the accumulation of stable heterologous associations, suggesting that the Rad54 ATPase is involved in preventing non-productive intermediates. We propose that Rad51/Rad54 form a functional unit operating in homology search, synaptic complex and D-loop formation.

Promotion of homology-directed DNA repair by polyamines

Polyamines, often elevated in cancer cells, have been shown to promote cell growth and proliferation. Whether polyamines regulate other cell functions remains unclear. Here, we explore whether and how polyamines affect genome integrity. When DNA double-strand break (DSB) is induced in hair follicles by ionizing radiation, reduction of cellular polyamines augments dystrophic changes with delayed regeneration. Mechanistically, polyamines facilitate homologous recombination-mediated DSB repair without affecting repair via non-homologous DNA end-joining and single-strand DNA annealing. Biochemical reconstitution and functional analyses demonstrate that polyamines enhance the DNA strand exchange activity of RAD51 recombinase. The effect of polyamines on RAD51 stems from their ability to enhance the capture of homologous duplex DNA and synaptic complex formation by the RAD51-ssDNA nucleoprotein filament. Our work demonstrates a novel function of polyamines in the maintenance of genome integrity via homology-directed DNA repair.

A mathematical modelling tool for unravelling the antibody-mediated effects on CTLA-4 interactions

BackgroundMonoclonal antibodies blocking the Cytotoxic T-lymphocyte antigen 4 (CTLA-4) receptor have revolutionized the field of anti-cancer therapy for the last few years. The human T-cell-based immune responses are modulated by two contradicting signals. CTLA-4 provides a T cell inhibitory signal through its interaction with B7 ligands (B7–1 and B7–2), while CD28 provides a stimulatory signal when interacting with the same ligands. A previous theoretical model has focused on understanding the processes of costimulatory and inhibitory complex formations at the synapse. Nevertheless, the effects of monoclonal antibody (mAb)-mediation on these complexes are relatively unexplored. In this work, we expand on the previous model to develop a new mathematical framework for studying the effects of anti-CTLA-4 mAbs on the co-stimulatory (CD28/B7 ligands) and the co-inhibitory (CTLA-4/B7 ligands) complex formation at the immunological synapse. In particular, we focus on two promising anti-CTLA-4 mAbs, tremelimumab (from AstraZeneca) and ipilimumab (from Bristol-Myers Squibb), which are currently in clinical trials and the market, respectively, for targeting multiple tumors.MethodsThe mathematical model in this work has been constructed based on ordinary differential equations and available experimental binding kinetics data for the anti-CTLA-4 antibodies from literature.ResultsThe numerical simulations from the current model are in agreement with a number of experimental data. Especially, the dose-curves for blocking the B7 ligand binding to CTLA-4 by ipilimumab are comparable with the results from a previous competitive binding assay by flow cytometry and ELISA. Our simulations predict the dose response and the relative efficacies of the two mAbs in blocking the inhibitory CTLA-4/B7 complexes.ConclusionsThe results show that different factors, such as multivalent interactions, mobility of molecules and competition effects, could impact the effects of antibody-mediation. The results, in particular, describe that the competitive effects could impact the dose-dependent inhibition by the mAbs very significantly. We present this model as a useful tool that can easily be translated to study the effects of any anti-CTLA-4 antibodies on immunological synaptic complex formation, provided reliable biophysical data for mAbs are available.

Lateral Fluid Percussion Injury Impairs Hippocampal Synaptic Soluble N-Ethylmaleimide Sensitive Factor Attachment Protein Receptor Complex Formation

Traumatic brain injury (TBI) and the activation of secondary injury mechanisms have been linked to impaired cognitive function, which, as observed in TBI patients and animal models, can persist for months and years following the initial injury. Impairments in neurotransmission have been well documented in experimental models of TBI, but the mechanisms underlying this dysfunction are poorly understood. Formation of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex facilitates vesicular docking and neurotransmitter release in the synaptic cleft. Published studies highlight a direct link between reduced SNARE complex formation and impairments in neurotransmitter release. While alterations in the SNARE complex have been described following severe focal TBI, it is not known if deficits in SNARE complex formation manifest in a model with reduced severity. We hypothesized that lateral fluid percussion injury (lFPI) reduces the abundance of SNARE proteins, impairs SNARE complex formation, and contributes to impaired neurobehavioral function. To this end, rats were subjected to lFPI or sham injury and tested for acute motor performance and cognitive function at 3 weeks post-injury. lFPI resulted in motor impairment between 1 and 5 days post-injury. Spatial acquisition and spatial memory, as assessed by the Morris water maze, were significantly impaired at 3 weeks after lFPI. To examine the effect of lFPI on synaptic SNARE complex formation in the injured hippocampus, a separate cohort of rats was generated and brains processed to evaluate hippocampal synaptosomal-enriched lysates at 1 week post-injury. lFPI resulted in a significant reduction in multiple monomeric SNARE proteins, including VAMP2, and α-synuclein, and SNARE complex abundance. The findings in this study are consistent with our previously published observations suggesting that impairments in hippocampal SNARE complex formation may contribute to neurobehavioral dysfunction associated with TBI.

