Multi-monoubiquitylation controls VASP-mediated actin dynamics.

The actin cytoskeleton performs multiple cellular functions, and as such, actin polymerization must be tightly regulated. We previously demonstrated that reversible, non-degradative ubiquitylation regulates the function of the actin polymerase VASP in developing neurons. However, the underlying mechanism of how ubiquitylation impacts VASP activity was unknown. Here, we show that mimicking multi-monoubiquitylation of VASP at K240 and K286 negatively regulates VASP interactions with actin. Using in vitro biochemical assays, we demonstrate the reduced ability of multi-monoubiquitylated VASP to bind, bundle, and elongate actin filaments. However, multi-monoubiquitylated VASP maintained the ability to bind and protect barbed ends from capping protein. Finally, we demonstrate the electroporation of recombinant multi-monoubiquitylated VASP protein altered cell spreading morphology. Collectively, these results suggest a mechanism in which ubiquitylation controls VASP-mediated actin dynamics.

pHusion - a robust and versatile toolset for automated detection and analysis of exocytosis.

Exocytosis is a fundamental process used by eukaryotes to regulate the composition of the plasma membrane and facilitate cell-cell communication. To investigate exocytosis in neuronal morphogenesis, previously we developed computational tools with a graphical user interface to enable the automatic detection and analysis of exocytic events from fluorescence timelapse images. Although these tools were useful, we found the code was brittle and not easily adapted to different experimental conditions. Here, we developed and validated a robust and versatile toolkit, named pHusion, for the analysis of exocytosis, written in ImageTank, a graphical programming language that combines image visualization and numerical methods. We tested pHusion using a variety of imaging modalities and pH-sensitive fluorophores, diverse cell types and various exocytic markers, to generate a flexible and intuitive package. Using this system, we show that VAMP3-mediated exocytosis occurs 30-times more frequently in melanoma cells compared with primary oligodendrocytes, that VAMP2-mediated fusion events in mature rat hippocampal neurons are longer lasting than those in immature murine cortical neurons, and that exocytic events are clustered in space yet random in time in developing cortical neurons.

Cerebellin-1 leads the way.

The cerebellin family of proteins influences synapse formation and function. In this issue of PLOS Biology, Han and colleagues identify a new role for Cerebellin-1 in axon growth and guidance.

Experience with cultured thymus tissue in 105 children.

BACKGROUND: Currently, there are no approved therapies to treat congenital athymia, a condition of immune deficiency resulting in high early mortality due to infection and immune dysregulation. Multiple syndromic conditions, such as complete DiGeorge syndrome, 22q11.2 deletion syndrome, CHARGE (coloboma, heart defects, choanal atresia, growth or mental retardation, genital hypoplasia, and ear anomalies and/or deafness) syndrome, diabetic embryopathy, other genetic variants, and FOXN1 deficiency, are associated with congenital athymia. OBJECTIVE: Our aims were to study 105 patients treated with cultured thymus tissue (CTT), and in this report, to focus on the outcomes of 95 patients with treatment-naive congenital athymia. METHODS: A total of 10 prospective, single-arm open-label studies with patient enrollment from 1993 to 2020 form the basis of this data set. Patients were tested after administration of CTT for T-cell development; all adverse events and infections were recorded. RESULTS: A total of 105 patients were enrolled and received CTT (the full analysis set). Of those patients, 10 had diagnoses other than congenital athymia and/or received prior treatments. Of those 105 patients, 95 patients with treatment-naive congenital athymia were included in the efficacy analysis set (EAS). The Kaplan-Meier estimated survival rates at year 1 and year 2 after administration of CTT in the EAS were 77% (95% CI = 0.670-0.844) and 76% (95% CI = 0.657-0.834), respectively. In all, 21 patients died in the first year before developing naive T cells and 1 died in the second year after receipt of CTT; 3 subsequent deaths were not related to immunodeficiency. A few patients developed alopecia, autoimmune hepatitis, psoriasis, and psoriatic arthritis after year 1. The rates of infections, autologous graft-versus-host-disease manifestations, and autoimmune cytopenias all decreased approximately 1 year after administration of CTT. CONCLUSION: Treatment with CTT led to development of naive T cells with a 1-year survival rate of 77% and a median follow-up time of 7.6 years. Immune reconstitution sufficient to prevent infections and support survival typically develops 6 to12 months after administration of CTT.

