Acute reorganization of postsynaptic GABAA receptors reveals the functional impact of molecular nanoarchitecture at inhibitory synapses.

Neurotransmitter receptors partition into nanometer-scale subdomains within the postsynaptic membrane that are precisely aligned with presynaptic neurotransmitter release sites. While spatial coordination between pre- and postsynaptic elements is observed at both excitatory and inhibitory synapses, the functional significance of this molecular architecture has been challenging to evaluate experimentally. Here we utilized an optogenetic clustering approach to acutely alter the nanoscale organization of the postsynaptic inhibitory scaffold gephyrin while monitoring synaptic function. Gephyrin clustering rapidly enlarged postsynaptic area, laterally displacing GABAA receptors from their normally precise apposition with presynaptic active zones. Receptor displacement was accompanied by decreased synaptic GABAA receptor currents even though presynaptic release probability and the overall abundance and function of synaptic GABAA receptors remained unperturbed. Thus, acutely repositioning neurotransmitter receptors within the postsynaptic membrane profoundly influences synaptic efficacy, establishing the functional importance of precision pre-/postsynaptic molecular coordination at inhibitory synapses.

Outcomes of Anterior Cruciate Ligament Reconstruction With Independently Tensioned Suture Tape Augmentation at 5-Year Follow-up.

BACKGROUND: Reconstruction using autograft remains the gold standard surgical treatment for anterior cruciate ligament (ACL) injuries. However, up to 10% to 15% of patients will suffer a graft failure in the future. Cadaveric studies have demonstrated that the addition of suture tape augmentation to ACL autograft constructs can increase graft strength and reduce elongation under cyclical loading. PURPOSE/HYPOTHESIS: This study aimed to investigate the clinical outcomes and rerupture rates after ACL reconstruction (ACLR) with suture tape augmentation. We hypothesized that augmentation with suture tape would lead to lower rerupture rates. STUDY DESIGN: Case series; Level of evidence, 4. METHODS: Patients undergoing primary ACLR using hamstring or patellar tendon autografts augmented with suture tape between 2015 and 2019 were recruited prospectively. Patients with multiligament injuries or a concomitant lateral extra-articular procedure were excluded. Patients were observed in person for 6 months, and patient-reported outcome measures (PROMs) were collected at 2 and 5 years postoperatively. All patients were contacted, and records were reviewed to determine the incidence of graft failure. PROMs collected were as follows: Knee injury and Osteoarthritis Outcome Score (KOOS), Veterans RAND 12-Item Health Survey (VR-12), Tegner and Marx activity scores, and visual analog scale for pain (VAS). RESULTS: A total of 97 patients, with a mean age of 34.7 (±13.4) years, were included (76% men; 52 hamstring and 45 patellar tendon grafts). The mean graft diameter was 8 (±1) mm. There was 1 rerupture (1.1%) out of the 90 patients who were contactable at a mean of 5 years postoperatively. Median KOOS scores at 2 years were as follows: Pain, 94; Symptoms, 86; Activities of Daily Living, 99; Sport and Recreation, 82; and Quality of Life, 81. The postoperative scores were significantly higher than the preoperative scores (P < .001). The VR-12 Physical score improved from 43 preoperatively to 55 at 2 years and remained at 56 at 5 years. The VAS pain, Tegner, and Marx scores were 0, 6, and 9, respectively, at 2 years postoperatively. There was no difference in PROMs between graft types. CONCLUSION: This study demonstrates encouraging results of suture tape augmentation of autograft ACLR for both hamstring and patellar tendon grafts. The failure rate of 1.1% at a mean follow-up of 5 years is lower than published rates for reconstruction, and PROMs results are satisfactory. The technique is safe to use and may permit a return to the preinjury sporting level with a lower chance of reinjury.

AMPA and GABAA receptor nanodomains assemble in the absence of synaptic neurotransmitter release.

