Unconditioned response to a naturally aversive stimulus is associated with sensitized defensive responding and self-reported fearful traits in a PTSD sample.

Unconditioned responding (UCR) to a naturally aversive stimulus is associated with defensive responding to a conditioned threat cue (CS+) and a conditioned safety cue (CS-) in trauma-exposed individuals during fear acquisition. However, the relationships of UCR with defensive responses during extinction training, posttraumatic stress disorder (PTSD) symptom severity, and fearful traits in trauma-exposed individuals are not known. In a sample of 100 trauma-exposed adults with a continuum of PTSD severity, we recorded startle responses and skin conductance responses (SCR) during fear acquisition and extinction training using a 140 psi, 250-ms air blast to the larynx as the unconditioned stimulus. We explored dimensional associations of two different measures of UCR (unconditioned startle and unconditioned SCR) with conditioned defensive responding to CS+ and CS-, conditioned fear (CS+ minus CS-), PTSD symptom severity, and a measure of fearful traits (composite of fear survey schedule, anxiety sensitivity index, and Connor-Davidson resilience scale). Unconditioned startle was positively associated with startle potentiation to the threat cue and the safety cue across both learning phases (CS+ Acquisition, CS- Acquisition, CS+ Extinction Training, CS- Extinction Training) and with fearful traits. Unconditioned SCR was positively associated with SCR to the CS+ and CS- and SCR difference score during Acquisition. Neither type of UCR was associated with PTSD symptom severity. Our findings suggest that UCR, particularly unconditioned startle to a naturally aversive stimulus, may inform research on biomarkers and treatment targets for symptoms of pervasive and persistent fear in trauma-exposed individuals.

Predicting Fear Extinction in Posttraumatic Stress Disorder.

Fear extinction is the basis of exposure therapies for posttraumatic stress disorder (PTSD), but half of patients do not improve. Predicting fear extinction in individuals with PTSD may inform personalized exposure therapy development. The participants were 125 trauma-exposed adults (96 female) with a range of PTSD symptoms. Electromyography, electrocardiogram, and skin conductance were recorded at baseline, during dark-enhanced startle, and during fear conditioning and extinction. Using a cross-validated, hold-out sample prediction approach, three penalized regressions and conventional ordinary least squares were trained to predict fear-potentiated startle during extinction using 50 predictor variables (5 clinical, 24 self-reported, and 21 physiological). The predictors, selected by penalized regression algorithms, were included in multivariable regression analyses, while univariate regressions assessed individual predictors. All the penalized regressions outperformed OLS in prediction accuracy and generalizability, as indexed by the lower mean squared error in the training and holdout subsamples. During early extinction, the consistent predictors across all the modeling approaches included dark-enhanced startle, the depersonalization and derealization subscale of the dissociative experiences scale, and the PTSD hyperarousal symptom score. These findings offer novel insights into the modeling approaches and patient characteristics that may reliably predict fear extinction in PTSD. Penalized regression shows promise for identifying symptom-related variables to enhance the predictive modeling accuracy in clinical research.

Circulating PACAP levels are associated with increased amygdala-default mode network resting-state connectivity in posttraumatic stress disorder.

The pituitary adenylate cyclase-activating polypeptide (PACAP) system is implicated in posttraumatic stress disorder (PTSD) and related amygdala-mediated arousal and threat reactivity. PTSD is characterized by increased amygdala reactivity to threat and, more recently, aberrant intrinsic connectivity of the amygdala with large-scale resting state networks, specifically the default mode network (DMN). While the influence of PACAP on amygdala reactivity has been described, its association with intrinsic amygdala connectivity remains unknown. To fill this gap, we examined functional connectivity of resting-state functional magnetic resonance imaging (fMRI) in eighty-nine trauma-exposed adults (69 female) screened for PTSD symptoms to examine the association between blood-borne (circulating) PACAP levels and amygdala-DMN connectivity. Higher circulating PACAP levels were associated with increased amygdala connectivity with posterior DMN regions, including the posterior cingulate cortex/precuneus (PCC/Precun) and left angular gyrus (lANG). Consistent with prior work, this effect was seen in female, but not male, participants and the centromedial, but not basolateral, subregions of the amygdala. Clinical association analyses linked amygdala-PCC/Precun connectivity to anxious arousal symptoms, specifically exaggerated startle response. Taken together, our findings converge with previously demonstrated effects of PACAP on amygdala activity in PTSD-related processes and offer novel evidence for an association between PACAP and intrinsic amygdala connectivity patterns in PTSD. Moreover, these data provide preliminary evidence to motivate future work ascertaining the sex- and subregion-specificity of these effects. Such findings may enable novel mechanistic insights into neural circuit dysfunction in PTSD and how the PACAP system confers risk through a disruption of intrinsic resting-state network dynamics.