Phase Transition in Postsynaptic Densities Underlies Formation of Synaptic Complexes and Synaptic Plasticity

Postsynaptic densities (PSDs) are membrane semi-enclosed, submicron protein-enriched cellular compartments beneath postsynaptic membranes, which constantly exchange their components with bulk aqueous cytoplasm in synaptic spines. Formation and activity-dependent modulation of PSDs is considered as one of the most basic molecular events governing synaptic plasticity in the nervous system. In this study, we discover that SynGAP, one of the most abundant PSD proteins and a Ras/Rap GTPase activator, forms a homo-trimer and binds to multiple copies of PSD-95. Binding of SynGAP to PSD-95 induces phase separation of the complex, forming highly concentrated liquid-like droplets reminiscent of the PSD. The multivalent nature of the SynGAP/PSD-95 complex is critical for the phase separation to occur and for proper activity-dependent SynGAP dispersions from the PSD. In addition to revealing a dynamic anchoring mechanism of SynGAP at the PSD, our results also suggest a model for phase-transition-mediated formation of PSD.

Mechanistic basis of MAGUK-organized complexes in synaptic development and signalling.

Membrane-associated guanylate kinases (MAGUKs) are a family of scaffold proteins that are highly enriched in synapses and are responsible for organizing the numerous protein complexes required for synaptic development and plasticity. Mutations in genes encoding MAGUKs and their interacting proteins can cause a broad spectrum of human psychiatric disorders. Here, we review MAGUK-mediated synaptic protein complex formation and regulation by focusing on findings from recent biochemical and structural investigations. These mechanistic-based studies show that the formation of MAGUK-organized complexes is often directly regulated by protein phosphorylation, suggesting a close connection between neuronal activity and the assembly of dynamic protein complexes in synapses.

Characterization of DNA binding and pairing activities associated with the native SFPQ·NONO DNA repair protein complex

Nonhomologous end joining (NHEJ) is a major pathway for repair of DNA double-strand breaks. We have previously shown that a complex of SFPQ (PSF) and NONO (p54nrb) cooperates with Ku protein at an early step of NHEJ, forming a committed preligation complex and stimulating end-joining activity by 10-fold or more. SFPQ and NONO show no resemblance to other repair factors, and their mechanism of action is uncertain. Here, we use an optimized microwell-based assay to characterize the in vitro DNA binding behavior of the native SFPQ·NONO complex purified from human (HeLa) cells. SFPQ·NONO and Ku protein bind independently to DNA, with little evidence of cooperativity and only slight mutual interference at high concentration. Whereas Ku protein requires free DNA ends for binding, SFPQ·NONO does not. Both Ku and SFPQ·NONO have pairing activity, as measured by the ability of DNA-bound protein to capture a second DNA fragment in a microwell-based assay. Additionally, SFPQ·NONO stimulates DNA-dependent protein kinase autophosphorylation, consistent with the ability to promote formation of a synaptic complex formation without occluding the DNA termini proper. These findings suggest that SFPQ·NONO promotes end joining by binding to internal DNA sequences and cooperating with other repair proteins to stabilize a synaptic pre-ligation complex.

Requirements for Site-Specific Recombination in the Tyrosine-Family Recombinase Active Site

Tyrosine recombinase (YR) shares the same active site and catalyzes the DNA rearrangement with identical phosphoryl transfer chemistry to topoisomerases IB (TopIBs). In order to find out the function of each residue within conserved catalytic center and what factor dictates the strikingly different kinetic characteristics, in cis for Cre and λ Int and in trans for Flp, the whole reaction process of tyrosine family recombinase-mediated site-specific recombinastion has been studied in detailed by using single-molecule TPM. For both systems, we found that the conserved His/Trp residue is strictly important to maintain the DNA binding capability and maintain the interaction in the protein-protein interface. The two conserved arginine residues are essential for the formation of catalytic synaptic complexes. Histidine residue participates in the strand cleavage for the Flp-FRT system but affect the synapsis for Cre-loxP system.

