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Vesicle and target membrane fusion involves tethering, docking and fusion. The GTPase SECRETORY4 (SEC4) positions the exocyst complex during vesicle membrane tethering, facilitating docking and fusion. Glycine max (soybean) Sec4 functions in the root during its defense against the parasitic nematode Heterodera glycines as it attempts to develop a multinucleate nurse cell (syncytium) serving to nourish the nematode over its 30-day life cycle. Results indicate that other tethering proteins are also important for defense. The G. max exocyst is encoded by 61 genes: 5 EXOC1 (Sec3), 2 EXOC2 (Sec5), 5 EXOC3 (Sec6), 2 EXOC4 (Sec8), 2 EXOC5 (Sec10) 6 EXOC6 (Sec15), 31 EXOC7 (Exo70) and 8 EXOC8 (Exo84) genes. At least one member of each gene family is expressed within the syncytium during the defense response. Syncytium-expressed exocyst genes function in defense while some are under transcriptional regulation by mitogen-activated protein kinases (MAPKs). The exocyst component EXOC7-H4-1 is not expressed within the syncytium but functions in defense and is under MAPK regulation. The tethering stage of vesicle transport has been demonstrated to play an important role in defense in the G. max-H. glycines pathosystem, with some of the spatially and temporally regulated exocyst components under transcriptional control by MAPKs.

Insulin promotes glucose uptake by triggering the translocation of glucose transporter type 4 (GLUT4) from intracellular vesicles to the plasma membrane through exocytosis. GLUT4 exocytosis is a vesicle fusion event involving fusion of GLUT4-containing vesicles with the plasma membrane. For GLUT4 vesicle fusion to occur, GLUT4 vesicles must first be tethered to the plasma membrane. A key tethering factor in exocytosis is a heterooctameric protein complex called the exocyst. The role of the exocyst in GLUT4 exocytosis, however, remains incompletely understood. Here we first systematically analyzed data from a genome-scale CRISPR screen in HeLa cells that targeted virtually all known genes in the human genome, including 12 exocyst genes. The screen recovered only a subset of the exocyst genes, including exocyst complex component 7 (Exoc7/Exo70). Other exocyst genes, however, were not isolated in the screen, likely because of functional redundancy. Our findings suggest that selection of an appropriate exocyst gene is critical for genetic studies of exocyst functions. Next we developed an inducible adipocyte genome editing system that enabled Exoc7 gene deletion in adipocytes without interfering with adipocyte differentiation. We observed that insulin-stimulated GLUT4 exocytosis was markedly inhibited in Exoc7 KO adipocytes. Insulin signaling, however, remained intact in these KO cells. These results indicate that the exocyst plays a critical role in insulin-stimulated GLUT4 exocytosis in adipocytes. We propose that the strategy outlined in this work could be instrumental in genetically dissecting other membrane-trafficking pathways in adipocytes.

Polyglutamine (polyQ) diseases comprise Huntington’s disease and several subtypes of spinocerebellar ataxia, including spinocerebellar ataxia type 3 (SCA3). The genomic expansion of coding CAG trinucleotide sequence in disease genes leads to the production and accumulation of misfolded polyQ domain-containing disease proteins, which cause cellular dysfunction and neuronal death. As one of the principal cellular protein clearance pathways, the activity of the ubiquitin–proteasome system (UPS) is tightly regulated to ensure efficient clearance of damaged and toxic proteins. Emerging evidence demonstrates that UPS plays a crucial role in the pathogenesis of polyQ diseases. Ubiquitin (Ub) E3 ligases catalyze the transfer of a Ub tag to label proteins destined for proteasomal clearance. In this study, we identified an E3 ligase, pre-mRNA processing factor 19 (Prpf19/prp19), that modulates expanded ataxin-3 (ATXN3-polyQ), disease protein of SCA3, induced neurodegeneration in both mammalian and Drosophila disease models. We further showed that Prpf19/prp19 promotes poly-ubiquitination and degradation of mutant ATXN3-polyQ protein. Our data further demonstrated the nuclear localization of Prpf19/prp19 is essential for eliciting its modulatory function towards toxic ATXN3-polyQ protein. Intriguingly, we found that exocyst complex component 7 (Exoc7/exo70), a Prpf19/prp19 interacting partner, modulates expanded ATXN3-polyQ protein levels and toxicity in an opposite manner to Prpf19/prp19. Our data suggest that Exoc7/exo70 exerts its ATXN3-polyQ-modifying effect through regulating the E3 ligase function of Prpf19/prp19. In summary, this study allows us to better define the mechanistic role of Exoc7/exo70-regulated Prpf19/prp19-associated protein ubiquitination pathway in SCA3 pathogenesis.