Schizophrenia-Linked Protein tSNARE1 Regulates Endosomal Trafficking in Cortical Neurons.

TSNARE1, which encodes the protein tSNARE1, is a high-confidence gene candidate for schizophrenia risk, but nothing is known about its cellular or physiological function. We identified the major gene products of TSNARE1 and their cytoplasmic localization and function in endosomal trafficking in cortical neurons. We validated three primary isoforms of TSNARE1 expressed in human brain, all of which encode a syntaxin-like Qa SNARE domain. RNA-sequencing data from adult and fetal human brain suggested that the majority of tSNARE1 lacks a transmembrane domain that is thought to be necessary for membrane fusion. Biochemical data demonstrate that tSNARE1 can compete with Stx12 for incorporation into an endosomal SNARE complex, supporting its possible role as an inhibitory SNARE. Live-cell imaging in cortical neurons from mice of both sexes demonstrated that brain tSNARE1 isoforms localized to the endosomal network. The most abundant brain isoform, tSNARE1c, localized most frequently to Rab7+ late endosomes, and endogenous tSNARE1 displayed a similar localization in human neural progenitor cells and neuroblastoma cells. In mature rat neurons from both sexes, tSNARE1 localized to the dendritic shaft and dendritic spines, supporting a role for tSNARE1 at the postsynapse. Expression of either tSNARE1b or tSNARE1c, which differ only in their inclusion or exclusion of an Myb-like domain, delayed the trafficking of the dendritic endosomal cargo Nsg1 into late endosomal and lysosomal compartments. These data suggest that tSNARE1 regulates endosomal trafficking in cortical neurons, likely by negatively regulating early endosomal to late endosomal trafficking.SIGNIFICANCE STATEMENT Schizophrenia is a severe and polygenic neuropsychiatric disorder. Understanding the functions of high-confidence candidate genes is critical toward understanding how their dysfunction contributes to schizophrenia pathogenesis. TSNARE1 is one of the high-confidence candidate genes for schizophrenia risk, yet nothing was known about its cellular or physiological function. Here we describe the major isoforms of TSNARE1 and their cytoplasmic localization and function in the endosomal network in cortical neurons. Our results are consistent with the hypothesis that the majority of brain tSNARE1 acts as a negative regulator to endolysosomal trafficking.

Care of Children with DiGeorge Before and After Cultured Thymus Tissue Implantation.

BACKGROUND: Children with complete DiGeorge anomaly (cDGA) have congenital athymia plus a myriad of other challenging clinical conditions. The term cDGA encompasses children with congenital athymia secondary to 22q11.2DS, CHARGE syndrome (coloboma, heart defects, choanal atresia, growth or mental retardation, genital abnormalities, and ear abnormalities and/or deafness), and other genetic abnormalities. Some children have no known genetic defects. Since 1993, more than 100 children with congenital athymia have been treated with cultured thymus tissue implantation (CTTI). Naïve T cells develop approximately 6 to 12 months after CTTI. Most of the children had significant comorbidities such as heart disease, hypoparathyroidism, and infections requiring complex clinical care post cultured thymus tissue implantation (CTTI). OBJECTIVE: The purpose of this guidance is to assist multidisciplinary teams in caring for children with cDGA both before and after CTTI. METHODS: Thirty-one specialists, in addition to the authors, were asked to share their experience in caring for children with cDGA at Duke University Health System, before and after CTTI. These specialists included physicians, nurses, dentists, therapists, and dieticians. RESULTS: The goal of a multidisciplinary approach is to have children in the best possible condition for receiving CTTI and provide optimal care post CTTI through development of naïve T cells and beyond. The CTT (cultured thymus tissue) must be protected from high doses of steroids which can damage CTT. Organs must be protected from adverse effects of immunosuppression. CONCLUSION: Creating a multidisciplinary team and a detailed plan of care for children with cDGA is important for optimal outcomes.

Glycosylation in Axonal Guidance.