Multiverse analyses of fear acquisition and extinction retention in posttraumatic stress disorder.

Persistent fear is a cardinal feature of posttraumatic stress disorder (PTSD), and deficient fear extinction retention is a proposed illness mechanism and target of exposure-based therapy. However, evidence for deficient fear extinction in PTSD has been mixed using laboratory paradigms, which may relate to underidentified methodological variation across studies. We reviewed the literature to identify parameters that differ across studies of fear extinction retention in PTSD. We then performed Multiverse Analysis in a new sample, to quantify the impact of those methodological parameters on statistical findings. In 25 PTSD patients (15 female) and 36 trauma-exposed non-PTSD controls (TENC) (20 female), we recorded skin conductance response (SCR) during fear acquisition and extinction learning (day 1) and extinction recall (day 2). A first Multiverse Analysis examined the effects of methodological parameters identified by the literature review on comparisons of SCR-based fear extinction retention in PTSD versus TENC. A second Multiverse Analysis examined the effects of those methodological parameters on comparisons of SCR to a danger cue (CS+) versus safety cue (CS-) during fear acquisition. Both the literature review and the Multiverse Analysis yielded inconsistent findings for fear extinction retention in PTSD versus TENC, and most analyses found no statistically significant group difference. By contrast, significantly elevated SCR to CS+ versus CS- was consistently found across all analyses in the literature review and the Multiverse Analysis of new data. We discuss methodological parameters that may most contribute to inconsistent findings of fear extinction retention deficit in PTSD and implications for future clinical research.

A multi-omic dissection of super-enhancer driven oncogenic gene expression programs in ovarian cancer.

The human genome contains regulatory elements, such as enhancers, that are often rewired by cancer cells for the activation of genes that promote tumorigenesis and resistance to therapy. This is especially true for cancers that have little or no known driver mutations within protein coding genes, such as ovarian cancer. Herein, we utilize an integrated set of genomic and epigenomic datasets to identify clinically relevant super-enhancers that are preferentially amplified in ovarian cancer patients. We systematically probe the top 86 super-enhancers, using CRISPR-interference and CRISPR-deletion assays coupled to RNA-sequencing, to nominate two salient super-enhancers that drive proliferation and migration of cancer cells. Utilizing Hi-C, we construct chromatin interaction maps that enable the annotation of direct target genes for these super-enhancers and confirm their activity specifically within the cancer cell compartment of human tumors using single-cell genomics data. Together, our multi-omic approach examines a number of fundamental questions about how regulatory information encoded into super-enhancers drives gene expression networks that underlie the biology of ovarian cancer.

Enhancer RNA Transcription Is Essential for a Novel CSF1 Enhancer in Triple-Negative Breast Cancer.

Enhancers are critical regulatory elements in the genome that help orchestrate spatiotemporal patterns of gene expression during development and normal physiology. In cancer, enhancers are often rewired by various genetic and epigenetic mechanisms for the activation of oncogenes that lead to initiation and progression. A key feature of active enhancers is the production of non-coding RNA molecules called enhancer RNAs, whose functions remain unknown but can be used to specify active enhancers de novo. Using a combination of eRNA transcription and chromatin modifications, we have identified a novel enhancer located 30 kb upstream of Colony Stimulating Factor 1 (CSF1). Notably, CSF1 is implicated in the progression of breast cancer, is overexpressed in triple-negative breast cancer (TNBC) cell lines, and its enhancer is primarily active in TNBC patient tumors. Genomic deletion of the enhancer (via CRISPR/Cas9) enabled us to validate this regulatory element as a bona fide enhancer of CSF1 and subsequent cell-based assays revealed profound effects on cancer cell proliferation, colony formation, and migration. Epigenetic silencing of the enhancer via CRISPR-interference assays (dCas9-KRAB) coupled to RNA-sequencing, enabled unbiased identification of additional target genes, such as RSAD2, that are predictive of clinical outcome. Additionally, we repurposed the RNA-guided RNA-targeting CRISPR-Cas13 machinery to specifically degrade the eRNAs transcripts produced at this enhancer to determine the consequences on CSF1 mRNA expression, suggesting a post-transcriptional role for these non-coding transcripts. Finally, we test our eRNA-dependent model of CSF1 enhancer function and demonstrate that our results are extensible to other forms of cancer. Collectively, this work describes a novel enhancer that is active in the TNBC subtype, which is associated with cellular growth, and requires eRNA transcripts for proper enhancer function. These results demonstrate the significant impact of enhancers in cancer biology and highlight their potential as tractable targets for therapeutic intervention.