Purpose The exocyst complex is a conserved protein complex that mediates fusion of intracellular vesicles to the plasma membrane and is implicated in processes including cell polarity, cell migration, ciliogenesis, cytokinesis, autophagy, and fusion of secretory vesicles. The essential role of these genes in human genetic disorders, however, is unknown. Methods We performed homozygosity mapping and exome sequencing of consanguineous families with recessively inherited brain development disorders. We modeled an EXOC7 splice variant in vitro and examined EXOC7 messenger RNA (mRNA) expression in developing mouse and human cortex. We modeled exoc7 loss-of-function in a zebrafish knockout. Results We report variants in exocyst complex members, EXOC7 and EXOC8, in a novel disorder of cerebral cortex development. In EXOC7, we identified four independent partial loss-of-function (LOF) variants in a recessively inherited disorder characterized by brain atrophy, seizures, and developmental delay, and in severe cases, microcephaly and infantile death. In EXOC8, we found a homozygous truncating variant in a family with a similar clinical disorder. We modeled exoc7 deficiency in zebrafish and found the absence of exoc7 causes microcephaly. Conclusion Our results highlight the essential role of the exocyst pathway in normal cortical development and how its perturbation causes complex brain disorders.

Colorectal cancer (CRC) is the third most frequent cancer and the fourth most lethal in the world. Recently, switches in specific alternative splicing (AS) events in cancer-associated genes, have been correlated with the acquisition of proliferative advantage for cells. Thus, using the public repository of The Cancer Genome Atlas (TCGA), the group of Nuno Barbosa-Morais at iMM have identified a signature of 46 AS events that significantly correlated with overall survival of colorectal cancer (CRC) patients, independent of gene expression. They found that inclusion of part of exon 8 from the EXOC7 gene (expression of isoform 5 rather than isoform 2) is one of these 46 AS events with a high prognostic value in CRC. EXOC7 is a key component of the exocyst complex and plays an important role in several cellular processes such as migration. Furthermore, differential expression of EXOC7 isoforms had already been correlated with breast tumour progression. In this project, we aim to validate the prognostic value of EXOC7 isoforms in a cohort of human CRC samples and explore its biological role in vitro. Overall, our results showed that levels of expression of EXOC7 isoform 5 are a good prognostic marker in CRC, independent of EXOC7 total expression. However, cellular overexpression of isoform 5 and 2 do not seem to affect biological processes such as exocytosis, epithelial-to-mesenchymal transition (EMT), or cytoskeleton organization. Finally, we believe that our study has allowed the validation of a good prognostic marker in CRC, not only, important in the identification of patients most likely to progress, but also, revealing a potential target of therapy. Nevertheless, more studies are needed in order to unravel the biological role of EXOC7 isoforms in vitro.

The octomeric exocyst complex governs the final step of exocytosis in both plants and animals. Its roles, however, extend beyond exocytosis and include organelle biogenesis, ciliogenesis, cell migration, and cell growth. Exo70 is a conserved component of the exocyst whose function in Drosophila is unclear. In this study, we characterized two mutant alleles of Drosophila exo70. exo70 mutants exhibit reduced synaptic growth, locomotor activity, glutamate receptor density, and mEPSP amplitude. We found that presynaptic Exo70 is necessary for normal synaptic growth at the neuromuscular junction (NMJ). At the neuromuscular junction, exo70 genetically interacts with the small GTPase ralA to regulate synaptic growth. Loss of Exo70 leads to the blockage of JNK signaling-, activity-, and temperature-induced synaptic outgrowths. We showed that this phenotype is associated with an impairment of integral membrane protein transport to the cell surface at synaptic terminals. In octopaminergic motor neurons, Exo70 is detected in synaptic varicosities, as well as the regions of membrane extensions in response to activity stimulation. Strikingly, mild thermal stress causes severe neurite outgrowth defects and pharate adult lethality in exo70 mutants. exo70 mutants also display defective locomotor activity in response to starvation stress. These results demonstrated that Exo70 is an important regulator of induced synaptic growth and is crucial for an organism9s adaptation to environmental changes. SIGNIFICANCE STATEMENT The exocyst complex is a conserved protein complex directing secretory vesicles to the site of membrane fusion during exocytosis, which is essential for transporting proteins and membranes to the cell surface. Exo70 is a subunit of the exocyst complex whose roles in neurons remain elusive, and its function in Drosophila is unclear. In Drosophila, Exo70 is expressed in both glutamatergic and octopaminergic neurons, and presynaptic Exo70 regulates synaptic outgrowth. Moreover, exo70 mutants have impaired integral membrane transport to the cell surface at synaptic terminals and block several kinds of induced synaptic growth. Remarkably, elevated temperature causes severe arborization defects and lethality in exo70 mutants, thus underpinning the importance of Exo70 functions in development and adaptation to the environment.