Postsynaptic neurotransmitter receptors and their associated scaffolding proteins assemble into discrete, nanometer-scale subsynaptic domains (SSDs) within the postsynaptic membrane at both excitatory and inhibitory synapses. Intriguingly, postsynaptic receptor SSDs are mirrored by closely apposed presynaptic active zones. These trans-synaptic molecular assemblies are thought to be important for efficient neurotransmission because they concentrate postsynaptic receptors near sites of presynaptic neurotransmitter release. While previous studies have characterized the role of synaptic activity in sculpting the number, size, and distribution of postsynaptic SSDs at established synapses, it remains unknown whether neurotransmitter signaling is required for their initial assembly during synapse development. Here, we evaluated synaptic nano-architecture under conditions where presynaptic neurotransmitter release was blocked prior to, and throughout synaptogenesis with tetanus neurotoxin (TeNT). In agreement with previous work, neurotransmitter release was not required for the formation of excitatory or inhibitory synapses. The overall size of the postsynaptic specialization at both excitatory and inhibitory synapses was reduced at chronically silenced synapses. However, both AMPARs and GABAARs still coalesced into SSDs, along with their respective scaffold proteins. Presynaptic active zone assemblies, defined by RIM1, were smaller and more numerous at silenced synapses, but maintained alignment with postsynaptic AMPAR SSDs. Thus, basic features of synaptic nano-architecture, including assembly of receptors and scaffolds into trans-synaptically aligned structures, are intrinsic properties that can be further regulated by subsequent activity-dependent mechanisms.

Molecular Determinants of Mouse Adaptation of Rat Hepacivirus.

The lack of robust immunocompetent animal models for hepatitis C virus (HCV) impedes vaccine development and studies of immune responses. Norway rat hepacivirus (NrHV) infection in rats shares HCV-defining characteristics, including hepatotropism, chronicity, immune responses, and aspects of liver pathology. To exploit genetic variants and research tools, we previously adapted NrHV to prolonged infection in laboratory mice. Through intrahepatic RNA inoculation of molecular clones of the identified variants, we here characterized four mutations in the envelope proteins responsible for mouse adaptation, including one disrupting a glycosylation site. These mutations led to high-titer viremia, similar to that observed in rats. In 4-week-old mice, infection was cleared after around 5 weeks compared to 2 to 3 weeks for nonadapted virus. In contrast, the mutations led to persistent but attenuated infection in rats, and they partially reverted, accompanied by an increase in viremia. Attenuated infection in rat but not mouse hepatoma cells demonstrated that the characterized mutations were indeed mouse adaptive rather than generally adaptive across species and that species determinants and not immune interactions were responsible for attenuation in rats. Unlike persistent NrHV infection in rats, acute resolving infection in mice was not associated with the development of neutralizing antibodies. Finally, infection of scavenger receptor B-I (SR-BI) knockout mice suggested that adaptation to mouse SR-BI was not a primary function of the identified mutations. Rather, the virus may have adapted to lower dependency on SR-BI, thereby potentially surpassing species-specific differences. In conclusion, we identified specific determinants of NrHV mouse adaptation, suggesting species-specific interactions during entry. IMPORTANCE A prophylactic vaccine is required to achieve the World Health Organization's objective for hepatitis C virus elimination as a serious public health threat. However, the lack of robust immunocompetent animal models supporting hepatitis C virus infection impedes vaccine development as well as studies of immune responses and viral evasion. Hepatitis C virus-related hepaciviruses were discovered in a number of animal species and provide useful surrogate infection models. Norway rat hepacivirus is of particular interest, as it enables studies in rats, an immunocompetent and widely used small laboratory animal model. Its adaptation to robust infection also in laboratory mice provides access to a broader set of mouse genetic lines and comprehensive research tools. The presented mouse-adapted infectious clones will be of utility for reverse genetic studies, and the Norway rat hepacivirus mouse model will facilitate studies of hepacivirus infection for in-depth characterization of virus-host interactions, immune responses, and liver pathology.