Exploring the impact of trauma type and extent of exposure on posttraumatic alterations in 5-HT1A expression.

The long-term behavioral, psychological, and neurobiological effects of exposure to potentially traumatic events vary within the human population. Studies conducted on trauma-exposed human subjects suggest that differences in trauma type and extent of exposure combine to affect development, maintenance, and treatment of a variety of psychiatric syndromes. The serotonin 1-A receptor (5-HT1A) is an inhibitory G protein-coupled serotonin receptor encoded by the HTR1A gene that plays a role in regulating serotonin release, physiological stress responding, and emotional behavior. Studies from the preclinical and human literature suggest that dysfunctional expression of 5-HT1A is associated with a multitude of psychiatric symptoms commonly seen in trauma-exposed individuals. Here, we synthesize the literature, including numerous preclinical studies, examining differences in alterations in 5-HT1A expression following trauma exposure. Collectively, these findings suggest that the impact of trauma exposure on 5-HT1A expression is dependent, in part, on trauma type and extent of exposure. Furthermore, preclinical and human studies suggest that this observation likely applies to additional molecular targets and may help explain variation in trauma-induced changes in behavior and treatment responsivity. In order to understand the neurobiological impact of trauma, including the impact on 5-HT1A expression, it is crucial to consider both trauma type and extent of exposure.

Transcriptional control by enhancers and enhancer RNAs.

The regulation of gene expression is a fundamental cellular process and its misregulation is a key component of disease. Enhancers are one of the most salient regulatory elements in the genome and help orchestrate proper spatiotemporal gene expression during development, in homeostasis, and in response to signaling. Notably, molecular aberrations at enhancers, such as translocations and single nucleotide polymorphisms, are emerging as an important source of human variation and susceptibility to disease. Herein we discuss emerging paradigms addressing how genes are regulated by enhancers, common features of active enhancers, and how non-coding enhancer RNAs (eRNAs) can direct gene expression programs that underlie cellular phenotypes. We survey the current evidence, which suggests that eRNAs can bind to transcription factors, mediate enhancer-promoter interactions, influence RNA Pol II elongation, and act as decoys for repressive cofactors. Furthermore, we discuss current methodologies for the identification of eRNAs and novel approaches to elucidate their functions.

A mouse model of Angelman syndrome imprinting defects.

Angelman syndrome, Prader-Will syndrome and Dup15q syndrome map to a cluster of imprinted genes located at 15q11-q13. Imprinting at this domain is regulated by an imprinting control region consisting of two distinct elements, the Angelman syndrome imprinting center (AS-IC) and the Prader-Willi syndrome imprinting center (PWS-IC). Individuals inheriting deletions of the AS-IC exhibit reduced expression of the maternally expressed UBE3A gene and biallelic expression of paternal-only genes. We have previously demonstrated that AS-IC activity partly consists of providing transcription across the PWS-IC in oocytes, and that these transcripts are necessary for maternal imprinting of Snrpn. Here we report a novel mouse mutation that truncates transcripts prior to transiting the PWS-IC and results in a domain-wide imprinting defect. These results confirm a transcription-based model for imprint setting at this domain. The imprinting defect can be preempted by removal of the transcriptional block in oocytes, but not by its removal in early embryos. Imprinting defect mice exhibit several traits often found in individuals with Angelman syndrome imprinting defects.

The Maize MID-COMPLEMENTING ACTIVITY Homolog CELL NUMBER REGULATOR13/NARROW ODD DWARF Coordinates Organ Growth and Tissue Patterning.