How millions of axons navigate accurately toward synaptic targets during development is a long-standing question. Over decades, multiple studies have enriched our understanding of axonal pathfinding with discoveries of guidance molecules and morphogens, their receptors, and downstream signalling mechanisms. Interestingly, classification of attractive and repulsive cues can be fluid, as single guidance cues can act as both. Similarly, guidance cues can be secreted, chemotactic cues or anchored, adhesive cues. How a limited set of guidance cues generate the diversity of axonal guidance responses is not completely understood. Differential expression and surface localization of receptors, as well as crosstalk and spatiotemporal patterning of guidance cues, are extensively studied mechanisms that diversify axon guidance pathways. Posttranslational modification is a common, yet understudied mechanism of diversifying protein functions. Many proteins in axonal guidance pathways are glycoproteins and how glycosylation modulates their function to regulate axonal motility and guidance is an emerging field. In this review, we discuss major classes of glycosylation and their functions in axonal pathfinding. The glycosylation of guidance cues and guidance receptors and their functional implications in axonal outgrowth and pathfinding are discussed. New insights into current challenges and future perspectives of glycosylation pathways in neuronal development are discussed.

TRIM9 and TRIM67 Are New Targets in Paraneoplastic Cerebellar Degeneration.

To describe autoantibodies (Abs) against tripartite motif-containing (TRIM) protein 9 and 67 in two patients with paraneoplastic cerebellar degeneration (PCD) associated with lung adenocarcinoma. Abs were characterized using immunohistochemistry, Western blotting, cultures of murine cortical, and hippocampal neurons, immunoprecipitation, mass spectrometry, knockout mice for Trim9 and 67, and cell-based assay. Control samples included sera from 63 patients with small cell lung cancer without any paraneoplastic neurological syndrome, 36 patients with lung adenocarcinoma and PNS, CSF from 100 patients with autoimmune encephalitis, and CSF from 165 patients with neurodegenerative diseases. We found Abs targeting TRIM9 and TRIM67 at high concentration in the serum and the cerebrospinal fluid (CSF) of a 78-year-old woman and a 65-year-old man. Both developed subacute severe cerebellar ataxia. Brain magnetic resonance imaging found no abnormality and no cerebellar atrophy. Both had CSF inflammation with mild pleiocytosis and a few oligoclonal bands. We identified a pulmonary adenocarcinoma, confirming the paraneoplastic neurological syndrome in both patients. They received immunomodulatory and cancer treatments without improvement of cerebellar ataxia, even though both were in remission of their cancer (for more than 10 years in one patient). Anti-TRIM9 and anti-TRIM67 Abs were specific to these two patients. All control serum and CSF samples tested were negative for anti-TRIM9 and 67. Anti-TRIM9 and anti-TRIM67 Abs appeared to be specific biomarkers of PCD and should be added to the panel of antigens tested when this is suspected.

Trim9 Deletion Alters the Morphogenesis of Developing and Adult-Born Hippocampal Neurons and Impairs Spatial Learning and Memory.

UNLABELLED: During hippocampal development, newly born neurons migrate to appropriate destinations, extend axons, and ramify dendritic arbors to establish functional circuitry. These developmental stages are recapitulated in the dentate gyrus of the adult hippocampus, where neurons are continuously generated and subsequently incorporate into existing, local circuitry. Here we demonstrate that the E3 ubiquitin ligase TRIM9 regulates these developmental stages in embryonic and adult-born mouse hippocampal neurons in vitro and in vivo Embryonic hippocampal and adult-born dentate granule neurons lacking Trim9 exhibit several morphological defects, including excessive dendritic arborization. Although gross anatomy of the hippocampus was not detectably altered by Trim9 deletion, a significant number of Trim9(-/-) adult-born dentate neurons localized inappropriately. These morphological and localization defects of hippocampal neurons in Trim9(-/-) mice were associated with extreme deficits in spatial learning and memory, suggesting that TRIM9-directed neuronal morphogenesis may be involved in hippocampal-dependent behaviors. SIGNIFICANCE STATEMENT: Appropriate generation and incorporation of adult-born neurons in the dentate gyrus are critical for spatial learning and memory and other hippocampal functions. Here we identify the brain-enriched E3 ubiquitin ligase TRIM9 as a novel regulator of embryonic and adult hippocampal neuron shape acquisition and hippocampal-dependent behaviors. Genetic deletion of Trim9 elevated dendritic arborization of hippocampal neurons in vitro and in vivo Adult-born dentate granule cells lacking Trim9 similarly exhibited excessive dendritic arborization and mislocalization of cell bodies in vivo These cellular defects were associated with severe deficits in spatial learning and memory.

Filopodia are required for cortical neurite initiation.