Regulation of cargo exocytosis by a Reps1-Ralbp1-RalA module.

Surface levels of membrane proteins are determined by a dynamic balance between exocytosis-mediated surface delivery and endocytosis-dependent retrieval from the cell surface. Imbalances in surface protein levels perturb surface protein homeostasis and cause major forms of human disease such as type 2 diabetes and neurological disorders. Here, we found a Reps1-Ralbp1-RalA module in the exocytic pathway broadly regulating surface protein levels. Reps1 and Ralbp1 form a binary complex that recognizes RalA, a vesicle-bound small guanosine triphosphatases (GTPase) promoting exocytosis through interacting with the exocyst complex. RalA binding results in Reps1 release and formation of a Ralbp1-RalA binary complex. Ralbp1 selectively recognizes GTP-bound RalA but is not a RalA effector. Instead, Ralbp1 binding maintains RalA in an active GTP-bound state. These studies uncovered a segment in the exocytic pathway and, more broadly, revealed a previously unrecognized regulatory mechanism for small GTPases, GTP state stabilization.

Dually innervated dendritic spines develop in the absence of excitatory activity and resist plasticity through tonic inhibitory crosstalk.

Dendritic spines can be directly connected to both inhibitory and excitatory presynaptic terminals, resulting in nanometer-scale proximity of opposing synaptic functions. While dually innervated spines (DiSs) are observed throughout the central nervous system, their developmental timeline and functional properties remain uncharacterized. Here we used a combination of serial section electron microscopy, live imaging, and local synapse activity manipulations to investigate DiS development and function in rodent hippocampus. Dual innervation occurred early in development, even on spines where the excitatory input was locally silenced. Synaptic NMDA receptor currents were selectively reduced at DiSs through tonic GABAB receptor signaling. Accordingly, spine enlargement normally associated with long-term potentiation on singly innervated spines (SiSs) was blocked at DiSs. Silencing somatostatin interneurons or pharmacologically blocking GABABRs restored NMDA receptor function and structural plasticity to levels comparable to neighboring SiSs. Thus, hippocampal DiSs are stable structures where function and plasticity are potently regulated by nanometer-scale GABAergic signaling.

miRNA-mediated control of gephyrin synthesis drives sustained inhibitory synaptic plasticity.

Activity-dependent protein synthesis is crucial for many long-lasting forms of synaptic plasticity. However, our understanding of the translational mechanisms controlling inhibitory synapses is limited. One distinct form of inhibitory long-term potentiation (iLTP) enhances postsynaptic clusters of GABAARs and the primary inhibitory scaffold, gephyrin, to promote sustained synaptic strengthening. While we previously found that persistent iLTP requires mRNA translation, the precise mechanisms controlling gephyrin translation during this process remain unknown. Here, we identify miR153 as a novel regulator of Gphn mRNA translation which controls gephyrin protein levels and synaptic clustering, ultimately impacting GABAergic synaptic structure and function. We find that iLTP induction downregulates miR153, reversing its translational suppression of Gphn mRNA and allowing for increased de novo gephyrin protein synthesis and synaptic clustering during iLTP. Finally, we find that reduced miR153 expression during iLTP is driven by an excitation-transcription coupling pathway involving calcineurin, NFAT and HDACs, which also controls the miRNA-dependent upregulation of GABAARs. Overall, this work delineates a miRNA-dependent post-transcriptional mechanism that controls the expression of the key synaptic scaffold, gephyrin, and may converge with parallel miRNA pathways to coordinate gene upregulation to maintain inhibitory synaptic plasticity.

Properties and modulation of excitatory inputs to the locus coeruleus.