Organogenesis occurs through cell division, expansion, and differentiation. How these cellular processes are coordinated remains elusive. The maize (Zea mays) leaf provides a robust system to study cellular differentiation due to its distinct tissues and cell types. The narrow odd dwarf (nod) mutant displays defects at both the cellular and tissue level that increase in severity throughout growth. nod mutant leaves have reduced size due to fewer and smaller cells compared with the wild type. The juvenile-to-adult transition is delayed, and proximal distal-patterning is abnormal in this mutant. Differentiation of specialized cells such as those forming stomata and trichomes is incomplete. Analysis of nod-1 sectors suggests that NOD plays a cell-autonomous function in the leaf. We cloned nod positionally and found that it encodes CELL NUMBER REGULATOR13 (CNR13), the maize MID-COMPLEMENTING ACTIVITY homolog. CNR13/NOD is localized to the membrane and is enriched in dividing tissues. Transcriptome analysis of nod mutants revealed overrepresentation of cell wall, hormone metabolism, and defense gene categories. We propose that NOD coordinates cell activity in response to intrinsic and extrinsic cues.

Keep on growing: building and patterning leaves in the grasses.

Monocot leaves have unique features that arise early in their development. Maturing leaves protectively enclose younger leaves and the meristem, the pool of founder cells from which a leaf emerges. Through the maturation process, proximal sheath and distal blade tissues differentiate and are separated by the ligule and auricle structures. Here we review current research focusing on the contribution of gene regulatory factors and phytohormones on the patterning and differentiation of monocot leaves primarily focusing on research in the grasses (Poaceae). The 10000 members of the grasses include the true grain cereals (wheat, rice, maize, etc.), biofuel crops such as sugarcane, pasture grasses, and bamboo. They are the most studied of the monocots due to their tremendous agricultural and agronomic importance.

Angelman syndrome imprinting center encodes a transcriptional promoter.

Clusters of imprinted genes are often controlled by an imprinting center that is necessary for allele-specific gene expression and to reprogram parent-of-origin information between generations. An imprinted domain at 15q11-q13 is responsible for both Angelman syndrome (AS) and Prader-Willi syndrome (PWS), two clinically distinct neurodevelopmental disorders. Angelman syndrome arises from the lack of maternal contribution from the locus, whereas Prader-Willi syndrome results from the absence of paternally expressed genes. In some rare cases of PWS and AS, small deletions may lead to incorrect parent-of-origin allele identity. DNA sequences common to these deletions define a bipartite imprinting center for the AS-PWS locus. The PWS-smallest region of deletion overlap (SRO) element of the imprinting center activates expression of genes from the paternal allele. The AS-SRO element generates maternal allele identity by epigenetically inactivating the PWS-SRO in oocytes so that paternal genes are silenced on the future maternal allele. Here we have investigated functional activities of the AS-SRO, the element necessary for maternal allele identity. We find that, in humans, the AS-SRO is an oocyte-specific promoter that generates transcripts that transit the PWS-SRO. Similar upstream promoters were detected in bovine oocytes. This result is consistent with a model in which imprinting centers become DNA methylated and acquire maternal allele identity in oocytes in response to transiting transcription.

Gene regulatory interactions at lateral organ boundaries in maize.

Maize leaves have distinct tissues that serve specific purposes. The blade tilts back to photosynthesize and the sheath wraps around the stem to provide structural support and protect young leaves. At the junction between blade and sheath are the ligule and auricles, both of which are absent in the recessive liguleless1 (lg1) mutant. Using an antibody against LG1, we reveal LG1 accumulation at the site of ligule formation and in the axil of developing tassel branches. The dominant mutant Wavy auricle in blade1 (Wab1-R) produces ectopic auricle tissue in the blade and increases the domain of LG1 accumulation. We determined that wab1 encodes a TCP transcription factor by positional cloning and revertant analysis. Tassel branches are few and upright in the wab1 revertant tassel and have an increased branch angle in the dominant mutant. wab1 mRNA is expressed at the base of branches in the inflorescence and is necessary for LG1 expression. wab1 is not expressed in leaves, except in the dominant mutant. The domain of wab1 expression in the Wab1-R leaf closely mirrors the accumulation of LG1. Although wab1 is not needed to induce lg1 expression in the leaf, LG1 is needed to counteract the severe phenotype of the dominant Wab1-R mutant. The regulatory interaction of LG1 and WAB1 reveals a link between leaf shape and tassel architecture, and suggests the ligule is a boundary similar to that at the base of lateral organs.

Regulatory modules controlling maize inflorescence architecture.