Summary Oncogene-induced senescence is a potent tumor-suppressive response. Paradoxically, senescence also induces an inflammatory secretome that promotes carcinogenesis and age-related pathologies. Consequently, the senescence-associated secretory phenotype (SASP) is a potential therapeutic target. Here, we describe an RNAi screen for SASP regulators. We identified 50 druggable targets whose knockdown suppresses the inflammatory secretome and differentially affects other SASP components. Among the screen candidates was PTBP1. PTBP1 regulates the alternative splicing of genes involved in intracellular trafficking, such as EXOC7, to control the SASP. Inhibition of PTBP1 prevents the pro-tumorigenic effects of the SASP and impairs immune surveillance without increasing the risk of tumorigenesis. In conclusion, our study identifies SASP inhibition as a powerful and safe therapy against inflammation-driven cancer., Graphical Abstract, Highlights • An RNAi screen identifies 50 specific SASP regulators • The splicing factor PTBP1 regulates a pro-inflammatory SASP subset • PTBP1 regulates alternative splicing of EXOC7 to control the SASP • PTBP1 depletion safely inhibits inflammation-driven cancer, By performing a genetic screen for regulators of the senescence-associated secretory phenotype (SASP), Georgilis et al. identify PTBP1, which controls SASP by regulating alternative splicing of genes involved in intracellular trafficking such as EXOC7. PTBP1 knockdown blocks the tumor-promoting functions of SASP.

Sec8 is one of the subunits of the exocyst, which is an evolutionarily conserved complex of eight proteins, comprising Sec3 (EXOC1), Sec5 (EXOC2), Sec6 (EXOC3), Sec8 (EXOC4), Sec10 (EXOC5), Sec15 (EXOC6), Exo70 (EXOC7), and Exo84 (EXOC8) subunits. Sec8 knockout mice embryos initiate gastrulation but are unable to progress beyond the primitive streak stage and die shortly. During embryonic development, the first epithelial-mesenchymal transition (EMT) event occurs at gastrulation. Sec8 may be involved in the early embryonic development through EMT. However, the function of Sec8 in EMT remains unclear. In the present study, it was found that Sec8 regulates N-cadherin expression by controlling Smad3 and Smad4 expression at the basal transcriptional level, thereby modulating cell migration and adhesion. Furthermore, Sec8 knockdown decreased CREB binding protein (CBP) expression at mRNA and protein levels. However, CBP knockdown did not affect Sec8 expression. These results indicated that Sec8 regulates N-cadherin expression by controlling Smad3 and Smad4 expression through CBP, thereby mediating the EMT.

The process of host cell invasion by Trypanosoma cruzi shares mechanistic elements with plasma membrane injury and repair. Both processes require Ca(2+)-triggered exocytosis of lysosomes, exocytosis of acid sphingomyelinase and formation of ceramide-enriched endocytic compartments. T. cruzi invades at peripheral sites, suggesting a need for spatial regulation of membrane traffic. Here, we show that Exo70 and Sec8 (also known as EXOC7 and EXOC4, respectively), components of the exocyst complex, accumulate in nascent T. cruzi vacuoles and at sites of mechanical wounding. Exo70 or Sec8 depletion inhibits T. cruzi invasion and Ca(2+)-dependent resealing of mechanical wounds, but does not affect the repair of smaller lesions caused by pore-forming toxins. Thus, T. cruzi invasion and mechanical lesion repair share a unique requirement for the exocyst, consistent with a dependence on targeted membrane delivery.

The exocyst is a protein complex that ensures spatial targeting of exocytotic vesicles to the plasma membrane. We present microarray data obtained from differentiating mouse embryonic stem cell cultures that identify an up-regulation of exocyst complex component 3-like 2 (exoc3l2) mRNA in sprouting blood vessels. Vascular expression of exoc3l2 is confirmed by qPCR analysis of different mouse tissues and immunofluorescence analyses of mouse brain sections. We detect an up-regulation of exoc3l2 mRNA synthesis in primary human endothelial cells in response to VEGFA, and this response is enhanced when the cells are grown on a three-dimensional collagen I matrix. Myc-tagged EXOC3L2 co-precipitates with the exocyst protein EXOC4, and immunofluorescence detection of EXOC3L2 shows partial subcellular colocalization with EXOC4 and EXOC7. Finally, we show that exoc3l2 silencing inhibits VEGF receptor 2 phosphorylation and VEGFA-directed migration of cultured endothelial cells.