Excitatory inputs drive burst firing of locus coeruleus (LC) noradrenaline (NA) neurons in response to a variety of stimuli. Though a small number of glutamatergic LC afferents have been investigated, the overall landscape of these excitatory inputs is largely unknown. The current study used an optogenetic approach to isolate three glutamatergic afferents: the prefrontal cortex (PFC), lateral hypothalamus (LH) and periaqueductal grey (PAG). AAV5-DIO-ChR2 was injected into each region in male and female CaMKII-Cre mice and the properties of excitatory inputs on LC-NA cells were measured. Notably we found differences among these inputs. First, the pattern of axonal innervation differed between inputs such that LH afferents were concentrated in the posterior portion of the LC-NA somatic region while PFC afferents were denser in the medial dendritic region. Second, basal intrinsic properties varied for afferents, with LH inputs having the highest connectivity and the largest amplitude excitatory postsynaptic currents while PAG inputs had the lowest initial release probability. Third, while orexin and oxytocin had minimal effects on any input, dynorphin strongly inhibited excitatory inputs originating from the LH and PAG, and corticotrophin releasing factor (CRF) selectively inhibited inputs from the PAG. Overall, these results demonstrate that individual afferents to the LC have differing properties, which may contribute to the modularity of the LC and its ability to mediate various behavioural outcomes. KEY POINTS: Excitatory inputs to the locus coeruleus (LC) are important for driving noradrenaline neuron activity and downstream behaviours in response to salient stimuli, but little is known about the functional properties of different glutamate inputs that innervate these neurons We used a virus-mediated optogenetic approach to compare glutamate afferents from the prefrontal cortex (PFC), the lateral hypothalamus (LH) and the periaqueductal grey (PAG). While PFC was predicted to make synaptic inputs, we found that the LH and PAG also drove robust excitatory events in LC noradrenaline neurons. The strength, kinetics, and short-term plasticity of each input differed as did the extent of neuromodulation by both dynorphin and corticotrophin releasing factor. Thus each input displayed a unique set of basal properties and modulation by peptides. This characterization is an important step in deciphering the heterogeneity of the LC.

Spatial and Temporal Control of Protein Secretion with Light.

How newly synthesized integral membrane proteins and secreted factors are sorted and trafficked to the appropriate location in different cell types remains an important problem in cell biology. One powerful approach for elucidating the trafficking route of a specific protein is to sequester it following synthesis in the endoplasmic reticulum and trigger its release with an externally applied cue. Combined with fluorescent probes, this approach can be used to directly visualize each trafficking step as cargo molecules progress through the different organelles of the secretory network. Here, we discuss design strategies and practical implementation of an inducible protein secretion system we recently developed (zapalog mediated ER trap: zapERtrap) that allows one to use light to initiate secretory trafficking from targeted cells or subcellular domains. We provide detailed protocols for experiments using this approach to visualize protein trafficking from the endoplasmic reticulum to the plasma membrane in fibroblast cell lines and primary cultured neurons.

Complementary Use of Super-Resolution Imaging Modalities to Study the Nanoscale Architecture of Inhibitory Synapses.

The nanoscale architecture of synapses has been investigated using multiple super-resolution methods, revealing a common modular structure for scaffolds, neurotransmitter receptors, and presynaptic proteins. This fundamental organization of proteins into subsynaptic domains (SSDs) is thought to be important for synaptic function and plasticity and common to many types of synapses. Using 3D super-resolution Structured Illumination Microscopy (3D-SIM), we recently showed that GABAergic inhibitory synapses exhibit this nanoscale organizational principle and are composed of SSDs of GABA A receptors (GABA A Rs), the inhibitory scaffold gephyrin, and the presynaptic active zone protein, RIM. Here, we have investigated the use of 3D-SIM and dSTORM to analyze the nanoscale architecture of the inhibitory synaptic adhesion molecule, neuroligin-2 (NL2). NL2 is a crucial mediator of inhibitory synapse formation and organization, associating with both GABA A Rs and gephyrin. However, the nanoscale sub-synaptic distribution NL2 remains unknown. We found that 3D-SIM and dSTORM provide complementary information regarding the distribution of NL2 at the inhibitory synapse, with NL2 forming nanoscale structures that have many similarities to gephyrin nanoscale architecture.