Genetic control of branching is a primary determinant of yield, regulating seed number and harvesting ability, yet little is known about the molecular networks that shape grain-bearing inflorescences of cereal crops. Here, we used the maize (Zea mays) inflorescence to investigate gene networks that modulate determinacy, specifically the decision to allow branch growth. We characterized developmental transitions by associating spatiotemporal expression profiles with morphological changes resulting from genetic perturbations that disrupt steps in a pathway controlling branching. Developmental dynamics of genes targeted in vivo by the transcription factor RAMOSA1, a key regulator of determinacy, revealed potential mechanisms for repressing branches in distinct stem cell populations, including interactions with KNOTTED1, a master regulator of stem cell maintenance. Our results uncover discrete developmental modules that function in determining grass-specific morphology and provide a basis for targeted crop improvement and translation to other cereal crops with comparable inflorescence architectures.

The SERK1 receptor-like kinase regulates organ separation in Arabidopsis flowers.

Through a sensitized screen for novel components of pathways regulating organ separation in Arabidopsis flowers, we have found that the leucine-rich repeat receptor-like kinase SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE1 (SERK1) acts as a negative regulator of abscission. Mutations in SERK1 dominantly rescue abscission in flowers without functional NEVERSHED (NEV), an ADP-ribosylation factor GTPase-activating protein required for floral organ shedding. We previously reported that the organization of the Golgi apparatus and location of the trans-Golgi network (TGN) are altered in nev mutant flowers. Disruption of SERK1 restores Golgi structure and the close association of the TGN in nev flowers, suggesting that defects in these organelles may be responsible for the block in abscission. We have also found that the abscission zones of nev serk1 flowers are enlarged compared to wild-type. A similar phenotype was previously observed in plants constitutively expressing a putative ligand required for organ separation, INFLORESCENCE DEFICIENT IN ABSCISSION (IDA), suggesting that signalling through IDA and its proposed receptors, HAESA and HAESA-LIKE2, may be deregulated in nev serk1 abscission zone cells. Our studies indicate that in addition to its previously characterized roles in stamen development and brassinosteroid perception, SERK1 plays a unique role in modulating the loss of cell adhesion that occurs during organ abscission.

The EVERSHED receptor-like kinase modulates floral organ shedding in Arabidopsis.

Plant cell signaling triggers the abscission of entire organs, such as fruit, leaves and flowers. Previously, we characterized an ADP-ribosylation factor GTPase-activating protein, NEVERSHED (NEV), that regulates membrane trafficking and is essential for floral organ shedding in Arabidopsis. Through a screen for mutations that restore organ separation in nev flowers, we have identified a leucine-rich repeat receptor-like kinase, EVERSHED (EVR), that functions as an inhibitor of abscission. Defects in the Golgi structure and location of the trans-Golgi network in nev abscission zone cells are rescued by a mutation in EVR, suggesting that EVR might regulate membrane trafficking during abscission. In addition to shedding their floral organs prematurely, nev evr flowers show enlarged abscission zones. A similar phenotype was reported for plants ectopically expressing INFLORESCENCE DEFICIENT IN ABSCISSION, a predicted signaling ligand for the HAESA/HAESA-LIKE2 receptor-like kinases, indicating that this signaling pathway may be constitutively active in nev evr flowers. We present a model in which EVR modulates the timing and region of abscission by promoting the internalization of other receptor-like kinases from the plasma membrane.

Lectin mapping reveals stage-specific display of surface carbohydrates in in vitro and haemolymph-derived cells of the entomopathogenic fungus Beauveria bassiana.

