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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.

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.

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.