DNA Origami Based Visualization System for Studying Site-Specific Recombination Events

Site-specific recombination involves reciprocal exchange between defined DNA sites. The reaction initiates from the formation of a recombinase-DNA synaptic complex, in which two recombination sites arrange in an appropriate configuration. However, there is incomplete information about how the topological state of the substrate influences the synapsis and outcome of the reaction. Here, we show that Cre-mediated recombination can be regulated by controlling the orientation and topology of the loxP substrate in a DNA frame nanoscaffold. High-speed atomic force microscopy analyses revealed that the loxP-containing substrate strands in the antiparallel orientation can be recombined only through formation of synaptic complexes. By tethering Holliday junction (HJ) intermediates to DNA frames in different connection patterns and using them as a starting substrate, we found that the topological state of the HJ intermediates dictates the outcome of the resolution. Our approach should provide a new platform for structural-functional studies of various DNA targeting enzymes, especially which require formation of synaptic complexes.

Concurrent V(D)J recombination and DNA end instability increase interchromosomal trans -rearrangements in ATM-deficient thymocytes

During the CD4−CD8− (DN) stage of T-cell development, RAG-dependent DNA breaks and V(D)J recombination occur at three T-cell receptor (TCR) loci: TCRβ, TCRγ and TCRδ. During this stage, abnormal trans-rearrangements also take place between TCR loci, occurring at increased frequency in absence of the DNA damage response mediator ataxia telangiectasia mutated (ATM). Here, we use this model of physiologic trans-rearrangement to study factors that predispose to rearrangement and the role of ATM in preventing chromosomal translocations. The frequency of DN thymocytes with DNA damage foci at multiple TCR loci simultaneously is increased 2- to 3-fold in the absence of ATM. However, trans-rearrangement is increased 10 000- to 100 000-fold, indicating that ATM function extends beyond timely resolution of DNA breaks. RAG-mediated synaptic complex formation occurs between recombination signal sequences with unequal 12 and 23 base spacer sequences (12/23 rule). TCR trans-rearrangements violate this rule, as we observed similar frequencies of 12/23 and aberrant 12/12 or 23/23 recombination products. This suggests that trans-rearrangements are not the result of trans-synaptic complex formation, but they are instead because of unstable cis synaptic complexes that form simultaneously at distinct TCR loci. Thus, ATM suppresses trans-rearrangement primarily through stabilization of DNA breaks at TCR loci.

Observation of Synapse Rotation of Engineered Recombinase HIN-H107Y

Site-specific recombination promotes integration, excision or inversion of DNA strands during gene transcription and chromosome replication. The recombination process generally involves the formation of synaptic complexes, DNA cleavage and rejoining, though the specific mechanism of each enzyme varies. The Hin recombinase is a member of the serine recombinase family which has an active serine residue that can catalyze the DNA cleavage and re-ligating. Hin functions to invert a 900 bp DNA segment between two hin-binding (hix) sites within the Salmonella chromosome that contains a promoter for downstream flagellar genes. The inversion requires the auxiliary protein Fis and two Fis-binding sites on the targeted DNA segment, which assemble together with hix sites and Hin to form an invertasome structure. The assembly is stimulated by the protein HU. A class of engineered Hin mutants Hin-H107Y (H107Y) is able to assemble oligonucleotide substrates containing hix sites into stable synaptic complexes that catalyze recombination without Fis and HU. Structural data suggest that Hin share with gamma-delta resolvase a similar helix-turn-helix domain at the 40-50 amino acid residues. We hypothesized that H107Y could act like resolvase to form four subunits synapse, cleave both strands of DNA within the center of hix site, rotate and rejoin. We therefore used single DNA assays to analyze the dynamics associated with H107Y catalyzed strand exchange.Enzyme-DNA binding and synapse formation was observed with a single DNA containing two hix sites. We further obtained data for subunits rotation and re-ligation from a braiding assay which consisted of double DNAs each having a single hix site, indicating that the rotation strand exchange mechanism observed recently for Bxb1 Int is also possessed by Hin.