Autologous versus synthetic bone grafts for the surgical management of tibial plateau fractures: a systematic review and meta-analysis of randomized controlled trials.

AIMS: Our objective was to conduct a systematic review and meta-analysis, to establish whether differences arise in clinical outcomes between autologous and synthetic bone grafts in the operative management of tibial plateau fractures. METHODS: A structured search of MEDLINE, EMBASE, the online archives of Bone & Joint Publishing, and CENTRAL databases from inception until 28 July 2021 was performed. Randomized, controlled, clinical trials that compared autologous and synthetic bone grafts in tibial plateau fractures were included. Preclinical studies, clinical studies in paediatric patients, pathological fractures, fracture nonunion, or chondral defects were excluded. Outcome data were assessed using the Risk of Bias 2 (ROB2) framework and synthesized in random-effect meta-analysis. The Preferred Reported Items for Systematic Review and Meta-Analyses guidance was followed throughout. RESULTS: Six studies involving 353 fractures were identified from 3,078 records. Following ROB2 assessment, five studies (representing 338 fractures) were appropriate for meta-analysis. Primary outcomes showed non-significant reductions in articular depression at immediate postoperative (mean difference -0.45 mm, p = 0.25, 95%confidence interval (CI) -1.21 to 0.31, I2 = 0%) and long-term (> six months, standard mean difference -0.56, p = 0.09, 95% CI -1.20 to 0.08, I2 = 73%) follow-up in synthetic bone grafts. Secondary outcomes included mechanical alignment, limb functionality, and defect site pain at long-term follow-up, perioperative blood loss, duration of surgery, occurrence of surgical site infections, and secondary surgery. Mean blood loss was lower (90.08 ml, p < 0.001, 95% CI 41.49 to 138.67) and surgery was shorter (16.17 minutes, p = 0.04, 95% CI 0.39 to 31.94) in synthetic treatment groups. All other secondary measures were statistically comparable. CONCLUSION: All studies reported similar methodologies and patient populations; however, imprecision may have arisen through performance variation. These findings supersede previous literature and indicate that, despite perceived biological advantages, autologous bone grafting does not demonstrate superiority to synthetic grafts. When selecting a void filler, surgeons should consider patient comorbidity, environmental and societal factors in provision, and perioperative and postoperative care provision. Cite this article: Bone Jt Open 2022;3(3):218-228.

Opioid Prescription Following Wrist and Ankle Fracture Fixation in Scotland-Tradition Prevails.

The American 'opioid crisis' is rapidly spreading internationally. Perioperative opioid use increases the risk of long-term opioid use. We review opioid use following wrist and ankle fracture fixation across Scotland, establishing prescribing patterns and associations with patient, injury, or perioperative factors. Six Scottish orthopedic units contributed. A total of 598 patients were included. Patient demographics were similar across all sites. There was variation in anesthetic practice, length of stay, and AO fracture type (p < 0.01). For wrist fractures, 85.6% of patients received a discharge opioid prescription; 5.0% contained a strong opioid. There was no significant variation across the six units in prescribing practice. For ankle fractures, 82.7% of patients received a discharge opioid prescription; 17% contained a strong opioid. Dundee and Edinburgh used more strong opioids; Inverness and Paisley gave the least opioids overall (p < 0.01). Younger patient age, location, and length of stay were independent predictors of increased prescription on binary regression. Despite variability in perioperative practices, discharge opioid analgesic prescription remains overwhelmingly consistent. We believe that the biggest influence lies with the prescriber-institutional 'standard practice'. Education of these prescribing clinicians regarding the risk profile of opioids is key to reducing their use following surgery, thus lowering long-term opioid dependence.

Stepwise disassembly of GABAergic synapses during pathogenic excitotoxicity.