The entomopathogenic fungus Beauveria bassiana and its insect host target represent a model system with which to examine host-pathogen interactions. Carbohydrate epitopes on the surfaces of fungal cells play diverse roles in processes that include adhesion, non-self recognition and immune invasion with respect to invertebrate hosts. B. bassiana produces a number of distinct cell types that include aerial conidia, submerged conidia, blastospores and haemolymph-derived cells termed in vivo blastospores or hyphal bodies. In order to characterize variations in the surface carbohydrate epitopes among these cells, a series of fluorescently labelled lectins, combined with confocal microscopy and flow cytometry to quantify the response, was used. Aerial conidia displayed the most diverse lectin binding characteristics, showing reactivity against concanavalin A (ConA), Galanthus nivalis (GNL), Griffonia simplicifolia (GSII), Helix pomatia (HPA), Griffonia simplicifolia isolectin (GSI), peanut agglutinin (PNA), Ulex europaeus agglutinin I (UEAI) and wheatgerm agglutinin (WGA), and weak reactivity against Ricinus communis I (RCA), Sambucus nigra (SNA), Limax flavus (LFA) and Sophora japonica (SJA) lectins. Lectin binding to submerged conidia was similar to that to aerial conidia, except that no reactivity against UEAI, HPA and SJA was noted, and WGA appeared to bind strongly at specific polar spots. In contrast, the majority of in vitro blastospores were not bound by ConA, GNL, GSII, GSI, SNA, UEAI, LFA or SJA, with PNA binding in large patches, and some polarity in WGA binding noted. Significant changes in lectin binding also occurred after aerial conidial germination and in cells grown on either lactose or trehalose. For germinated conidia, differential lectin binding was noted between the conidial base, the germ tube and the hyphal tip. Fungal cells isolated from the haemolymph of the infected insect hosts Manduca sexta and Heliothis virescens appeared to shed most carbohydrate epitopes, displaying binding only to the GNL, PNA and WGA lectins. Ultrastructural examination of the haemolymph-derived cells revealed the presence of a highly ordered outermost brush-like structure not present on any of the in vitro cells. Haemolymph-derived hyphal bodies placed into rich broth medium showed expression of several surface carbohydrate epitopes, most notably showing increased PNA binding and strong binding by the RCA lectin. These data indicate robust and diverse production of carbohydrate epitopes on different developmental stages of fungal cells and provide evidence that surface carbohydrates are elaborated in infection-specific patterns.

Uptake of the fluorescent probe FM4-64 by hyphae and haemolymph-derived in vivo hyphal bodies of the entomopathogenic fungus Beauveria bassiana.

The entomopathogenic fungus Beauveria bassiana is under intensive study as a pest biological control agent. B. bassiana produces several distinct single-cell types that include aerial conidia, in vitro blastospores and submerged conidia. Under appropriate nutrient conditions these cells can elaborate germ tubes that form hyphae, which in turn lead to the formation of a fungal mycelium. In addition, B. bassiana displays a dimorphic transition, producing in vivo specific yeast-like hyphal bodies during growth in the arthropod haemolymph. The amphiphilic styryl dye FM4-64 was used to investigate internalization and morphological features of in vitro and in vivo insect haemolymph-derived B. bassiana cells. In vitro blastospores and submerged conidia displayed a punctate pattern of internal labelling, whereas aerial conidia failed to internalize the dye under the conditions tested. FM4-64 was also taken up into both apical and subapical compartments of living hyphae in a time-dependent manner, with clearly observable vesicle labelling. Internalization, where occurring, was reversibly disrupted by lowering the temperature of the assay or by treatment with azide/fluoride and latrunculin A. Treatment with cytochalasin D and monensin also caused abnormal vesicle trafficking, although some staining of vesicles was noted. Fungal cells derived from infected Heliothis virescens haemolymph (in vivo cells) actively internalized FM4-64. The in vivo blastospores or hyphal bodies displayed bright membrane and internal vesicle staining, although diffuse staining of internal structures was also visible. These results suggest active uptake by different developmental stages of B. bassiana, including haemolymph-derived cells that can evade the insect immune system.

Regulation of membrane trafficking and organ separation by the NEVERSHED ARF-GAP protein.

Cell separation, or abscission, is a highly specialized process in plants that facilitates remodeling of their architecture and reproductive success. Because few genes are known to be essential for organ abscission, we conducted a screen for mutations that alter floral organ shedding in Arabidopsis. Nine recessive mutations that block shedding were found to disrupt the function of an ADP-ribosylation factor-GTPase-activating protein (ARF-GAP) we have named NEVERSHED (NEV). As predicted by its homology to the yeast Age2 ARF-GAP and transcriptional profile, NEV influences other aspects of plant development, including fruit growth. Co-localization experiments carried out with NEV-specific antiserum and a set of plant endomembrane markers revealed that NEV localizes to the trans-Golgi network and endosomes in Arabidopsis root epidermal cells. Interestingly, transmission electron micrographs of abscission zone regions from wild-type and nev flowers reveal defects in the structure of the Golgi apparatus and extensive accumulation of vesicles adjacent to the cell walls. Our results suggest that NEV ARF-GAP activity at the trans-Golgi network and distinct endosomal compartments is required for the proper trafficking of cargo molecules required for cell separation.