Extension of neurites from a cell body is essential to form a functional nervous system; however, the mechanisms underlying neuritogenesis are poorly understood. Ena/VASP proteins regulate actin dynamics and modulate elaboration of cellular protrusions. We recently reported that cortical axon-tract formation is lost in Ena/VASP-null mice and Ena/VASP-null cortical neurons lack filopodia and fail to elaborate neurites. Here, we report that neuritogenesis in Ena/VASP-null neurons can be rescued by restoring filopodia formation through ectopic expression of the actin nucleating protein mDia2. Conversely, wild-type neurons in which filopodia formation is blocked fail to elaborate neurites. We also report that laminin, which promotes the formation of filopodia-like actin-rich protrusions, rescues neuritogenesis in Ena/VASP-deficient neurons. Therefore, filopodia formation is a key prerequisite for neuritogenesis in cortical neurons. Neurite initiation also requires microtubule extension into filopodia, suggesting that interactions between actin-filament bundles and dynamic microtubules within filopodia are crucial for neuritogenesis.

Clinical course and outcome predictors of critically ill infants with complete DiGeorge anomaly following thymus transplantation.

OBJECTIVES: To identify risk factors for PICU admission and mortality of infants with complete DiGeorge anomaly treated with thymus transplantation. We hypothesized that age at transplantation and the presence of congenital heart disease would be risk factors for emergent PICU admission, and these factors plus development of septicemia would increase morbidity and mortality. DESIGN: Retrospective review. SETTING: Academic medical-surgical PICU. PATIENTS: All infants with complete DiGeorge anomaly treated with thymus transplantation between January 1, 1993, and July 1, 2010. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Consent was obtained from 71 infants with complete DiGeorge anomaly for thymus transplantation, and 59 infants were transplanted. Median age at transplantation was 5.0 months (range, 1.1-22.1 mo). After transplantation, 12 of 59 infants (20%) required 25 emergent PICU admissions. Seven of 12 infants (58%) survived to PICU discharge with six surviving 6 months posttransplantation. Forty-two of 59 infants (71%) transplanted had congenital heart disease, and 9 of 12 (75%) who were admitted to the PICU had congenital heart disease. In 15 of 25 admissions (60%), intubation and mechanical ventilation were necessary. There was no difference between median ventilation-free days between infants with and without congenital heart disease (33 d vs 23 d, p = 0.544). There was also no correlation between ventilation-free days and age of transplantation (R, 0.17; p = 0.423). Age at transplantation and the presence of congenital heart disease were not associated with risk for PICU admission (odds ratio, 0.95; 95% CI, 0.78-1.15 and odds ratio, 1.27; 95% CI, 0.30-5.49, respectively) or PICU mortality (odds ratio, 0.98; 95% CI, 0.73-1.31 and odds ratio, 0.40; 95% CI, 0.15-1.07, respectively). CONCLUSIONS: Most transplanted infants did not require emergent PICU admission. Age at transplantation and the presence of congenital heart disease were not associated with PICU admission or mortality.

Loss of the E3 ubiquitin ligase TRIM67 alters the post-synaptic density proteome.

The E3 ubiquitin ligase TRIM67 is enriched in the central nervous system and is required for proper neuronal development. Previously we demonstrated TRIM67 coordinates with the closely related E3 ubiquitin ligase TRIM9 to regulate cytoskeletal dynamics downstream of the netrin-1 during axon guidance and axon branching in early neuronal morphogenesis. Interestingly, loss of Trim67 impacts cognitive flexibility in a spatial learning and memory task. Despite this behavioral phenotype, it was previously uninvestigated if TRIM67 was involved in synapse formation or function. Here we demonstrate TRIM67 localizes to the post-synaptic density (PSD) within dendritic spines. Furthermore, we show that loss of Trim67 significantly changes the PSD proteome, including changes in the regulation of the actin and microtubule cytoskeletons. Collectively, our data propose a synaptic role for TRIM67.

Loss of the E3 ubiquitin ligase TRIM67 alters the post-synaptic density proteome.

The E3 ubiquitin ligase TRIM67 is enriched in the central nervous system and is required for proper neuronal development. Previously we demonstrated TRIM67 coordinates with the closely related E3 ubiquitin ligase TRIM9 to regulate cytoskeletal dynamics downstream of the netrin-1 during axon guidance and axon branching in early neuronal morphogenesis. Interestingly, loss of Trim67 impacts cognitive flexibility in a spatial learning and memory task. Despite this behavioral phenotype, it was previously uninvestigated if TRIM67 was involved in synapse formation or function. Here we demonstrate TRIM67 localizes to the post-synaptic density (PSD) within dendritic spines. Furthermore, we show that loss of Trim67 significantly changes a subset of proteins within the PSD proteome, including changes in the regulation of the actin and microtubule cytoskeletons. Collectively, our data propose a synaptic role for TRIM67.