GABAergic synaptic inhibition controls neuronal firing, excitability, and synaptic plasticity to regulate neuronal circuits. Following an acute excitotoxic insult, inhibitory synapses are eliminated, reducing synaptic inhibition, elevating circuit excitability, and contributing to the pathophysiology of brain injuries. However, mechanisms that drive inhibitory synapse disassembly and elimination are undefined. We find that inhibitory synapses are disassembled in a sequential manner following excitotoxicity: GABAARs undergo rapid nanoscale rearrangement and are dispersed from the synapse along with presynaptic active zone components, followed by the gradual removal of the gephyrin scaffold, prior to complete elimination of the presynaptic terminal. GABAAR nanoscale reorganization and synaptic declustering depends on calcineurin signaling, whereas disassembly of gephyrin relies on calpain activation, and blockade of both enzymes preserves inhibitory synapses after excitotoxic insult. Thus, inhibitory synapse disassembly occurs rapidly, with nanoscale precision, in a stepwise manner and most likely represents a critical step in the progression of hyperexcitability following excitotoxicity.

Neurexin-3 defines synapse- and sex-dependent diversity of GABAergic inhibition in ventral subiculum.

Ventral subiculum (vSUB) is integral to the regulation of stress and reward; however, the intrinsic connectivity and synaptic properties of the inhibitory local circuit are poorly understood. Neurexin-3 (Nrxn3) is highly expressed in hippocampal inhibitory neurons, but its function at inhibitory synapses has remained elusive. Using slice electrophysiology, imaging, and single-cell RNA sequencing, we identify multiple roles for Nrxn3 at GABAergic parvalbumin (PV) interneuron synapses made onto vSUB regular-spiking (RS) and burst-spiking (BS) principal neurons. Surprisingly, we find that intrinsic connectivity of vSUB and synaptic function of Nrxn3 in vSUB are sexually dimorphic. We reveal that PVs make preferential contact with RS neurons in male mice, but BS neurons in female mice. Furthermore, we determine that despite comparable Nrxn3 isoform expression in male and female PV neurons, Nrxn3 knockout impairs synapse density, postsynaptic strength, and inhibitory postsynaptic current (IPSC) amplitude at PV-RS synapses in males, but enhances presynaptic release and IPSC amplitude in females.

Precision Mapping of Amyloid-ß Binding Reveals Perisynaptic Localization and Spatially Restricted Plasticity Deficits.

Secreted amyloid-ß (Aß) peptide forms neurotoxic oligomeric assemblies thought to cause synaptic deficits associated with Alzheimer's disease (AD). Soluble Aß oligomers (Aßo) directly bind to neurons with high affinity and block plasticity mechanisms related to learning and memory, trigger loss of excitatory synapses and eventually cause cell death. While Aßo toxicity has been intensely investigated, it remains unclear precisely where Aßo initially binds to the surface of neurons and whether sites of binding relate to synaptic deficits. Here, we used a combination of live cell, super-resolution and ultrastructural imaging techniques to investigate the kinetics, reversibility and nanoscale location of Aßo binding. Surprisingly, Aßo does not bind directly at the synaptic cleft as previously thought but, instead, forms distinct nanoscale clusters encircling the postsynaptic membrane with a significant fraction also binding presynaptic axon terminals. Synaptic plasticity deficits were observed at Aßo-bound synapses but not closely neighboring Aßo-free synapses. Thus, perisynaptic Aßo binding triggers spatially restricted signaling mechanisms to disrupt synaptic function. These data provide new insight into the earliest steps of Aßo pathology and lay the groundwork for future studies evaluating potential surface receptor(s) and local signaling mechanisms responsible for Aßo binding and synapse dysfunction.

GluN2B S1303 phosphorylation by CaMKII or DAPK1: no indication for involvement in ischemia or LTP.

Binding of two different CaM kinases, CaMKII and DAPK1, to the NMDA-type glutamate receptor (NMDAR) subunit GluN2B near S1303 has been implicated in excitotoxic/ischemic neuronal cell death. The GluN2BΔCaMKII mutation (L1298A, R1300Q) is neuroprotective but abolishes only CaMKII but not DAPK1 binding. However, both kinases can additionally phosphorylate GluN2B S1303. Thus, we here tested S1303 phosphorylation for possible contribution to neuronal cell death. The GluN2BΔCaMKII mutation completely abolished phosphorylation by CaMKII and DAPK1, suggesting that the mutation could mediate neuroprotection by disrupting phosphorylation. However, S1303 phosphorylation was not increased by excitotoxic insults in hippocampal slices or by global cerebral ischemia induced by cardiac arrest and cardiopulmonary resuscitation in vivo. In hippocampal cultures, S1303 phosphorylation was induced by chemical LTD but not LTP stimuli. These results indicate that the additional effect of the GluN2BΔCaMKII mutation on phosphorylation needs to be considered only in LTD but not in LTP or ischemia/excitotoxicity.

zapERtrap: A light-regulated ER release system reveals unexpected neuronal trafficking pathways.

Here we introduce zapalog-mediated endoplasmic reticulum trap (zapERtrap), which allows one to use light to precisely trigger forward trafficking of diverse integral membrane proteins from internal secretory organelles to the cell surface with single cell and subcellular spatial resolution. To demonstrate its utility, we use zapERtrap in neurons to dissect where synaptic proteins emerge at the cell surface when processed through central (cell body) or remote (dendrites) secretory pathways. We reveal rapid and direct long-range trafficking of centrally processed proteins deep into the dendritic arbor to synaptic sites. Select proteins were also trafficked to the plasma membrane of the axon initial segment, revealing a novel surface trafficking hotspot. Proteins locally processed through dendritic secretory networks were widely dispersed before surface insertion, challenging assumptions for precise trafficking at remote sites. These experiments provide new insights into compartmentalized secretory trafficking and showcase the tunability and spatiotemporal control of zapERtrap, which will have broad applications for regulating cell signaling and function.

Measuring Transcellular Interactions through Protein Aggregation in a Heterologous Cell System.

Protein interactions at cellular interfaces dictate a multitude of biological outcomes ranging from tissue development and cancer progression to synapse formation and maintenance. Many of these fundamental interactions occur in trans and are typically induced by heterophilic or homophilic interactions between cells expressing membrane anchored binding pairs. Elucidating how disease relevant mutations disrupt these fundamental protein interactions can provide insight into a myriad of cell biology fields. Many protein-protein interaction assays do not typically disambiguate between cis and trans interactions, which potentially leads to an overestimation of the extent of binding that is occurring in vivo and involve labor intensive purification of protein and/or specialized monitoring equipment. Here, we present an optimized simple protocol that allows for the observation and quantification of only trans interactions without the need for lengthy protein purifications or specialized equipment. The HEK cell aggregation assay involves the mixing of two independent populations of HEK cells, each expressing membrane-bound cognate ligands. After a short incubation period, samples are imaged and the resulting aggregates are quantified.

Architecture and Dynamics of the Neuronal Secretory Network.

Regulated synthesis and movement of proteins between cellular organelles are central to diverse forms of biological adaptation and plasticity. In neurons, the repertoire of channel, receptor, and adhesion proteins displayed on the cell surface directly impacts cellular development, morphology, excitability, and synapse function. The immensity of the neuronal surface membrane and its division into distinct functional domains present a challenging landscape over which proteins must navigate to reach their appropriate functional domains. This problem becomes more complex considering that neuronal protein synthesis is continuously refined in space and time by neural activity. Here we review our current understanding of how integral membrane and secreted proteins important for neuronal function travel from their sites of synthesis to their functional destinations. We discuss how unique adaptations to the function and distribution of neuronal secretory organelles may facilitate local protein trafficking at remote sites in neuronal dendrites to support diverse forms of synaptic plasticity.