TRIM9 controls growth cone responses to netrin through DCC and UNC5C.

The guidance cue netrin-1 promotes both growth cone attraction and growth cone repulsion. How netrin-1 elicits these diverse axonal responses, beyond engaging the attractive receptor DCC and repulsive receptors of the UNC5 family, remains elusive. Here we demonstrate that murine netrin-1 induces biphasic axonal responses in cortical neurons: attraction at lower concentrations and repulsion at higher concentrations using both a microfluidic-based netrin-1 gradient and bath application of netrin-1. TRIM9 is a brain-enriched E3 ubiquitin ligase previously shown to bind and cluster the attractive receptor DCC at the plasma membrane and regulate netrin-dependent attractive responses. However, whether TRIM9 also regulated repulsive responses to netrin-1 remained to be seen. In this study, we show that TRIM9 localizes and interacts with both the attractive netrin receptor DCC and the repulsive netrin receptor, UNC5C, and that deletion of murine Trim9 alters both attractive and repulsive responses to murine netrin-1. TRIM9 was required for netrin-1-dependent changes in surface levels of DCC and total levels of UNC5C in the growth cone during morphogenesis. We demonstrate that DCC at the membrane regulates growth cone area and show that TRIM9 negatively regulates FAK activity in the absence of netrin-1. We investigate membrane dynamics of the UNC5C receptor using pH-mScarlet fused to the extracellular domain of UNC5C. Minutes after netrin addition, levels of UNC5C at the plasma membrane drop in a TRIM9-independent fashion, however TRIM9 regulated the mobility of UNC5C in the plasma membrane in the absence of netrin-1. Together this work demonstrates that TRIM9 interacts with and regulates both DCC and UNC5C during attractive and repulsive axonal responses to netrin-1.

The E3 ubiquitin ligase TRIM9 regulates synaptic function and actin dynamics in response to netrin-1.

During neuronal development, dynamic filopodia emerge from dendrites and mature into functional dendritic spines during synaptogenesis. Dendritic filopodia and spines respond to extracellular cues, influencing dendritic spine shape and size as well as synaptic function. Previously, the E3 ubiquitin ligase TRIM9 was shown to regulate filopodia in early stages of neuronal development, including netrin-1-dependent axon guidance and branching. Here, we demonstrate that TRIM9 also localizes to dendritic filopodia and spines of murine cortical and hippocampal neurons during synaptogenesis and is required for synaptic responses to netrin. In particular, TRIM9 is enriched in the postsynaptic density (PSD) within dendritic spines and loss of Trim9 alters the PSD proteome, including the actin cytoskeleton landscape. While netrin exposure induces accumulation of the Arp2/3 complex and filamentous actin in dendritic spine heads, this response is disrupted by genetic deletion of Trim9. In addition, we document changes in the synaptic receptors associated with loss of Trim9. These defects converge on a loss of netrin-dependent increases in neuronal firing rates, indicating TRIM9 is required downstream of synaptic netrin-1 signaling. We propose that TRIM9 regulates cytoskeletal dynamics in dendritic spines and is required for the proper response to synaptic stimuli.

The E3 ubiquitin ligase TRIM9 regulates synaptic function and actin dynamics.

During neuronal development, dynamic filopodia emerge from dendrites and mature into functional dendritic spines during synaptogenesis. Dendritic filopodia and spines respond to extracellular cues, influencing dendritic spine shape and size as well as synaptic function. Previously, the E3 ubiquitin ligase TRIM9 was shown to regulate filopodia in early stages of neuronal development, including netrin-1 dependent axon guidance and branching. Here we demonstrate TRIM9 also localizes to dendritic filopodia and spines of murine cortical and hippocampal neurons during synaptogenesis and is required for synaptic responses to netrin. In particular, TRIM9 is enriched in the post-synaptic density (PSD) within dendritic spines and loss of Trim9 alters the PSD proteome, including the actin cytoskeleton landscape. While netrin exposure induces accumulation of the Arp2/3 complex and filamentous actin in dendritic spine heads, this response is disrupted by genetic deletion of Trim9. In addition, we document changes in the synaptic receptors associated with loss of Trim9. These defects converge on a loss of netrin-dependent increases in neuronal firing rates, indicating TRIM9 is required downstream of synaptic netrin-1 signaling. We propose TRIM9 regulates cytoskeletal dynamics in dendritic spines and is required for the proper response to synaptic stimuli.

Proteomic analysis reveals a PLK1-dependent G2/M degradation program and a role for AKAP2 in coordinating the mitotic cytoskeleton.

Ubiquitination is an essential regulator of cell division. The kinase Polo-like kinase 1 (PLK1) promotes protein degradation at G2/M phase through the E3 ubiquitin ligase Skp1-Cul1-F box (SCF)ßTrCP. However, the magnitude to which PLK1 shapes the mitotic proteome is uncharacterized. Combining quantitative proteomics with pharmacologic PLK1 inhibition revealed a widespread, PLK1-dependent program of protein breakdown at G2/M. We validated many PLK1-regulated proteins, including substrates of the cell-cycle E3 SCFCyclin F, demonstrating that PLK1 promotes proteolysis through at least two distinct E3 ligases. We show that the protein-kinase-A-anchoring protein A-kinase anchor protein 2 (AKAP2) is cell-cycle regulated and that its mitotic degradation is dependent on the PLK1/ßTrCP signaling axis. Expression of a non-degradable AKAP2 mutant resulted in actin defects and aberrant mitotic spindles, suggesting that AKAP2 degradation coordinates cytoskeletal organization during mitosis. These findings uncover PLK1's far-reaching role in shaping the mitotic proteome post-translationally and have potential implications in malignancies where PLK1 is upregulated.

Cytoskeleton: Septin wreaths regulate actin in neuritogenesis.

The shape of a neuron changes dramatically during development. New work reports a novel septin cytoskeleton network that is important in establishing proper neuronal morphology.

Multi-monoubiquitination controls VASP-mediated actin dynamics.

The actin cytoskeleton performs multiple cellular functions, and as such, actin polymerization must be tightly regulated. We previously demonstrated that reversible, non-degradative ubiquitination regulates the function of the actin polymerase VASP in developing neurons. However, the underlying mechanism of how ubiquitination impacts VASP activity was unknown. Here we show that mimicking multi-monoubiquitination of VASP at K240 and K286 negatively regulates VASP interactions with actin. Using in vitro biochemical assays, we demonstrate the reduced ability of multi-monoubiquitinated VASP to bind, bundle, and elongate actin filaments. However, multi-monoubiquitinated VASP maintained the ability to bind and protect barbed ends from capping protein. Lastly, we demonstrate the introduction of recombinant multi-monoubiquitinated VASP protein altered cell spreading morphology. Collectively, these results suggest a mechanism in which ubiquitination controls VASP-mediated actin dynamics.

pHusion: A robust and versatile toolset for automated detection and analysis of exocytosis.

Exocytosis is a fundamental process used by eukaryotic cells to regulate the composition of the plasma membrane and facilitate cell-cell communication. To investigate the role exocytosis plays in neuronal morphogenesis, previously we developed computational tools with a graphical user interface (GUI) to enable the automatic detection and analysis of exocytic events (ADAE GUI) from fluorescence timelapse images. Though these tools have proven useful, we found that the code was brittle and not easily adapted to different experimental conditions. Here, we have developed and validated a robust and versatile toolkit, named pHusion, for the analysis of exocytosis written in ImageTank, a graphical programming language that combines image visualization and numerical methods. We tested this method using a variety of imaging modalities and pH-sensitive fluorophores, diverse cell types, and various exocytic markers to generate a flexible and intuitive package. Using pHusion, we show that VAMP3-mediated exocytosis occurs 30-times more frequently in melanoma cells compared with primary oligodendrocytes, that VAMP2-mediated fusion events in mature rat hippocampal neurons are longer lasting than those in immature murine cortical neurons, and that exocytic events are clustered in space yet random in time in developing cortical neurons. Summary Statement: Exocytosis is an essential process by which cells change shape, alter membrane composition, and communicate with other cells. Though all eukaryotic cells carry out exocytosis, the regulation of vesicle fusion, the cargo of vesicles, and the role exocytosis plays in cell fate differ greatly across cell types. Here, we developed a flexible and robust set of tools to enable automatic identification and analysis of exocytic events across a wide range of cell types, vesicle types, and imaging conditions.